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 -LRB- TEA -RRB- 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 -LRB- Kv2 .1 -RRB- 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 -LRB- like Shaker -RRB- 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 -LRB- Hille , 2001 -RRB- . 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 -LRB- Tamargo et al. , 2004 ; Yost , 1999 -RRB- . 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 -LRB- TEA -RRB- . 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 -LRB- Doyle et al. , 1998 ; Jiang et al. , 2002 , 2003 -RRB- and a mammalian voltage-gated flavor -LRB- Long , Campbell & MacKinnon , 2005 -RRB- . 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 -LRB- Kavanaugh et al. , 1991 ; MacKinnon & Yellen , 1990 -RRB- . 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 -LRB- Heginbotham & MacKinnon , 1992 ; Kavanaugh et al. , 1992 -RRB- . This amino acid -LRB- position 449 of the prototypical voltage-gated K channel , Shaker -RRB- 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 -LRB- Jarolimek et al. , 1995 , 1996 -RRB- . In addition , amino acids other than that at position 449 are involved in TEA binding -LRB- Pascual et al. , 1995 -RRB- . A molecular dynamics study -LRB- Crouzy , Berneche & Roux , 2001 -RRB- 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 -LRB- Andalib et al. , 2004 -RRB- . 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 -LRB- Mozhayeva & Naumov , 1972 -RRB- . 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 -LRB- MacKinnon , Latorre & Miller , 1989 -RRB- . The pH-dependent TEA block of Kv1 .1 channels is sensitive to changes in bulk solution ionic strength -LRB- Bretschneider et al. , 1999 -RRB- . 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 -LRB- gallamine -RRB- is even more sensitive -LRB- Quinn & Begenisich , 2001 -RRB- . 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 -LRB- Immke & Korn , 2000 ; Immke et al. , 1999 -RRB- . 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 -LRB- Grissmer et al. , 1994 ; Taglialatela et al. , 1991 -RRB- . 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 -LRB- Chan Test Inc. , Cleveland , OH -RRB- . 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 -LRB- PCR -RRB- protocol and the resulting mutants analyzed by DNA sequencing . OOCYTE ISOLATION AND MICROINJECTION Xenopus laevis oocytes were maintained as described by Goldin -LRB- 1992 -RRB- . Isolated ovarian lobes were rinsed with Ca2 + - free OR-2 solution -LRB- in mM -RRB- : 82.5 NaCl , 2.5 KCl , 1 MgCl2 and 5 4 - -LRB- 2-hydroxyethyl -RRB- -1-piperazineethanesulfonic acid -LRB- HEPES ; pH 7.6 , NaOH -RRB- and then defolliculated by incubation for 60 -- 90 min with 2 mg/ml collagenase type 1A -LRB- Sigma , St. Louis , MO -RRB- . Cleaned oocytes were transferred and maintained for 2 h in ND-96 solution -LRB- in mM -RRB- : 96 NaCl , 2 KCl , 1.8 CaCl2 , 1 MgCl2 , 5 HEPES and 2.5 Na-pyruvate -LRB- pH 7.6 , NaOH -RRB- 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 -LRB- frog -RRB- 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 -LRB- 20 -- 22 °C -RRB- using the cut-open oocyte voltage-clamp apparatus -LRB- model CA-1B ; Dagan , Minneapolis , MN -RRB- . 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 -LRB- ELV-1 , Dagan -RRB- was modified to include a low-volume -LRB- 80 μl -RRB- chamber insert . The low chamber volume and high flow rates -LRB- ∼ 1 ml/min -RRB- produced efficient solution exchange . The measured difference in junction potential for the two experimental solutions -LRB- see below -RRB- was < 2 mV , so no correction to membrane voltages was made . Recording electrodes were made either of 1 BBL glass with filament -LRB- 1.5 mm outer diameter ; World Precision Instruments , Sarasota , Fl -RRB- or of GC-150F glass -LRB- Warner Instrument , Hamden , CT -RRB- . 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 -LRB- standard error of them mean , SEM -RRB- kΩ in normal-ionic strength solutions and 2.2 ± 0.11 kΩ in reduced-ionic strength solutions -LRB- n = 50 -RRB- . Electrical access to the internal compartment was achieved by 0.125 % saponin treatment in a solution of -LRB- in mM -RRB- 100 KCl and 10 HEPES -LRB- pH 7.4 , N-methyl-D-glucamine -LSB- NMDG -RSB- -RRB- . The external solution was -LRB- in mM -RRB- 140 NaCl , 2 CaCl2 , 10 KCl , 10 HEPES -LRB- pH 7.4 , NMDG -RRB- . An external solution of reduced ionic strength was used that consisted of -LRB- in mM -RRB- 35 NaCl , 0.5 CaCl2 , 10 KCl , 10 HEPES -LRB- pH 7.4 , NMDG -RRB- . 