ISCEV Standard for Clinical Electro-oculography (EOG) 2006

The Clinical Electro-oculogram (EOG) is an electrophysiological test of function of the outer retina and retinal pigment epithelium (RPE) in which the change in the electrical potential between the cornea and the ocular fundus is recorded during successive periods of dark and light adaptation. This document sets out a Standard Method for performance of the test, and also gives detailed guidance on technical and practical issues, and on reporting test results. The main object of the Standard is to promote consistent quality of testing and reporting within and between centres. This 2006 Standard, from the International Society for Clinical Electrophysiology of Vision (ISCEV: www.iscev.org ), is a revision of the previous Standard published in 1993, and reviewed and re-issued in 1998.
Purpose and use of this Standard
This Standard is one of a series of Standards and Guidelines for electrophysiology of vision [1–8] available for download from www.iscev.org, and is a revision of the ISCEV Standard for Clinical Electro-oculography (first issued 1993 – re-approved 1998) [1, 2], which provides a Standard Method for measurement of the electro-oculogram (EOG). Also included is advice on possible variations on the Standard Method, additional tests, and comment on the procedures recommended. The major change in this current standard compared with the earlier EOG standards is that it recommends one single standard testing protocol and measurement strategy.
Clinical and research users of the clinical EOG are encouraged to use the current Standard Method where possible, to achieve consistency of results within and between test centres. Reports of EOG recordings performed to the Standard Method given here should cite this 2006 Standard. Where a method is used which deviates from the Standard Method, the deviations should be stated, together with any normative or reference data. Where the method used conforms to a previous EOG Standard, this may be cited instead.
EOG origins, pathological effects, and principles of measurement
Electrophysiology of the RPE in dark and light adaptation
The eye has a standing electrical potential between front and back, sometimes called the corneo-fundal potential. The potential is mainly derived from the retinal pigment epithelium (RPE), and it changes in response to retinal illumination. The potential decreases for 8–10 min in darkness. Subsequent retinal illumination causes an initial fall in the standing potential over 60–75 s (the fast oscillation (FO)), followed by a slow rise for 7–14 min (the light response). These phenomena arise from ion permeability changes across the basal RPE membrane. The clinical electro-oculogram (EOG) makes an indirect measurement of the minimum amplitude of the standing potential in the dark and then again at its peak after the light rise. This is usually expressed as a ratio of ‘light peak to dark trough’ and referred to as the Arden ratio. The behaviour of the corneo-fundal potential in the normal eye is predictable in defined conditions, such as those described in this Standard, but changing from dark to light actually initiates a triggered response extending for about 2 h in the form of a diminishing sinusoidal oscillation.
Diseases affecting the light response of the EOG
The light response is affected in diffuse disorders of the RPE and the photoreceptor layer of the retina including some characterised by rod dysfunction, or chorio-retinal atrophic and inflammatory diseases. In most of these there is correlation with the electroretinogram (ERG), except notably in the case of Best’s vitelliform maculopathy, in which the clinical EOG is usually highly abnormal in the presence of a normal ERG.
Measurement of the clinical EOG
The potential across the RPE causes the front of the eye to be electrically positive compared to the back. As a result, potentials measured between two electrodes placed on the skin at each side of an eye will change as the eye turns from left to right. The EOG method is used widely to record eye movements, on the assumption of unchanging corneo-fundal potentials. In the clinical EOG described here, we use defined eye movements to monitor the changes in corneo-fundal potential. If the test subject looks alternately at targets a fixed angle apart, the potential recorded from the skin resembles a square wave whose amplitude will be a fixed proportion of the corneo-fundal potential. During a light/dark cycle, this indirectly measured potential will change in the same way as the source potentials, so that the Arden ratios (and timing of peaks etc.) will be a close approximation to the average changes occurring across the RPE.
The Standard Method
This section outlines the Standard Clinical EOG method, definitions, explanations, and instrument specifications. Further notes regarding testing strategies are given in later sections.
Pupils Apply dilating drops before any other action to allow pupil dilation to be relatively complete by the start of the test (see notes below). Their size should be checked at the start and end of the test and recorded at the end of the test.
Electrodes After suitable skin preparation, place small recording electrodes, close to the canthi of each eye as in Fig. 1. Connect the electrodes from each eye to separate channels of a differential amplifier. The ‘ground’ electrode can be placed on the forehead. The impedance between any pair of electrodes should not exceed 5 kΩ. The electrodes, amplifier and impedance meter must be approved for medical use.
Fig. 1(a and b). Recording electrode positions
Amplifier This should have a band pass of either 0 (d.c.) to 30 Hz, or 0.1 to 30 Hz, to provide recordings of the saccades which appear as square waves.
Full field (Ganzfeld) stimulator This should have a comfortable head/chin rest, and two red fixation lights 15 degrees left and right of centre. The fixation lights should be bright when the light adapting background is on, and as dim as practical in the dark.
Pre-adaptation The test subject should be in stable indoor lighting for as long as possible before the test, and should not be exposed to any large changes in lighting (lighter/darker) during this period, such as indirect ophthalmoscopy. As near as is practical, the pre-test light exposure should be the same for all test subjects.
Preparing the test subject Explain the procedure including, ‘chin/head on rest/restraint in stimulator, 15 min dark, 15 min light, fixation lights alternate in simple rhythmic manner, for 10 s each minute, when lights change, move eyes in single sweep to next one, do not turn head, do not anticipate the changes’. Practice the procedure with the recording system on and coach the subject if there is head movement, overshoot, stepping, or anticipation.
Dark phase In total darkness for 15 min, except for the dim fixation lights, alternate the fixation lights every 1 s for 10 s every 1 min, and record the resulting EOG potentials. The test subject should remain looking into the stimulator the whole time if possible, and should be warned of the start of each measurement sequence to ensure attention. The operator must have an instant view of the recordings to check for patient compliance, and errors such as overshoot.
Light phase Bring on the ganzfeld background light of 100 photopic cd/m2 . If necessary, bring this on gradually over a short period (e.g. 20 s) for patient comfort, especially in cases of photophobia. Continue recording every 1 min as above. Keep the test subject forward in the stimulator bowl for the whole time and with eyes open.
This completes the procedure for the test subject.
Measure the amplitude of the EOG Taking care to remove the effects of overshoot (see Fig. 2 and notes below) and stepped saccades, measure the EOG amplitude in μV, either manually or by a computer algorithm. Calculate the average of the amplitudes in each 10 s trial.
Fig. 2Idealised saccadic recording with d.c. amplifier (top) and example a.c. coupled amplifier with high pass filter at 0.5 Hz and 0.1 Hz. Overshoot is hard to recognise using 0.5 Hz
Plot the change in the responses Plot the average amplitude of the EOG in μV on a graph representing the 30 min of the test. Ignore any points known to be incorrect (e.g. subject did not follow the fixation lights properly), and identify the underlying curve (see notes) as in Fig. 3 (lower panel).
Fig. 3Idealised (underlying) EOG response (top) and practical response with noise and trial/trial variability. Arrows show the dark trough (DT) and light peak (LP). The underlying curve must be estimated before recording the Arden ratio (LP/DT)
Calculate the Arden ratio This is the ratio between the peak and trough of the underlying curve, not the maximum and minimum recorded values.
Report As a basic factual report state the Arden ratio, the first (dark) trough amplitude in μV, the time from start of the light phase to the peak (if present), the pupil size, and the type of adapting light source. The report should also state any difficulties which were encountered during the test session which may affect confidence in the results, such as the test subject’s difficulty in performing the test.
Deviation from the Standard
This Standard represents a basic or core procedure. If a laboratory chooses a procedure which varies from the Standard Method above, it is critical to cite this Standard, but specify the deviations from the Standard Method, such as different luminance level for the adapting light. If a statistical report is given, then this must be supported by reference data obtained under the same conditions.
Additional tests
Some centres measure the ‘Fast Oscillation’ (FO), often in conjunction with the Clinical EOG.
The fast oscillations:
The fast oscillations (FOs) have the opposite polarity to the light rise of the Standard Clinical EOG. At light onset there is a decrease in the standing potential that recovers 30–40 s after light onset. This decrease, the ‘light-trough’ (LT), is caused by a decrease in intracellular Cl- that results in a decrease of Cl- transport across the basolateral membrane resulting in a hyperpolarisation of the RPE basal membrane.
The FO is recorded using the same parameters as for the clinical EOG (amplification, electrode placement and stimulus light intensity). However, recordings should be made continuously as the subject executes regular horizontal saccades at 1/s. Alternating light and dark for 60 s each induces the FOs, which have a near sinusoidal appearance. During the light interval a light trough (LT) develops and begins to rise again after 30–40 s. The subsequent interval of darkness results in a dark rise (DR) at 30–40 s following the onset of darkness. The next interval of light induces another light trough. The total number of light-dark intervals should be at least 4 with 60 s periods of light and dark, making a total test time of 8 min. Pre-adaptation does not affect the FO, and so this test can be performed either independently or in conjunction with the clinical EOG, provided pre-adaptation conditions for the latter are consistent within the lab.
Figure 4 shows a schematic idealised representation of the FO cycle, from which the ratio of DP:LT should be reported. The normal amplitude ratio is typically between 1.05 and 1.30. However, at present each laboratory should obtain and use their own reference data. It should be noted that high blood glucose levels increase the FO amplitudes.
Fig. 4Idealised representation of fast oscillations (FO). In the dark intervals (black bars) the standing potential increases to a dark rise maximum (DR). Following light onset the standing potential falls to a light trough (LT). The FO ratio of the DR:LT standing potentials should be recorded
Practical notes, instruments and definitions (alphabetical)
Amplifiers For a 30 degree saccade, the typical EOG amplitudes are between 250 and 1000 μV with essential frequency content of 0 to 30 Hz. With such signal amplitudes, no special shielding nor interference protection should be necessary. Medical grade amplifiers intended for human physiological recording are required to be ‘electrically isolated’ to prevent any risk of electric shock to the patient via the recording electrodes. Laboratory grade amplifiers may not have this protection. To obtain recordings of saccadic movements as a ‘square wave’ (the ideal) requires a d.c. amplifier (i.e. no high pass filter) but there are operational penalties in terms of base line shift due to electrode polarisation and movement, which can be partially eliminated by the use a high pass filter (e.g. 0.1 Hz). If a higher frequency is used (e.g. 0.5 Hz) there is considerable ‘droop’ of the square wave, making identification of overshoot and stepped saccades more difficult (see Fig. 2). For the low pass filter, 30 Hz is sufficient. Including higher frequencies adds noise to the response (e.g. EMG) and since most automatic cursor placement regimes find the lowest and highest parts of the wave, this exaggerates the recorded amplitudes. Since both the peak and the trough amplitudes will be equally exaggerated the Arden ratio will be underestimated. For guidance on verifying amplifier performance, see reference [3].
Amplifier saturation EOG potentials measured during saccadic eye movements as in the Standard Clinical Method can vary by about 5:1 in amplitude between subjects, which, with the light rise, may mean a total amplitude range of up to 15:1. Thus, the operator must be able to see the recordings of the saccades to ensure saturation does not occur, and to adjust the amplifier gain settings accordingly.
Arden ratio Using the Standard clinical EOG method described here, the Arden ratio is the peak EOG amplitude occurring in the light phase, divided by the minimum amplitude during the dark phase. The peak and trough values must be measured from the ‘underlying curve’ as shown on Fig. 3, not from individual measurements.
Compliance of the patient Test subjects will have difficulty performing the saccadic movements if they do not have good central vision, or suffer from diplopia or dysconjugate eye movements, have nystagmus, are very young, or are infirm and are unable to take up the physical positions required for the duration of the tests. Some patients also suffer claustrophobia or fear of the dark, and so the testing must be performed in such a way as to minimise these fears. Real-time view of the EOG potentials, and infrared camera view of the patient’s eyes, are very useful in ensuring full compliance. In most cases, coaching under observation can remedy poor co-operation. A common issue is the patient closing eyes during the light phase, which can be seen by a camera.
The cost of unreliable performance is that the margins of error are widened. This does not mean that no information can be gleaned. If the suspected disorder is one with a clear test outcome, it may be possible to make the diagnosis on one or two reliable trials performed near the time of the trough and the peak, to ensure that there is indeed a light rise of some amplitude, or not.
Diplopia Test subjects often find the fixation lights too near for them to achieve convergence. The subject may be advised to look between the pair of images in cases of intractable diplopia. Subjects with ocular motility problems are likely to give unreliable results. If the suspected retinal disorder is bilateral, it is acceptable to patch one eye to perform the tests (see the notes on interaction between eyes below).
Electrodes Recording the EOG is relatively undemanding as regards the electrodes. These should be relatively non-polarisable such as standard medical EEG or ECG electrodes, of a size appropriate for attachment to the side of the nose. If baseline drift is excessive, less polarisable electrodes may be necessary.
Fixation targets These should be small, red, and adjustable in brightness so that they can be seen clearly by all subjects above the background illumination, and to be dim (just visible to the test subject) in darkness.
Full field (Ganzfeld) stimulator This should be as large as practicable to allow adequate distance from eye to fixation lights. It should have a chin rest and forehead bar to ensure stable head position, and it should have fixation lights 15 degree each side of centre. The background intensity should be stable and ‘visibly white’ (see notes on Light below).
Interaction between eyes The EOG potentials from one eye will contaminate the response from the other. This is approximately 15% with electrodes placed on each side of the nose close to the inner canthi, rising to about 40% as they come close together and then touch (e.g. becoming a common central electrode placed on the bridge of the nose). This can give misleading results in cases of an electrically inactive (e.g. total retinal detachment), or absent eye, in which case the defective eye appears to have the same Arden ratio as for the fellow eye, albeit on a much smaller apparent standing potential. More confusing is when the eyes have similar standing potentials but different Arden ratios. In these cases, the measured Arden ratio from the better eye is enhanced at the expense of that from the weaker eye, which can in fact appear to have an Arden ratio of less than 1.0, solely due to interaction.
Light
Luminance For the light phase the luminance of the full field adapting light should be 100 photopic cd/m2 as measured with a photometer (photopic filter) with a traceable calibration (see also notes on pupil dilation below). The calibration of the ganzfeld stimulator should be carried out periodically, e.g. once a year, and corrective action applied (see ref [3] for guidance). Modest room lighting may be turned on during the light phase when the impact on the effective luminance in the bowl should be negligible with the test subject looking into the bowl.
Darkness The dark phase should take place in total darkness, and the fixation lights should be dimmed to the minimum necessary to enable fixation.
Colour There are several possible sources of adapting light such as tungsten, halogen, LED, and fluorescent. For a commercial recording system, the type will be stated in the manufacturer’s literature. Spectrally these are quite different, and the spectra for tungsten and halogen also change with brightness. This issue makes the definition of a single Standard Method for recording the clinical EOG difficult at present, and therefore all reports should state the type of light source used. If the user has additional data, such as colour temperature, or scotopic luminance, then this should be stated.
Normative data/reference range At present there is no standard international reference range for the clinical EOG. With the introduction of this Standard (The Standard Clinical EOG Method 2006) there is a greater possibility of developing and comparing normative data (see notes on reporting).
Photophobia The EOG ‘light rise’ is a ‘triggered’ response, and is assumed to start from the onset of the light phase. Some test subjects will find the light too bright and it may be necessary to bring the adapting light up gradually over, say, a 20-second period. If so, this variation should be stated. If the adapting light is brought on very slowly (e.g. minutes), the response is changed.
Plotting The average EOG amplitude calculated from each 10-second trial should be plotted (see Fig. 3). The first critical step is that the underlying physiologic curve is recognised and drawn for derivation of the trough and peak. This may require a curve fitting algorithm or a traditional ‘flexicurve’ (curve fitting ruler) on paper. Only then should the Arden ratio be calculated. It is helpful if any uncertain values have been identified and marked at the time of recording, so that they can be ignored when identifying the underlying curve.
Pupil dilation Having dilated pupils means less variability in the light entering the eye. There may be occasions when full pupil dilation is impossible or undesirable. If pupils are not artificially dilated, then the report should state this. An attempt should be made to adjust the adapting luminance to accommodate smaller or variable pupils, and this luminance should be quoted (it is usual to try to achieve the same value in Trolands, as for dilated pupils). In many cases useful diagnostic information can be obtained without dilated pupils, especially with extreme results (no light rise, strong light rise). Intermediate results for the Arden ratio will obviously be of less value with undilated and therefore variable pupil sizes.
Reporting
Basic factual report This should include the Arden ratio, the first (dark) trough amplitude in μV, the time from the start of the light phase to the light peak (if present), the pupil size at the end of the test, and the type of adapting light source.
Statistical report This should show how the calculated Arden ratio relates to a quoted reference range. If the reference range was obtained with any different test parameters, this should be explained.
No fully authenticated normal reference data is available currently for the Standard Clinical EOG method given above. However, from a review of existing published data, Arden ratios <1.5 are reported as being abnormally low, and those >2.0 are reported as probably normal, and in between as borderline. This guidance may be useful in the absence of local normative data, but the values do not constitute validated diagnostic criteria.
Saccade measurement Use a scale or electronic calliper to measure the change in EOG potential resulting from each saccade, and calculate an average for each 10 second trial. The average should include only those measurements judged to be reliable. Alternative measurement methods may be used, including computer algorithms.
If a computer algorithm is used there is a need to ensure that the values returned properly represent the true EOG amplitudes. Each algorithm is likely to make some mistakes. Fortunately, the final dark/light response curve will form an ‘average of averages’ in which the influence of a few errors should be swamped by the remaining data.
Particular causes of unreliability are overshoot (see Fig. 2), stepped saccades, missing saccades, inverse saccades (eyes go opposite way to fixation lights), and eccentric fixation in which the saccade length switches between two or more values. When measuring manually, these difficulties are fairly obvious.
Standing potentials This Standard recommends the reporting of the minimum standing potential (dark trough minimum), taken from the underlying response curve, not the minimum recorded value. This value is not often used in diagnosis at present, but if the value is abnormally low it may indicate an inactive retina (e.g. total retinal detachment), and the calculated Arden ratio may be unreliable because of the low value of the divisor in the ratio. Normal values for the minimum standing potential should be established, but as a guide, minimum standing potentials less than 150 μV should be treated as low, and more likely to produce unreliable Arden ratios.
Warning of start of each trial There should be a warning, verbal or automatic, of the impending start of each trial, to ensure readiness of both test subject and operator. Some users have sounds (beeps) to co-incide with the alternations of the fixation lights. However, it may be better to rely solely on the visual stimuli, to avoid the possibility of alternation without actual fixation.