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 -LRB- Wescor , Logan , UT -RRB- . Addition of TEA-Cl was made by substitution with NaCl , which maintained solution ionic strength . Gallamine-I3 -LRB- 1,2,3-tris -LSB- 2-triethylammonium ethoxy -RSB- benzene triiodide ; Sigma -RRB- 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 -LSB- B -RSB- 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 -LRB- RCSB Protein Data Bank entry 1J95 -RRB- as a template for the Kv2 .1 channel -LRB- Doyle et al. , 1998 -RRB- . The KcsA residues in the outer vestibule were mutated to their Kv2 .1 counterparts with the DeepView/Swiss-PdbViewer , version 3.7 -LRB- http://www.ca.expasy.org/spdbv/ -RRB- . The molecular views of the K channel structures were generated with ViewerLite 4.2 for Windows -LRB- Accelrys , San Diego , CA ; http://www.accelrys.com/ -RRB- . 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 -LRB- Long et al. , 2005 -RRB- . 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 -LRB- Kavanaugh et al. , 1991 ; MacKinnon & Yellen , 1990 -RRB- . Both Kv2 .1 and Kv3 .1 channels have a tyrosine at this position -LRB- shown in green -RRB- . 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 -LRB- K356 and K382 -RRB- in Kv2 .1 channels that are a major focus of this study . Kv2 .1 and Kv3 .1 channels have a tyrosine amino acid -LRB- shown in green -RRB- at the position that is important for high-affinity TEA block -LRB- see text -RRB- . In red are several amino acids -LRB- aspartate and glutamate -RRB- 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 -LRB- in ribbon form -RRB- is apparent in this view . The Kv2 .1 K356 and K382 lysine residues are shown with space-filling atoms -LRB- without hydrogens -RRB- . 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 -LRB- indicated in red in the alignment -RRB- . Kv2 .1 channels are unique in having two positively charged amino acids -LRB- K382 and K356 -RRB- in the outer vestibule -LRB- blue in the alignment and illustrated as space-filling atoms in the structural diagrams -RRB- . 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 -LRB- Quinn & Begenisich , 2001 -RRB- . 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 -LRB- 50 μM -RRB- produced a small -LRB- near 25 % -RRB- block of Kv3 .1 channels in a normal-ionic strength solution -LRB- see Materials and Methods -RRB- 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 -LRB- Grissmer et al. , 1994 ; Jarolimek et al. , 1995 ; Taglialatela et al. , 1991 -RRB- . 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 . -LRB- A -RRB- 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 - -LRB- ■ -RRB- and low - -LRB- ○ -RRB- ionic strength solutions . Mean values from three to five measurements and SEM limits -LRB- if larger than symbol -RRB- 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 . -LRB- B -RRB- 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 - -LRB- ■ -RRB- and low - -LRB- ○ -RRB- 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 -LRB- < 20 % -RRB- , 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 -LRB- Quinn & Begenisich , 2001 -RRB- . 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 - -LRB- ■ -RRB- and low - -LRB- ○ -RRB- ionic strength solutions . Mean values from 4 -- 10 measurements and SEM limits -LRB- if larger than symbols -RRB- 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 -LRB- Immke et al. , 1999 ; Taglialatela et al. , 1991 -RRB- 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 -LRB- T449Y -RRB- 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 -LRB- n = 3 -RRB- . 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 -LRB- e.g. , Begenisich , 1975 ; Frankenhaeuser & Hodgkin , 1957 ; Mozhayeva & Naumov , 1972 -RRB- . 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 -LRB- Hartmann et al. , 1991 ; Taglialatela et al. , 1991 -RRB- . 