History and acknowledgements
This Standard forms part of series of Standards, Recommendations and Guidelines prepared by the International Society for Clinical Electrophysiology of Vision (ISCEV):
Participants in the review: ISCEV EOG Standard Revision Committee 2006; Malcolm Brown (Chairman), Royal Liverpool University Hospital, Liverpool UK
Michael Marmor, Stanford University, California USA; Vaegan (ISCEV Board Member-at-Large) University of New South Wales, Australia, Eberhart Zrenner University Eye Hospital, Tuebingen, Germany
EOG Standard Advisory Panel 2006 includes the members above plus: Michael Bach, (ISCEV President), Universität-Augenklinik, Freiburg, Germany: Mitchell Brigell, (ISCEV Director of Standard), Pfizer Global. R&D, Ann Arbor, Michigan USA: Graham Holder, (ISCEV Director of Education), Moorfields Eye Hospital, London UK
Paul Constable, City University, London UK: Masao Yoshikawa, Mayo Corporation, Aichi, Japan; Carol Westall, Hospital for Sick Children, Toronto Canada; Geoffrey Arden, (Honorary ISCEV member) City University, London UK

electro-oculogram
retinal pigment epithelium
light adaptation
arden ratio
clinical standards


Pharmacology and Surface Electrostatics of the K Channel Outer Pore Vestibule

In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block. Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology. The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge, with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged vestibule with pharmacological properties approaching those of other types of K channels.
Introduction
More than 80 mammalian genes code for K channel protein subunits, so it is not surprising that the physiological roles of K channels include such diverse actions as controlling action potential duration, action potential firing patterns and salt and water movement (Hille, 2001). Given the important, complex physiological processes controlled by K channels, it is not surprising that considerable effort has been expended on understanding their pharmacological properties (Tamargo et al., 2004; Yost, 1999). K channels are blocked by many chemical agents, including many types of complex peptides, especially peptide toxins, and by simple amine compounds, including the quaternary amine tetraethyl ammonium (TEA).
The study of the pharmacology of K channels has been greatly aided by mutational analysis of cloned K channels and the solution of the crystal structures of several bacterial K channels (Doyle et al., 1998; Jiang et al., 2002, 2003) and a mammalian voltage-gated flavor (Long, Campbell & MacKinnon, 2005). This work has revealed a remarkable conservation of the general structure of the outer vestibule and selectivity filter of K channels. In spite of this structural conservation, there are considerable differences in the pharmacological properties of K channels.
Compared to the peptide toxins and other agents that inhibit K channels, the simple quaternary amine TEA ought to be the easiest to understand. Early mutational analysis revealed a “hot spot” for external TEA block: the affinity for this compound changes over more than two orders of magnitude depending on the amino acid at this position (Kavanaugh et al., 1991; MacKinnon & Yellen, 1990). Additional studies revealed that an aromatic amino acid side chain at this hot spot on all four subunits of the homotetrameric channels contributes to high-affinity TEA binding (Heginbotham & MacKinnon, 1992; Kavanaugh et al., 1992). This amino acid (position 449 of the prototypical voltage-gated K channel, Shaker) is located just outside the entrance to the pore selectivity filter, and all the data appeared consistent with the idea that TEA was stabilized in its blocking site through cation π-electron interaction with this aromatic side chain.
More recent studies, however, have suggested that the mechanism of TEA and other quaternary ion block of K channels is more complex. Analysis of the structure-activity relations for K channel block of quaternary amines revealed contributions from hydrophobic interactions as well as a role for dehydration of the blocking ions (Jarolimek et al., 1995, 1996). In addition, amino acids other than that at position 449 are involved in TEA binding (Pascual et al., 1995). A molecular dynamics study (Crouzy, Berneche & Roux, 2001) showed that K channel proteins do not have the three-dimensional geometry necessary for TEA: π-electron interaction. Finally, studies of the ability of TEA to alter the chemical modification of introduced cysteine residues indicate that TEA likely blocks at a location more external than position 449 (Andalib et al., 2004).
One factor that has received little attention in the study of the quaternary ammonium block in K channels is the involvement of electrostatic charges on the channel surface, which can certainly affect the interaction of charged blockers like quaternary amines. Indeed, TEA block of frog node K channels is increased in low-ionic strength solutions or in solutions with reduced Ca2+ concentration (Mozhayeva & Naumov, 1972). TEA block of a Ca2+-activated K channel is sensitive to solution ionic strength, and a chemically induced neutralization of negative surface charges reduces this ionic strength dependence (MacKinnon, Latorre & Miller, 1989). The pH-dependent TEA block of Kv1.1 channels is sensitive to changes in bulk solution ionic strength (Bretschneider et al., 1999). Finally, we have shown that TEA block of Shaker K channels is sensitive to bulk solution ionic strength and that block by a trivalent TEA analog (gallamine) is even more sensitive (Quinn & Begenisich, 2001). The results of these various studies suggest a role for surface electrostatics in quaternary ammonium block of K channels.
Kv2.1 channels are rather unique among K channels in that they have a relatively low affinity for TEA in spite of possessing an aromatic amino acid at the TEA hot spot that confers high-affinity block to other K channels. They are unique also in having two lysine residues in the outer vestibule that are not conserved in other K channels. These lysine residues have a powerful effect on TEA block of these channels (Immke & Korn, 2000; Immke et al., 1999). We have investigated the mechanism by which the positively charged side chains of these lysines affect TEA block. We tested the ionic strength dependence of block by TEA and the trivalent TEA analog gallamine as a way to probe for surface electrostatic effects. Kv3.1 channels have a high affinity for TEA and do not contain the Kv2.1 lysine residues noted above (Grissmer et al., 1994; Taglialatela et al., 1991). We found that, like Shaker K channels, quaternary amine block of Kv3.1 channels was ionic strength-dependent but block of wild-type Kv2.1 channels with the lysine residues was not. We also found that Kv2.1 channels were insensitive to gallamine. Neutralization of one or the other of the nonconserved lysine residues in Kv2.1 channels conferred ionic strength sensitivity to TEA block of Kv2.1 channels. Neutralization of both lysine side chains produced channels that were blocked by gallamine, and this block was more sensitive to solution ionic strength than block by TEA. Thus, surface electrostatics appears to play a role in TEA block of some, but not all, K channels depending on the location of some particular charged residues.
Materials and Methods
K CHANNEL CONSTRUCTS
Several K channel constructs were used in this study, including wild-type Kv2.1 and Kv3.1 channels (Chan Test Inc., Cleveland, OH). Three mutant Kv2.1 channels were also investigated: K356F, K382Q and the double mutant K356F/K382Q. The replacement amino acids were introduced into the Kv2.1 clone using a two-step polymerase chain reaction (PCR) protocol and the resulting mutants analyzed by DNA sequencing.
OOCYTE ISOLATION AND MICROINJECTION
Xenopus laevis oocytes were maintained as described by Goldin (1992). Isolated ovarian lobes were rinsed with Ca2+-free OR-2 solution (in mM): 82.5 NaCl, 2.5 KCl, 1 MgCl2 and 5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.6, NaOH) and then defolliculated by incubation for 60–90 min with 2 mg/ml collagenase type 1A (Sigma, St. Louis, MO). Cleaned oocytes were transferred and maintained for 2 h in ND-96 solution (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES and 2.5 Na-pyruvate (pH 7.6, NaOH) before injection of mRNA coding for the channel construct of interest. Injected oocytes were transferred to multiwell tissue culture plates and incubated at 18°C in ND-96 solution supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Animal (frog) handling and care utilized methods consistent with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.
ELECTROPHYSIOLOGICAL RECORDINGS
K channel currents were recorded 1–5 days after mRNA injection. Recordings were made at room temperature (20–22°C) using the cut-open oocyte voltage-clamp apparatus (model CA-1B; Dagan, Minneapolis, MN). Connections to the different compartments were made with glass capillaries containing 75 μm platinum wires and filled with a 1 M NaCl, 3% agar solution. The experimental chamber (ELV-1, Dagan) was modified to include a low-volume (80 μl) chamber insert. The low chamber volume and high flow rates (∼1 ml/min) produced efficient solution exchange. The measured difference in junction potential for the two experimental solutions (see below) was <2 mV, so no correction to membrane voltages was made.
Recording electrodes were made either of 1 BBL glass with filament (1.5 mm outer diameter; World Precision Instruments, Sarasota, Fl) or of GC-150F glass (Warner Instrument, Hamden, CT). Electrodes had tip diameters of ∼2 μm and were filled with a 3 M KCl solution. Data acquisition was performed using a 12-bit analog/digital converter controlled by a personal computer. Current records were filtered at 5 kHz. Series resistance compensation was used: a mean value of 0.74 ± 0.05 (standard error of them mean, SEM) kΩ in normal-ionic strength solutions and 2.2 ± 0.11 kΩ in reduced-ionic strength solutions (n = 50).