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 . -LRB- A -RRB- Fraction of Kv3 .1 channel current blocked by 100 μM TEA at the indicated membrane potentials in normal - -LRB- ■ -RRB- and low - -LRB- ○ -RRB- ionic strength solutions . -LRB- B -RRB- Fraction of Kv2 .1 channel current blocked by 5 mM TEA at the indicated membrane potentials in normal - -LRB- ■ -RRB- and low - -LRB- ○ -RRB- 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 -LRB- see Fig. 1 -RRB- . This issue has been discussed previously -LRB- Taglialatela et al. , 1991 -RRB- , and one possibility that those authors suggested is that the positively charged side chain of the nearby K382 -LRB- see Fig. 1 -RRB- 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 . -LRB- A -RRB- Dose-response relation for TEA block of current -LRB- at +40 mV -RRB- from Kv2 .1 K382Q channels in normal - -LRB- ■ -RRB- and low - -LRB- ○ -RRB- ionic strength solutions . Mean values from three measurements and SEM limits -LRB- if larger than symbols -RRB- 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 . -LRB- B -RRB- Dose-response relation for TEA block of current -LRB- at +40 mV -RRB- from Kv2 .1 K356F channels in normal - -LRB- ■ -RRB- and low - -LRB- ○ -RRB- 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 -LRB- not significantly different from zero , n = 3 -RRB- . Inspection of the likely topology of the outer pore of Kv2 .1 channels -LRB- Fig. 1 -RRB- suggests that the other positively charged amino acid unique to Kv2 .1 -LRB- K356 -RRB- 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 -LRB- phenylalanine -RRB- . 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 -LRB- Fig. 2B -RRB- . Figure 6 . Ionic strength-dependent TEA and gallamine block of K356F/K382Q Kv2 .1 channels . -LRB- A -RRB- Dose-response relation for TEA block of current -LRB- at +40 mV -RRB- in normal - -LRB- ■ -RRB- and low - -LRB- ○ -RRB- 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 . -LRB- B -RRB- Dose-response relation for gallamine block of current -LRB- at +40 mV -RRB- in normal - -LRB- ■ -RRB- and low - -LRB- ○ -RRB- ionic strength solutions . Mean values from three to six measurements and SEM limits -LRB- if larger than symbols -RRB- 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 -LRB- MacKinnon et al. , 1989 ; Quinn & Begenisich , 2001 -RRB- , 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 -LRB- Kavanaugh et al. , 1991 ; MacKinnon & Yellen , 1990 -RRB- . 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 -LRB- see Fig. 1 -RRB- 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 -LRB- Fig. 5 -RRB- , 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 -LRB- Fig. 6 -RRB- . 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 -LRB- Fig. 4 -RRB- . 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 -LRB- K382 and K356 -RRB- and the one farther away -LRB- K350 -RRB- are sufficient to balance the electric field from the four negatively charged amino acids in the vicinity -LRB- D351 , E352 , D353 , D354 -RRB- . 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 -LRB- K336 and K382 -RRB- 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 -LRB- as observed -RRB- 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 -LRB- K382V , K356G -RRB- does , in fact , increase TEA affinity -LRB- Immke et al. , 1999 -RRB- . 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 can not 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 -LRB- a total of eight positive charges on these tetrameric channels -RRB- 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 -LRB- Fig. 5B -RRB- . Another feature of the gallamine block of the double mutant Kv2 .1 channels was the apparent saturation of block at levels well below 100 % -LRB- Fig. 6B -RRB- . 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 -LRB- Hartmann et al. , 1991 ; Immke & Korn , 2000 ; Immke et al. , 1999 -RRB- . 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 -LRB- Zhou et al. , 2001 -RRB- , and Shaker K channels exhibit both high - and low-affinity TEA block configurations also controlled by the K + levels -LRB- Thompson & Begenisich , 2005 -RRB- . Future experiments will be required to test if Kv2 .1 channels -LRB- or mutant Kv2 .1 channels -RRB- 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 -LRB- MacKinnon et al. , 1989 -RRB- , exhibits strong charge-dependent effects -LRB- Hui et al. , 2002 -RRB- . 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 can not 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 .