Electrical access to the internal compartment was achieved by 0.125% saponin treatment in a solution of (in mM) 100 KCl and 10 HEPES (pH 7.4, N-methyl-D-glucamine [NMDG]). The external solution was (in mM) 140 NaCl, 2 CaCl2, 10 KCl, 10 HEPES (pH 7.4, NMDG). An external solution of reduced ionic strength was used that consisted of (in mM) 35 NaCl, 0.5 CaCl2, 10 KCl, 10 HEPES (pH 7.4, NMDG). The osmolarity of this solution was matched to that of normal ionic strength by addition of 216 mM glucose. In order to test for possible effects of glucose beyond simply maintaining solution osmolarity, we also used mannitol and sucrose. We found that the ionic strength sensitivity of both TEA and gallamine block was the same regardless of which agent was used for osmotic balance. Osmolarity of all solutions was verified using a vapor pressure osmometer (Wescor, Logan, UT). Addition of TEA-Cl was made by substitution with NaCl, which maintained solution ionic strength. Gallamine-I3 (1,2,3-tris[2-triethylammonium ethoxy]benzene triiodide; Sigma) was added to the external solution with no compensation for changes in solution ionic strength.
Oocytes were clamped at a holding potential of −80 mV, and 80-ms test pulses were applied to elicit channel current. Channel block by TEA or gallamine was computed by calculating the steady-state current recorded at the end of each pulse as a fraction of the average current recorded before application and following washout of the blocker. Only those results with at least 90% recovery from block were considered. Concentration-response relationships were constructed by plotting the mean fraction of current blocked at a test potential of +40 mV. These were fit by the standard binding isotherm:
where [B] is the concentration of TEA or gallamine, Bmax is the maximal fraction of current blocked and Kapp is the concentration that blocks half the maximal current.
We used the crystal structure of the KcsA K channel (RCSB Protein Data Bank entry 1J95) as a template for the Kv2.1 channel (Doyle et al., 1998). The KcsA residues in the outer vestibule were mutated to their Kv2.1 counterparts with the DeepView/Swiss-PdbViewer, version 3.7 (http://www.ca.expasy.org/spdbv/). The molecular views of the K channel structures were generated with ViewerLite 4.2 for Windows (Accelrys, San Diego, CA; http://www.accelrys.com/). The electrostatic surface view was also generated with ViewerLite using Gasteiger charges.
Results
CHARGED RESIDUESINTHE OUTER VESTIBULE OF K CHANNELS
Figure 1 illustrates two views of the outer vestibule of K channels. Shown are schematics based on the crystal structure of the bacterial K channel KcsA, but all crystallized K channels, including the mammalian voltage-gated K channel Kv1.2, share a common structure for this outer part of the channel (Long et al., 2005). On the left is a view from above the channel, and the right part of the figure illustrates a side view with two of the subunits removed. Shown below the structures is an alignment of the amino acids in the outer vestibule and selectivity filter of Shaker, Kv2.1 and Kv3.1 channels. As noted in the Introduction, an aromatic residue at Shaker position 449 confers high-affinity TEA block. Replacement of the native threonine at position 449 in Shaker channels to a tyrosine renders the channel highly sensitive to TEA (Kavanaugh et al., 1991; MacKinnon & Yellen, 1990). Both Kv2.1 and Kv3.1 channels have a tyrosine at this position (shown in green).
Figure 1.Sequence and structure of the outer vestibule of K channels. Bottom, Amino acid sequence alignment of three K channels in the region that forms much of the channel pore outer vestibule. In blue are the two lysine residues (K356 and K382) in Kv2.1 channels that are a major focus of this study. Kv2.1 and Kv3.1 channels have a tyrosine amino acid (shown in green) at the position that is important for high-affinity TEA block (see text). In red are several amino acids (aspartate and glutamate) in the vestibule area that have negatively charged side chains. Top left, Structures of gallamine and TEA. Bottom left, Top-view structure of KcsA. The fourfold symmetry of the channel (in ribbon form) is apparent in this view. The Kv2.1 K356 and K382 lysine residues are shown with space-filling atoms (without hydrogens). The tyrosine important for TEA binding is shown with “ball and stick” atoms in green. Right, Side view of the channel with two subunits removed for clarity. Apparent are the outer vestibule and narrow selectivity filter. As in the top view, the relevant lysines and tyrosines are shown with space-filling and ball-and-stick atoms, respectively.
All three channels contain several negative amino acids in this vestibule region (indicated in red in the alignment). Kv2.1 channels are unique in having two positively charged amino acids (K382 and K356) in the outer vestibule (blue in the alignment and illustrated as space-filling atoms in the structural diagrams). The influence of these lysine residues on TEA block is an important part of the present study.
IONIC STRENGTH-DEPENDENT BLOCK OF KV3.1 CHANNELS BY TEA+ AND GALLAMINE3+
As described above, TEA block of Shaker K channels is sensitive to solution ionic strength, and block by a trivalent TEA analog, gallamine, is even more sensitive (Quinn & Begenisich, 2001). These results demonstrate that negative charges on the surface of the channel contribute to an electrostatic potential that influences block by these charged molecules. As a first step toward identifying the amino acids that could contribute to this type of surface electrostatics, we tested the ionic strength dependence of TEA and gallamine block of two other K channels: Kv3.1 and Kv2.1.
Shown in the upper part of Figure 2A are raw current traces from an oocyte expressing Kv3.1 channels. TEA (50 μM) produced a small (near 25%) block of Kv3.1 channels in a normal-ionic strength solution (see Materials and Methods) but almost a 50% block when applied in a solution of low ionic strength. The lower panel illustrates the concentration dependence of current block, measured at +40 mV, at normal and low ionic strength. The lines are fits of equation 1 to the data with a Kapp value of 0.14 mM for TEA block in the normal-ionic strength solution, well within the 0.09–0.2 mM range previously reported for TEA block of this K channel (Grissmer et al., 1994; Jarolimek et al., 1995; Taglialatela et al., 1991). TEA was more effective in the low-ionic strength solution with a Kapp value of 0.055 mM, a 2.5-fold increase in apparent TEA affinity in the low-ionic strength solution.
Figure 2.Ionic strength-dependent TEA and gallamine block of Kv3.1 channels. (A) Top, Raw currents as indicated in response to step depolarizations of 80 ms to −20, 0, +20 and +40 mV from a –80-mV holding voltage. Calibration, 1 μA, 20 ms. Bottom, Dose-response relation for TEA block of current at +40 mV in normal- (■) and low- (○) ionic strength solutions. Mean values from three to five measurements and SEM limits (if larger than symbol) are plotted. Lines are fits of equation 1 to the data with Kapp values of 0.14 and 0.055 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.91 and 0.89 for normal- and low-ionic strength conditions, respectively. (B) Top, Raw currents as indicated in response to step depolarizations of 80 ms to −20, 0, +20 and +40 mV from a –80-mV holding voltage. Calibration, 0.5 μA, 20 ms. Bottom, Dose-response relation for gallamine block of current at +40 mV in normal- (■) and low- (○) ionic strength solutions. Mean values from three to five measurements and SEM limits are plotted. Lines are fits of equation 1 to the data with Kapp values of 0.066 and 0.017 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.91 and 0.94 for normal- and low ionic-strength conditions, respectively.
The upper part of Figure 2B contains raw currents illustrating block by 20 μM gallamine at normal and low ionic strength. This concentration of gallamine blocked very little current at normal ionic strength (<20%), but more than half the current was blocked when the gallamine was applied in the low-ionic strength solution. The lower part of Figure 2B shows the concentration dependence of block by gallamine, measured at +40 mV, at normal and low ionic strength. The lines are fits of equation 1 to the data with Kapp values of 0.066 mM at normal and 0.017 mM at low ionic strength, a 3.9-fold increase in apparent gallamine affinity in the low-ionic strength solution. Similar results have been observed for gallamine block of Shaker K channels (Quinn & Begenisich, 2001).
Thus, quaternary amine block of Kv3.1 channels, like Shaker K channels, was sensitive to solution ionic strength and this sensitivity was greater for trivalent than monovalent blocking ions. These results demonstrate that Kv3.1, like Shaker channels, contain charged residues that contribute to a negative electrostatic potential that influences block by these charged molecules. Our further investigations indicated that not all channels share this property, as demonstrated in Figure 3.
Figure 3.Ionic strength-dependent TEA block of Kv2.1 channels. Top, Raw currents as indicated in response to step depolarizations of 80 ms to −20, 0, +20 and +40 mV from a −80-mV holding voltage. Calibration, 2 μA, 20 ms. Bottom, Dose-response relation for TEA block of current at +40 mV in normal- (■) and low- (○) ionic strength solutions. Mean values from 4–10 measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the pooled normal- and low-ionic strength data with Kapp and Bmax values of 5.1 mM and 0.83, respectively.
TEA+ BLOCK OF KV2.1 CHANNELS WAS NOT SENSITIVE TO IONIC STRENGTH
The upper part of Figure 3 illustrates TEA block of currents recorded from Kv2.1 channels. The raw current records show that, in this oocyte, block by 5 mM TEA was about 35%, independent of the solution ionic strength. The lower part of the figure demonstrates the dose-response relation for block of the ionic current at +40 mV from many oocytes in the normal- and low-ionic strength solutions. Clearly, TEA block of Kv2.1 channels was not ionic strength-dependent and well described by a Kapp value of 5.1 mM. This Kapp value is quite similar to the 4.2–4.5 mM values previously reported for these channels (Immke et al., 1999; Taglialatela et al., 1991) and illustrates the fact that these Kv2.1 channels are almost 40-fold less sensitive to TEA block than other K channels in spite of the fact that they contain the aromatic amino acids normally associated with high-affinity TEA block. In addition, unlike Shaker and Kv3.1 channels, TEA block of Kv2.1 channels was not sensitive to solution ionic strength.
Kv2.1 channels also differed from Shaker (T449Y) and Kv3.1 channels with respect to block by gallamine. We could detect no significant block of Kv2.1 channels by this trivalent TEA analog. In the normal-ionic strength solution, there was an average 3.5% increase in current in the presence of 5 mM gallamine, but this was not significantly different from zero (n = 3). Similar results showing no significant block were obtained with 10 mM gallamine and with the low-ionic strength solution.
Our low-ionic strength solution contained one-fourth the normal Na+ and Ca2+ concentrations than the normal-ionic strength solution. In addition to changes in ionic strength induced by alterations in the monovalent ion concentrations, changes in Ca2+ concentrations are known to shift the voltage dependence of K channel gating but do not block these channels (e.g., Begenisich, 1975; Frankenhaeuser & Hodgkin, 1957; Mozhayeva & Naumov, 1972). It is because of this effect of alterations in Na+ and Ca2+ concentrations that we measured TEA and gallamine block at +40 mV, a voltage at which there are minimal gating changes. Nevertheless, it is worthwhile to demonstrate that these gating shifts do not affect our measurement of TEA block in the normal- and low-ionic strength solutions. Thus, in a separate set of experiments, we measured TEA block of Kv3.1 and Kv2.1 channels at several voltages in the two ionic strength solutions. As can be seen in Figure 4, there is a slight voltage dependence to the TEA block of Kv3.1 channels and little or no voltage dependence to block of Kv2.1 channels, consistent with previous observations on these channels (Hartmann et al., 1991; Taglialatela et al., 1991). Importantly, these results show that the ionic strength dependence of TEA block was independent of test potential, so our use of +40 mV introduced no bias into the analysis.
Figure 4.Voltage dependence of TEA block of Kv3.1 and Kv2.1 channels. (A) Fraction of Kv3.1 channel current blocked by 100 μM TEA at the indicated membrane potentials in normal- (■) and low- (○) ionic strength solutions. (B) Fraction of Kv2.1 channel current blocked by 5 mM TEA at the indicated membrane potentials in normal- (■) and low- (○) ionic strength solutions.
As noted in Introduction, the low Kv2.1 sensitivity to TEA is surprising since, like Kv3.1, this channel contains the tyrosine residue at the “hot spot” necessary for high-affinity TEA binding (see Fig. 1). This issue has been discussed previously (Taglialatela et al., 1991), and one possibility that those authors suggested is that the positively charged side chain of the nearby K382 (see Fig. 1) could “repulse TEA” and so reduce Kv2.1 TEA sensitivity. If there were a significant through-space electrostatic repulsion of TEA by the charged side chain of K382, then that repulsion should increase in a low-ionic strength solution. The data presented in Figure 3, however, show that this did not occur.
We suggest that there is little or no net surface charge on wild-type Kv2.1 channels in a position to influence TEA block, so ionic strength should have no effect, as the data demonstrated. If this is correct, then removing some of the positive charge should shift the balance to a net negative surface charge and confer ionic strength dependence to TEA block. Thus, we mutated K382 in Kv2.1 to the neutral glutamine that occupies the analogous location in Kv3.1 channels. The action of TEA on these mutant channels is illustrated in Figure 5A.
Figure 5.Ionic strength-dependent TEA block of mutant Kv2.1 channels. (A) Dose-response relation for TEA block of current (at +40 mV) from Kv2.1 K382Q channels in normal- (■) and low- (○) ionic strength solutions. Mean values from three measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the data with Kapp values of 4.9 and 2.1 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.96 and 0.91 for normal- and low-ionic strength conditions, respectively. (B) Dose-response relation for TEA block of current (at +40 mV) from Kv2.1 K356F channels in normal- (■) and low- (○) ionic strength solutions. Mean values from four to six measurements and SEM limits are plotted. Line is a fit of equation 1 to the data with Kapp values of 6.1 and 2.8 mM for normal- and low- ionic strength conditions, respectively. Bmax values of 0.73 and 0.80 for normal- and low-ionic strength conditions, respectively.
POSITIVE CHARGE NEUTRALIZATION IN KV2.1 CHANNELS
As illustrated in Figure 5A, TEA blocked K382Q Kv2.1 channels with an apparent Kapp value near 5 mM in the normal-ionic strength solution. TEA was more effective when applied in the low-ionic strength solution: block was described by a Kapp value near 2 mM. Thus, as predicted, removal of positive charge altered the electrostatic environment in the vestibule such that TEA block became sensitive to solution ionic strength. These K382Q channels remained resistant to block by 5 mM gallamine (not significantly different from zero, n = 3).
Inspection of the likely topology of the outer pore of Kv2.1 channels (Fig. 1) suggests that the other positively charged amino acid unique to Kv2.1 (K356) may also be in a position to electrostatically effect TEA block. Thus, we tested TEA block of Kv2.1 channels with the lysine at position 356 mutated to the equivalent residue in Shaker (phenylalanine). Figure 5B shows TEA block of these K356F channels. As can be seen, replacement of the charged K356 residue, like neutralization of K382, resulted in a channel with ionic strength-dependent TEA block: Kapp values of 6.1 and 2.8 mM in the normal- and low-ionic strength solutions, respectively. This construct was also quite insensitive to block by gallamine: 5 mM inhibited only 4.4 ± 0.5% of the current at +40 mV.
We also tested the ionic strength dependence of block of channels with both charged lysine residues neutralized. Figure 6A shows that TEA block of the double mutant K356F/K382Q channels was sensitive to solution ionic strength. Figure 6B demonstrates that these double mutant channels, unlike wild-type channels or channels with only a single neutralization, could be blocked by millimolar concentrations of gallamine. In normal-ionic strength solution, the Kapp for gallamine block was about 1.2 mM; in reduced-ionic strength solution, it was about 0.13 mM. This almost 10-fold increase in gallamine block with ionic strength change was even larger than that seen in Kv3.1 channels (Fig. 2B).
Figure 6.Ionic strength-dependent TEA and gallamine block of K356F/K382Q Kv2.1 channels. (A) Dose-response relation for TEA block of current (at +40 mV) in normal- (■) and low- (○) ionic strength solutions. Mean values from three to six measurements and SEM limits are plotted. Line is a fit of equation 1 to the data with Kapp values of 5.6 and 2.8 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.80 and 0.91 for normal- and low-ionic strength conditions, respectively. (B) Dose-response relation for gallamine block of current (at +40 mV) in normal- (■) and low- (○) ionic strength solutions. Mean values from three to six measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the data with Kapp values of 1.2 and 0.13 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.47 and 0.56 for normal- and low-ionic strength conditions, respectively.
Discussion
TEA block of some K channels is sensitive to ionic strength changes (MacKinnon et al., 1989; Quinn & Begenisich, 2001), indicating that surface electrostatics plays a role in the permeation and pharmacological properties of such channels. We showed here that this property is shared by Kv3.1 channels. In contrast, TEA block of wild-type Kv2.1 channels was insensitive to solution ionic strength. Kv2.1 channels are also unusual in having a relatively low TEA affinity in spite of having the type of amino acid at the critical position that normally confers high TEA affinity (Kavanaugh et al., 1991; MacKinnon & Yellen, 1990). We showed that Kv2.1 channels were also unusual in being insensitive to the trivalent TEA analog gallamine.
Inspection of the crystal structures of the outer vestibule of K channels and the specific amino acid composition of Shaker, Kv2.1 and Kv3.1 channels (see Fig. 1) reveals that the noted pharmacological differences of Kv2.1 channels could result from the presence of charged lysine residues not represented in Kv3.1 or Shaker channels. Neutralization of either K356 or K382 in Kv2.1 conferred an ionic strength-sensitive TEA block to these channels (Fig. 5), but these mutant channels remained very insensitive to block by gallamine. Kv2.1 channels with both positively charged lysines neutralized attained a reasonable sensitivity to gallamine block, which was particularly sensitive to solution ionic strength (Fig. 6).
The lack of ionic strength sensitivity of TEA block of Kv2.1 channels indicates that these channels, in contrast to Shaker and Kv3.1 channels, have little or no net charge on the outer vestibule in a position to affect TEA block. If so, then neutralization of the charged lysines not represented in Shaker and Kv3.1 channels should result in a net negative surface charge and thus confer ionic strength sensitivity to TEA block on the mutant Kv2.1 channels. This was the result that was obtained (Fig. 4). Figure 7 illustrates the outer entrance of the KcsA channel pore which has been “mutated” to contain Kv2.1 amino acids. Shown is the electrostatic potential arising from the charged amino acids as it is mapped onto the protein. Negative potentials are in red and positive potentials are in blue. The most prominent charged side chains are identified, and it is clear that K356 and K382 sit quite close to the pore entrance but that several negatively charged amino acids are also nearby. The lack of ionic strength dependence of TEA block of Kv2.1 indicates a negligible surface potential in a position to affect TEA block. Thus, it is likely that that the two lysine residues near the pore (K382 and K356) and the one farther away (K350) are sufficient to balance the electric field from the four negatively charged amino acids in the vicinity (D351, E352, D353, D354). According to this picture, mutating either or both of K356/K382 would cause the near zero electrostatic potential of the wild-type channel to shift negative, accounting for the ionic strength dependence of the TEA block of these mutant channels.