O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

k channel
surface potential
channel block
tetraethylammonium
gallamine


Storage hexamer utilization in two lepidopterans : differences correlated with the timing of egg formation Most insects produce two or more storage hexamers whose constituents and developmental profiles are sufficiently different to suggest specialization in the ways that they support metamorphosis and reproduction . Hexamerin specializations are compared here in the Cecropia moth -LRB- Hyalophora cecropia -RRB- , which produces eggs during the pupal-adult molt , and the Monarch butterfly -LRB- Danaus plexippus -RRB- , which produces eggs under long-day conditions after adult eclosion . In both sexes of both species , reserves of arylphorin -LRB- ArH -RRB- were exhausted by the end of metamorphosis . In Cecropia , the same was true for the high-methionine hexamerins , V-MtH and M-MtH . But in short day Monarch females 20 -- 30 % of the pupal reserves of V-MtH and M-MtH survived metamorphosis , persisting until long-day conditions were imposed to stimulate egg formation . Differences in storage sites have been documented in other lepidopterans , with MtH reserves being found primarily in fat body protein granules and the ArH reserve being found primarily in the hemolymph . Similar differences could explain how a fraction of the MtH 's , but not of ArH , escapes utilization during metamorphosis in a species with post-eclosion egg formation . No differences in utilization schedules were detected between V - and M-MtH , despite divergent compositions and antigenic reactivity . Introduction Insects prepare for the synthetic demands of molting , metamorphosis and reproduction by accumulating hexamerins in their hemolymph and fat body . First to be described were two soluble storage hexamerins isolated from larvae of Calliphora erythrocephala -LRB- Munn and Greville , 1969 -RRB- . Two or more hexamerins were later reported in the hemolymph and fat body of many other insects , with some lepidopterans having as many as four . Hexamerins occurring within a single species can differ in amino acid composition , stage of synthesis , distribution between hemolymph and fat body , timing of clearance from the hemolymph , and antigenic reactivity -LRB- reviewed by Wyatt and Pan , 1978 ; Riddiford and Law , 1983 ; Kanost et al. , 1990 ; Telfer and Kunkle , 1991 -RRB- . The first storage proteins to be described in lepidopterans were two methionine-rich hexamerins from Hyalophora cecropia -LRB- Tojo et al. , 1978 -RRB- . They differed in electrophoretic mobility in native PAGE and also in amino acid composition , methionine contents being 7.0 % for the more slowly migrating and 4.9 % for the faster . -LRB- We adopt here descriptive acronyms , V-MtH and M-MtH , for very and moderately methionine-rich hexamerins . -RRB- The two appear in the hemolymph late in the last larval instar , rise to maximum concentrations early in the larval-pupal molt , and are then largely endocytosed by the cells of the fat body , which store them in crystalline form until they are utilized during adult development . MtH 's have been identified in a variety of moths , either as isolated proteins or from cDNA sequencing -LRB- Tojo et al. , 1978 ; 1980 ; 1985 ; Ryan et al. , 1985 ; Bean and Silhacek , 1988 ; Jones et al. , 1990 ; Jones et al. , 1993 ; Memmel et al. , 1994 -RRB- . They are identified here in a butterfly by antigenic cross-reactions . They tend to be more abundant in female pupae than in males , and this has led to a prediction that they play special roles in egg formation . A third lepidopteran hexamerin , first described in Manduca sexta -LRB- Kramer et al. , 1980 -RRB- , has a high aromatic amino acid content , resembling in this regard one of the prototypical hexamerins of Calliphora . The importance of aromatic amino acids in sclerotization led to the suggestion that hexamerins in this class , now known as arylphorins -LRB- ArH -RRB- -LRB- Telfer et al. , 1983 -RRB- , are adapted to support cuticle deposition . While many lepidopterans produce just these three , some also contain a riboflavin-binding hexamerin , RbH -LRB- Telfer and Massey , 1987 ; Miller and Silhacek , 1992 ; Magee et al. , 1994 -RRB- . Like ArH , RbH is a major component of pupal hemolymph that disappears during adult development -LRB- Pan and Telfer , 1999 -RRB- . Labeling experiments in saturniid moths failed to confirm the targeting of hexamerins for special developmental processes . In Actias luna M-MtH and ArH proved to be equivalent sources of labeled amino acids for the production of eggs and cuticle , as well as other adult tissues -LRB- Pan and Telfer , 1996 -RRB- , and RbH and ArH were found to be similarly equivalent in H. cecropia -LRB- Pan and Telfer , 1999 -RRB- . These tests were performed in species that produce eggs at the same time that they form the soma of moths . They thus left open the question of which of the hexamerins remain available to support egg formation when the latter is delayed until after adult eclosion . To investigate this we compared the timing of hexamerin depletion in two lepidopterans that form eggs at different times relative to eclosion . The Cecropia silkmoth , Hyalophora cecropia , ecloses with a full complement