Figure 7.Electrostatic potential map of the outer vestibule of Kv2.1 channels. Top view of the channel with the pore in the middle; the fourfold symmetry of these K channels is apparent. Electrostatic potentials computed as described in Materials and Methods and mapped onto the surface of the Kv2.1 channel. Negatively charged amino acid side chains are red, and blue represents positive charges. The two lysine residues that are the focus of this work (K336 and K382) as well as the other exposed charged amino acids are indicated in one of the four channel subunits.
The “uncovering” of negative charges caused by replacing the lysines with uncharged amino acids would be expected not only to confer ionic strength dependence on TEA block (as observed) but also to increase the apparent affinity for TEA block. While these charge-mutant channels did exhibit an ionic strength-dependent TEA block, their TEA sensitivities were essentially the same as the wild-type channel. It is possible that the mutations also produced some minor structural alteration in the outer vestibule that decreased the TEA affinity, masking the apparent increase expected with a more negatively charged vestibule. This view is supported by the fact that replacement of these lysines with neutral amino acids different from those used here (K382V, K356G) does, in fact, increase TEA affinity (Immke et al., 1999).
Our data also suggest that at least one component of the gallamine sensitivity of K channels may be the net charge in the outer vestibule. The intrinsic gallamine affinity for K channels may be relatively low, but the negatively charged vestibules intrinsic to, for example, Shaker and Kv3.1 channels would increase the local gallamine concentration and thus overcome the intrinsic low affinity. The essentially neutral vestibule of wild-type Kv2.1 channels cannot be occupied by gallamine at millimolar levels. Even the mild negativity of K356F and K382Q Kv2.1 channels is not sufficient to allow reasonable block by gallamine. However, removal of both positively charged lysines (a total of eight positive charges on these tetrameric channels) apparently produces a sufficiently negative vestibule to provide for gallamine block. Consistent with this strongly negatively charged vestibule is the very large ionic strength dependence of gallamine block of these double mutant channels (Fig. 5B).
Another feature of the gallamine block of the double mutant Kv2.1 channels was the apparent saturation of block at levels well below 100% (Fig. 6B). We do not know the reason for this effect, but it is worth noting that, under some circumstances, TEA block of Kv2.1 channels displays this phenomenon (Hartmann et al., 1991; Immke & Korn, 2000; Immke et al., 1999). Such a result would be observed if the channels had both high and very low affinity for these blocking ions. The outer vestibule of KcsA channels exists in two conformations controlled by K+ levels (Zhou et al., 2001), and Shaker K channels exhibit both high- and low-affinity TEA block configurations also controlled by the K+ levels (Thompson & Begenisich, 2005). Future experiments will be required to test if Kv2.1 channels (or mutant Kv2.1 channels) exhibit such behavior with respect to gallamine block.
Finally, it should be noted that electrostatic potentials contribute to the block of other types of ion channels. For example, snail toxin peptide block of voltage-gated Na channels, like scorpion toxin block of K channels (MacKinnon et al., 1989), exhibits strong charge-dependent effects (Hui et al., 2002). While the details of block are different in these two examples, these and the results of the present study demonstrate the widespread importance of electrostatic potentials in the outer vestibule of ion channels.
In summary, the results reported here indicate that surface electrostatics may play a significant role in the pharmacology of K channels. Shaker and Kv3.1 channels have negative charges in the outer vestibule that contribute to TEA and gallamine block. Wild-type Kv2.1 channels have a lower TEA affinity than Kv3.1 and other channels even though these channels possess the aromatic amino acid necessary for high-affinity TEA block. Our results suggest that at least one reason for this is that Kv2.1 channels do not have the exposed negative charges that contribute to TEA block in Kv3.1 and Shaker channels, likely because these negative charges are rendered ineffective by the nearby positively charged side chains of lysines at positions 356 and 382. Gallamine cannot block Kv2.1 channels unless both lysines are neutralized, likely because this TEA analog has an intrinsically low affinity but is particularly sensitive to electrostatic potentials owing to its trivalent nature. A full understanding of the pharmacology of the outer vestibule of K channels will have to take into account the electrostatic charges in this structural region.

k channel
tetraethylammonium
gallamine
channel block
surface potential
quaternary ammonium ion


Erosion—diagnosis and risk factors

Dental erosion is a multifactorial condition: The interplay of chemical, biological and behavioural factors is crucial and helps explain why some individuals exhibit more erosion than others. The erosive potential of erosive agents like acidic drinks or foodstuffs depends on chemical factors, e.g. pH, titratable acidity, mineral content, clearance on tooth surface and on its calcium-chelation properties. Biological factors such as saliva, acquired pellicle, tooth structure and positioning in relation to soft tissues and tongue are related to the pathogenesis of dental erosion. Furthermore, behavioural factors like eating and drinking habits, regular exercise with dehydration and decrease of salivary flow, excessive oral hygiene and, on the other side, an unhealthy lifestyle, e.g. chronic alcoholism, are predisposing factors for dental erosion. There is some evidence that dental erosion is growing steadily. To prevent further progression, it is important to detect this condition as early as possible. Dentists have to know the clinical appearance and possible signs of progression of erosive lesions and their causes such that adequate preventive and, if necessary, therapeutic measures can be initiated. The clinical examination has to be done systematically, and a comprehensive case history should be undertaken such that all risk factors will be revealed.
Introduction
Erosion has, for many years, been a condition of little interest to clinicians and researchers. This has changed during the last years, and there is some evidence that the presence of dental erosion is growing steadily. In the UK, the prevalence of erosion was shown to have increased from the time of the children’s dental health survey in year 1993 compared to 1996/1997 [82]. In another UK study, the progression of erosion was investigated: 1,308 children were examined at the age of 12 years and 2 years later. In this study, 4.9% of the subjects at baseline and 13.1% 2 years later had deep-enamel or dentine lesions. Approximately 12% of erosion-free children at 12 years developed the condition over the subsequent 2 years. New or more advanced lesions were seen in 27% of the children over the study period [20]. The progression of erosion seems to be greater in older adults (52–56 years) compared to younger (32–36 years) and has a skewed distribution [64]. In this study, the group with high-erosion progression was found to have four or more dietary acid intakes per day, a low buffering capacity of stimulated saliva and used a hard-bristle toothbrush. Intake frequency of the same magnitude was also associated with an increased risk for erosion in children. In this investigation, the erosion group ate fruits significantly more frequently and had different drinking habits, such as swishing, sucking or holding drinks in their mouths [83].
Dental erosion is a multifactorial condition. To prevent further progression, it is important to detect this condition as early as possible. It is fundamental to diagnose the possible risk factors such that preventive measures can be initiated. This overview is aimed to give some basic aspects about the diagnosis and the risk factors of erosion. A more detailed description can be found elsewhere [67].
Diagnosis
Diagnosis of early forms of erosion is difficult, as it is accompanied by few signs and fewer if any symptoms. There is no device available in routine dental practice for the specific detection of dental erosion and its progression. Therefore, clinical appearance is the most important feature for dental professionals to diagnose this condition. This is of particular importance in the early stage of dental erosion [68]. At a more advanced stage, it can be very difficult to determine if dentine is exposed or not [31]. It is possible to use disclosing agents to render dentine involvement visible.
The appearance of smooth silky-glazed, sometimes dull, enamel surface with the absence of perikymata and intact enamel along the gingival margin are some typical signs of enamel erosion on facial and oral sites. It has been hypothesized that the preserved enamel band along the oral and facial gingival margin could be due to some plaque remnants, which could act as a diffusion barrier for acids. This phenomenon could also be due to an acid-neutralizing effect of the sulcular fluid [66]. In the more advanced stages, further changes in the morphology can be found. These changes result in developing a concavity in enamel, the width of which clearly exceeds its depth. Facial erosion should be distinguished from wedge-shaped defects which are located at or apical to the enamel–cementum junction. The coronal part of wedge-shaped defects ideally has a sharp margin and cuts at right angle into the enamel surface, whereas the apical part bottoms out to the root surface. Thereby, the depth of the defect exceeds its width.
The initial features of erosion on occlusal and incisal surfaces are the same as described above. Further progression of occlusal erosion leads to a rounding of the cusps and restorations rising above the level of the adjacent tooth surfaces. In severe cases, the whole occlusal morphology disappears. Erosive lesions have to be distinguished from attrition and abrasion. The latter are often flat, have glossy areas with distinct margins and corresponding features at the antagonistic teeth. It is sometimes challenging to distinguish between the influences of erosion, attrition or abrasion during a clinical examination. Indeed, they may occur simultaneously with sometimes similar shape. The most commonly reported areas with this condition are occlusal surfaces [6]. Figures 1, 2, 3, and 4 show typical pattern of dental erosion process.
Fig. 1Facial erosion: The intact enamel border along the gingival margin of tooth 12 and some plaque remnants are clearly visible. Note the smooth silky-glazed appearance and the absence of perikymata on the enamel surfaceFig. 2Advanced facial erosion of teeth 43, 44 and 45 with dentinal involvement. The width of the lesions exceeds its depthFig. 3Erosion with involvement of dentine on the oral surface of tooth 13, 12 and 11. Intact enamel borders along the gingival marginFig. 4a–c Typical pattern of advanced occlusal erosion of teeth 45 and 46 of three different patients: The whole occlusal morphology disappears, and extensive exposed dentinal areas are visible
The clinical examination should be done systematically using a simple but accurate index. This is a difficult task to achieve as an index with a too-fine grading shows a small inter- and intraexaminer reliability [60]. For a dental practitioner, the most important part is to recognize the condition and to describe its dimension and severity. It is important to search for a general pattern and not to over-interpret one single sign. For epidemiological purpose, an index with high detection capability and reliability is most important. Whenever possible, the clinical examination should be accomplished by a thorough history taking with respect to general health, diet and habits and by the assessment of saliva flow rates and buffer capacity. People who show signs and symptoms of erosion are often not aware of this condition. Only when a comprehensive case history is undertaken will all the risk factors be revealed.
It is difficult to judge the activity and progression of dental erosion. One tool is the comparison of clinical photographs of tooth surfaces to estimate the possible substance loss over time. Thereby, the discoloration of the lesions and their sensitivity state may give some information about the activity of the tooth surface. Further, study casts as well as the examination of dental radiography, especially bitewings longitudinally taken, can provide information about the substance loss over time. For research purpose, computed controlled mapping [13] or profilometric measurements using acid-resistant markers [6, 92] are tools to monitor progression.
Risk factors
When an acidic solution comes in contact with enamel, it has to diffuse first through the acquired pellicle, and only thereafter can it interact with enamel. The acquired pellicle is an organic film, free of bacteria, covering oral hard and soft tissues. It is composed of mucins, glycoproteins and proteins, including several enzymes [41]. On the surface of enamel, the hydrogen ion component of the acid will start to dissolve the enamel crystal. First, the prism sheath area and then the prism core are dissolved, leaving the well-known honey comb appearance [74]. Thereafter, fresh, unionized acid will eventually diffuse into the interprismatic areas of enamel and dissolve further mineral in the region underneath the surface [23, 27, 65]. This will lead to an outflow of ions (dissolution) and subsequently to a local pH rise in the tooth substance immediately below and in the liquid surface layer adjacent to the enamel surface [65]. The events in dentine are, in principle, the same but are even more complex.
There are different predisposing factors and aetiologies of the erosive condition. The interplay of chemical, biological and behavioural factors is crucial and helps explain why some individuals exhibit more erosion than others, even if they are exposed to the same acid challenge in their diets. Comprehensive knowledge of the different risk factors is a prerequisite to initiate adequate preventive (non-interventive) and, if necessary, therapeutic (interventive) measures. When a restoration becomes inevitable, in all situations, the preparations have to follow the principles of minimally invasive treatment.
Figure 5 is an attempt to reveal the multifactorial predisposing factors and aetiologies of the erosive condition.
Fig. 5Interactions of the different factors for the development of dental erosion (adapted from [68])
Chemical factors
Several in vitro and in situ studies show that the erosive potential of an acidic drink or foodstuff is not exclusively dependent on its pH value but is also strongly influenced by its mineral content, its titratable acidity (‘the buffering capacity’) and by the calcium-chelation properties [7, 8, 37, 44, 61, 72, 73, 76, 77, 80, 86, 88, 94, 95, 96]. The pH value, calcium, phosphate and fluoride content of a drink or foodstuff determine the degree of saturation with respect to the tooth mineral, which is the driving force for dissolution. Solutions oversaturated with respect to dental hard tissue will not dissolve it. A low degree of undersaturation with respect to enamel or dentine leads to a very initial surface demineralization which is followed by a local rise in pH and increased mineral content in the liquid surface layer adjacent to the tooth surface. This layer will then become saturated with respect to enamel (or dentine) and will not demineralize further.
Acids such as citric acid exist in water as a mixture of hydrogen ions, acid anions (e.g. citrate) and undissociated acid molecules, with the amounts of each determined by the acid-dissociation constant and the pH of the solution. The hydrogen ion directly attacks the crystal surface. Over and above the effect of the hydrogen ion, the citrate anion may complex with calcium, also removing it from the crystal surface. Each acid anion has a different strength of calcium complexation dependent on the structure of the molecule and how easily it can attract the calcium ion [28]. Consequently, acids such as citric acids have double actions and may be very damaging to the tooth surface. Up to 32% of the calcium in saliva can be complexed by citrate at concentrations common in fruit juices, thus reducing the super-saturation of saliva and increasing the driving force for dissolution with respect to tooth minerals [75].
The dissolution with water of drinks containing organic acids with high buffering capacity will hardly reduce the pH but will reduce the titratable acidity. This is of some importance, as the greater the buffering capacity of the drink, the longer it will take for saliva to neutralize the acid. But dilution will also reduce concentrations of Ca and P (if present), which have a protective effect [10, 61, 62].
The calcium and phosphate content of a foodstuff or beverage are important factors for the erosive potential as they influence the concentration gradient within the local environment of the tooth surface. The addition of calcium (and phosphate) salts to erosive drinks showed promising results. Addition of calcium to a low-pH blackcurrant juice drink has been shown to reduce the erosive effect of the drink [48]. In a follow-up study, a blackcurrant drink with added calcium was compared to a conventional orange drink in situ. Servings of 250 ml of each drink were consumed four times per day during 20 working days. Measurements of enamel loss were made by profilometry on enamel samples for up to 20 days. The experimental carbonated blackcurrant drink supplemented with calcium caused significantly less enamel loss than the conventional carbonated orange drink at all time points measured [98]. When Ca was added to a sports drink, a reduction of the erosive potential was found [46]. Today, several Ca-enriched orange juices and sports drinks are on the market which hardly soften the enamel surface. Yoghurt is another example of food with a low pH (~4.0), yet it has hardly any erosive effect due to its high calcium and phosphate content, which makes it supersaturated with respect to apatite. A yoghurt or another milk-based food may have an erosive potential when it has a low content of Ca and/or P and a low pH. It has to be kept in mind that, with the added mineral, enamel dissolution could not always be completely prevented. But the progression can be retarded which has some implications for the patient and the clinician.
Theoretically, fluoride has some protective effect in a drink with a pH higher than that indicated by the saturation curve of fluorapatite at given Ca and P concentrations. Lussi et al. [61, 62] and Mahoney et al. [71] found an inverse correlation of the erosive potential of different beverages with their fluoride content. It is unlikely that fluoride at the concentration present in beverages alone has any great beneficial effect on erosion because the challenge is high. However, it is possible that, under conditions in which the other erosive factors are not excessive, fluoride in solution may exert some protective effect [71]. It appears that topical fluoride application can positively affect the tooth-wear process. The influence of immersion in fluoride solutions on brushing abrasion of eroded dentine slabs was investigated by Attin et al. [3]. Bovine dentine specimens were alternately stored in a demineralising and in a remineralising solution and brushed five times. Before each immersion in the remineralising solution, the specimens were not treated, treated with 250 ppm or treated with 2,000 ppm neutral sodium fluoride solution for 1 min. The least wear was found in the uneroded controls, whereas the greatest wear was found in the group without fluoride treatment. Significantly greater wear resistance was found as fluoride concentration increased. The same group evaluated the abrasion resistance of eroded enamel brushed with an acidified fluoride gel [4]. They found that treatment with a slightly acidic amine/sodium fluoride gel increased the abrasion resistance of bovine enamel compared to an unfluoridated gel. They speculated that the fluoride is incorporated into and deposited on the enamel during treatment with the acidic gel, which could be one reason for the higher resistance.
The adhesiveness and displacement of the liquid are other factors to be considered in the erosive process. There appear to be differences in the ability of beverages to adhere to enamel based on their thermodynamic properties, e.g. the thermodynamic work of adhesion [50].
In summary, the two very often-cited parameters, the pH and the titratable acidity, do not readily explain the erosive potential of food and drink. The mineral content is also an important parameter, as is the ability of any of the components to complex calcium and to remove it from the mineral surface.
Biological factors
Biological factors such as saliva, acquired pellicle, tooth structure and positioning in relation to soft tissues and tongue are related to dental erosion development.
A very important biological parameter is saliva. Several salivary protective mechanisms come into play during an erosive challenge: dilution and clearance of an erosive agent from the mouth, neutralisation and buffering of acids, and slowing down the rate of enamel dissolution through the common ion effect by salivary calcium and phosphate [104]. Practical experience demonstrates the importance of saliva in patients suffering from salivary-flow impairment. Studies have shown that erosion may be associated with low salivary flow or/and low buffering capacity [53, 64, 89]. The dry mouth condition is usually related to aging [18, 81, 84], even though some other studies have not found this correlation [9, 43]. It is well established that patients taking medication can also present decreased saliva output [101], as well as those who have received radiation therapy for neck and head cancer [19]. Tests of the stimulated and unstimulated flow rate as well as of the buffer capacity of saliva may provide some information about the susceptibility of an individual to dental erosion. However, it has to be kept in mind that these parameters are two of a multifactorial condition. Sialometric evaluations should be carried at a fixed time point or in a limited time interval in the morning, avoiding intra-individual variations due to the circadian cycle [29]. Studies have shown that sour foodstuff has a strong influence on the anticipatory salivary flow [15, 55], which can be significantly increased when compared to the normal unstimulated flow rate [25]. Hypersalivation also occurs in advance of vomiting as a response from the ‘vomiting center’ of the brain [56], as frequently seen in individuals suffering from anorexia and bulimia nervosa, rumination or chronic alcoholism. It is suggested that this could minimize the erosion caused by acids of gastric origin. On the other hand, patients with symptoms of gastro-esophageal reflux disease (GERD) should not expect the salivary output to increase before the gastric juice regurgitation because this is an involuntary response not co-coordinated by the autonomic nervous system [42, 90]. Therefore, there may be insufficient time for saliva to act before erosion occurs. The influence of saliva on the remineralisation/rehardening of erosive damaged dental hard tissue is discussed controversially. There is evidence that acid-softened enamel can reharden after exposure to saliva or remineralisation solution and that dietary products and fluoride can enhance the rehardening process [2, 26, 33]. Other investigations could not find a significant rehardening effect of saliva in situ [16, 32, 34, 58]. Some limited increase of the abrasion resistance of softened enamel was found after intraoral exposure to saliva [5, 51]. It seems that in vitro, some rehardening could be expected when supersaturated solution or saliva with no protein added are used [23, 24], whereas in situ, this is only the case to a very small amount.
The salivary acquired pellicle is a protein-based layer which is rapidly formed on dental surfaces after its removal by tooth brushing with dentifrice, other prophylaxis, measures or chemical dissolution. This organic layer becomes detectable on dental surfaces after few minutes of exposure to the oral environment [39, 93]. Enzymatic activity is also detectable at early stages of pellicle formation [40, 41]. It is suggested that it grows until reaching an equilibrium between protein adsorption and de-sorption within 2 h [57].
The acquired pellicle may protect against erosion by acting as a diffusion barrier or a perm-selective membrane preventing the direct contact between the acids and the tooth surface [39, 102, 103], reducing the dissolution rate of hydroxyapatite [57].
Millward et al. [78] monitored the pH at the surface of teeth of healthy volunteers after drinking 1% citric acid. They observed that the pH recovered to above pH 5.5 within 2 min from a site adjacent to the palatal surface of the upper central incisor and within 4–5 min from another palatal surface of the upper first molar. Other observations have revealed a longer clearance time on upper incisors for patients with active erosions and normal saliva values compared to patients with no erosion (Lussi, unpublished data). These differences could be due to the anatomy of the teeth and soft tissues that may influence the retention/clearance pattern of erosive agents. Furthermore, soft tissue movements by the tongue and buccal mucosa and swallowing pattern can influence clearance rate. The importance of the tongue in modifying the tooth-wear process has long been the subject of speculation. Holst and Lange [45] considered mechanical abrasion caused by tongue to be a contributing factor in erosion caused by vomiting. Observations from animal studies also provide support in that beverages produced erosion mainly on the lingual surfaces of rat molar teeth in areas where the tongue contacts the teeth [96]. Dugmore and Rock [21] found that orthodontic anomaly appeared to confer a protective effect.
Behavioural factors
During and after an erosive challenge, behavioural factors play a role in modifying the extent of tooth wear. The manner that dietary acids are introduced into the mouth will affect which teeth are contacted by the erosive challenge and possibly the clearance pattern. As lifestyles have changed through the decades, the total amount and frequency of consumption of acidic foods and drinks have also changed. Soft drink consumption in the USA increased by 300% in 20 years [11], and serving sizes increased from 185 g (6.6 oz) in the 1950s to 340 g (12 oz) in the 1960s and to 570 g (20 oz) in the late 1990s. Around the year 1995, between 56 and 85% of children at school in the USA consumed at least one soft drink daily, with the highest amounts ingested by adolescent males. Of this group, 20% consumed four or more servings daily [36]. Studies in children and adults have shown that this number of servings per day is associated with the presence and the progression of erosion when other risk factors exist [64, 83].
High erosion was associated with a method of drinking whereby the drink was kept in the mouth for a longer period [54]. One study investigated a randomly selected group of Swiss adults and diagnosed the cause of tooth wear. It showed that the consumption of erosive drinks and foodstuffs was strongly associated with erosion on facial and occlusal surfaces. Severe palatal erosions were scarce and highly associated with chronic vomiting [60]. The presence of calculus (odds ratio 0.48) or eating fruit other than apples or citrus fruit (0.48) reduced the chances of erosion. High consumption of carbonated drinks increased the odds of erosion being present at 12 years by 252% and was a strong predictor of the amount of erosion found at age 14 [21]. In a sample of 987 children (2 to 5 year-olds), consumption of vitamin C supplements, carbonated drinks and fruit syrup from a feeding bottle at bed- or naptime were related to erosion [1]. Considerable risk of erosion was found when citrus fruits were eaten more than twice a day and soft drinks were drunk daily [53].
Excessive consumption of acidic candies combined with a low salivary buffering capacity may aggravate erosive lesions [17, 63]. The high intake of herbal teas, widely perceived as a healthy drink, may have an erosive potential exceeding that of orange juice [85]. An increase in agitation (e.g. when a patient is swishing his/her drink in the mouth) will enhance the dissolution process because the solution on the surface layer adjacent to tooth mineral will be readily renewed. Further, the amount of the drink in the mouth in relation to the amount of saliva present will modify the dissolution process [69]. Several authors have suggested that using a straw is beneficial, since the straw directs drinks past the anterior teeth and towards the pharynx [22, 38, 49]. However, the placing of a straw labial to the anterior teeth can be destructive [70]. Nighttime exposure to erosive agents may be particularly destructive because of the absence of salivary flow. Two examples of this are bedtime baby bottle-feeding with acidic beverages and gastroesophageal reflux with regurgitation during sleeping.
Healthier lifestyle paradoxically can lead to dental health problems in the form of dental erosion, as it often involves regular exercise and what is considered healthy diets with more fruits and vegetables. A lactovegetarian diet, which includes the consumption of acidic foods, has been associated with a higher prevalence of dental erosion [30]. The benefits of exercise are well-proven; however, exercise increases the loss of body fluids and may lead to dehydration and decreased salivary flow. A few case reports and studies have reported an association between sports activities and dental erosion. The cause could be direct acid exposure or strenuous exercise which may increase gastroesophageal reflux. Risk groups are swimmers exercising in water with low pH and athletes consuming frequently erosive sport drinks. In a study with 25 swimmers and 20 cyclists, the latter showed significantly more tooth wear into dentine. However, no association between erosion and sports drink consumption was found [79]. Professional swimmers train several hours in the water, which should have proper pH regulation. The main disinfection techniques used are gas chlorination and sodium hypochlorite. In the Netherlands, where the sodium hypochlorite method is used, only 0.14% of the tested pools were found in the year 2001 to have low pH values [59]. Another case report confirmed these findings [91]. In a review by Geurtsen [35], an increased prevalence of dental erosion among intensive swimmers due to low-pH gas-chlorinated pool water was described. The recommended pH for swimming pools is between pH 7.2 and 8.0. Swimming activities in pH-adjusted pools do not harm the teeth [100]. However, erosion among competitive swimmers was found in 39% of swim-team members who trained in a pool with a pH of 2.7 which is a H concentration 100,000 times higher than that recommended for swimming pools [12]. Sports drinks are often erosive [46, 47, 95, 97], and when consumed during strenuous activity when the person is in a state of some dehydration, the possible destructive effects may be enhanced further.
Health-conscious individuals also tend to have better than average oral hygiene. While good oral hygiene is of proven value in the prevention of periodontal disease and dental caries, frequent tooth brushing with abrasive oral hygiene products may enhance dental erosion.
At the other end of the spectrum, an unhealthy lifestyle may also be associated with dental erosion [105]. Alcoholics may be at particular risk for dental erosion and tooth wear. Robb and Smith [87] reported significantly more tooth wear in 37 alcoholic patients than in age- and sex-matched controls. Tooth wear was most pronounced in men and those with frequent alcohol consumption.
Wine has properties such as low pH and low content of P and Ca, which renders it to have an erosive potential. Professional wine tasting is very common all over the world. In some countries (e.g. Sweden, Finland), wine tasters are employed by the state to support their state-owned wine shops. Full-time Swedish wine tasters test on average 20–50 different wines nearly 5 days a week. Wiktorsson et al. [99] investigated the prevalence and severity of tooth erosion in 19 qualified wine tasters in relation to number of years of wine tasting, salivary flow rate and buffer capacity. Salivary flow rate and buffer capacity of unstimulated and stimulated saliva were measured. Data on occupational background and dental and medical histories were collected. Fourteen subjects had tooth erosion mainly on the labio-cervical surfaces of maxillary incisors and canines. The severity of the erosion tended to increase with years of occupational exposure. Caries activity in all subjects was low. It was concluded that full-time wine tasting is an occupation associated with an increased risk for tooth erosion. In a cross-sectional comparative study, the prevalence and severity of tooth-surface loss between winemakers (exposed) and their spouses (non-exposed) was examined. There was a difference in the prevalence and severity of tooth surface loss between the two groups [14]. On the other hand, many other studies were not able to find an association between dental erosion and behavioural factors [30, 52], or they found only a weak association [82]. One can only speculate about the reasons. A possible explanation is the mode of questioning the persons (orally vs written questionnaire), the statistics employed (multivariate vs univariate) and the population group under study (selected vs randomly).
Although no detrimental effects were described on a population level, one has to keep in mind that factors like sports-drink consumption and occupation can be, for some patients, a cofactor in the development of or in the increase in dental erosion when other factors are present. It is unlikely that one or two isolated factors (e.g. sports drink, dehydration) will be responsible for a multifactorial condition like erosion.
Conclusion
This overview shows the importance of early diagnosis of dental erosion and of accurate detection of possible risk factors and their interplay. These facts are prerequisites to initiate adequate preventive (and therapeutic) measures.

erosion
chemical
clinical appearance
biological and behavioural risk factors