of eggs that are ready to be fertilized and laid . The Monarch butterfly , Danaus plexippus , delays egg formation until it experiences a long-day photoperiod after eclosion -LRB- reviewed by Ackery and Vane-Wright , 1984 -RRB- . The strategy we used was to time in each species the disappearance of V-MtH , M-MtH and ArH during metamorphosis and egg formation . The accumulation of vitellogenin -LRB- Vg -RRB- was simultaneously measured , since synthesis of this abundant product by the adult fat body can be correlated with hexamerin depletion -LRB- Wheeler and Buck , 1996 ; Wheeler et al. , 1999 -RRB- . RbH was not included because we could not detect it with rabbit antibodies in Monarch larvae or pupae -LRB- Pan and Telfer , 1999 -RRB- . Materials and Methods 1 . Experimental Insects Diapausing pupae of field-reared Hyalophora cecropia were stored at 6 °C for at least five months and then allowed to initiate adult development by transfer to 25 °C . At this temperature development from apolyis to eclosion requires 23 days in females and 21 days in males . Migrating butterflies of Danaus plexippus were captured during mid-October in eastern Tennessee . Most of the catches were sufficiently fresh to suggest recent emergence in nearby regions . They were kept in cages at outdoor temperature and day length , and fed once per day with a 30 % honey solution . After two to three weeks under these conditions the ovaries contained only small , translucent follicles , indicating an absence of yolk formation . Their fat bodies , relative to those of freshly caught butterflies , were greatly enlarged and full of lipid droplets . To obtain pupae and second generation adults , some captured Monarchs were kept at 30 °C under long day conditions , and fed twice daily with the honey solution . They terminated reproductive diapause and began mating in 1 to 2 weeks . Potted milkweed , Asclepias curasavica , was provided for egg laying . Hatched caterpillars were left on the plants until they reached the 3rd instar , and were then transferred to field-collected cuttings of Asclepias syriaca . Additional eggs and caterpillars were generously provided by Mr. Paul deMarrais of Afton , Tennessee . 2 . Antibodies V-MtH , M-MtH , ArH and Vg were measured in both species with rabbit antibodies against the corresponding proteins of H. cecropia . The reactions with Monarch proteins , though weaker than those with Cecropia , were still strong enough to be useful . This relationship is shown in Figure 1 for antibodies to Cecropia V-MtH . ArH , Vg and what we identify below as M-MtH have been isolated from the hemolymph of Cecropia pupae by chromatographic methods , and the reactions of the antisera employed to measure them have already been described -LRB- Telfer et al. , 1983 ; Telfer and Pan , 1988 ; Pan and Telfer , 1992 -RRB- . Each of the three antisera reacted with its homologous isolate to produce a single zone of heavy precipitation in immunodiffusion tests and showed no additonal reactions when combined with unfractionated hemolymph , whole-body extracts , or isolates other than the one used for immunization . The antiserum against V-MtH has not been described before . We used native PAGE to isolate this hexamerin from pupal male hemolymph . Slab gels -LRB- 0.3 × 12 × 17 cm -RRB- containing 4 % acrylamide and lacking lane dividers were overlain with 40 µl of pupal male hemolymph diluted with twice its volume of native PAGE sample buffer . After electrophoresis a vertical strip from the center of the gel was stained with Coomassie Blue -LRB- Figure 2 , lane 1 -RRB- . Horizontal strips corresponding to the positions of the two MtH 's were cut from the unstained remainder of the gel and stored by freezing . The two MtH 's were identified by N-terminal amino acid microsequencing at a facility of the University of Pennsylvania School of Veterinary Medicine . For the faster of the two in native PAGE , the N-terminal sequence was RPDNDDVNFVVSM and for the slower band was SVVNDANYSF -LRB- 40 % of the fast component lacked the N-terminal RP but was otherwise identical to the other 60 % . -RRB- These sequences matched N-terminal sequences deduced from cDNA clones of , respectively , M-MtH and V-MtH after signal polypeptide deletion -LRB- Massey , 1995 ; accession number AF032398 for M-MtH and AF032399 for V-MtH -RRB- . The faster band corresponded to a 738 amino acid polypeptide with a methionine content of 4.76 % , while the slower corresponded to a 737 amino acid polypeptide with a methionine content of 8.54 % . Protein extracted from the slower band , thus identified as V-MtH , was used to immunize a rabbit -LRB- Harlow and Lane , 1988 -RRB- . In a variety of immunodiffusion tests with pupal hemolymph and extracts from H. cecropia , anti-V-MtH produced a single band of precipitation -LRB- Figure 1 -RRB- . 3 . Tissue Preparation Hemolymph was drained through a dorsal slit into a pre-chilled , graduated test tube containing 0.2 ml of a buffer solution -- either , pH 7.2 phosphate-buffered saline -LRB- PBS -RRB- for Cecropia or 0.15 M Tris-citrate , pH 8.2 , for the Monarch . -LRB- The Tris-citrate buffer used for the Monarch extraction was 6X that of the running buffer strength employed for Rocket Immunoelectrophoresis -LRB- Pan and Telfer , 1990 -RRB- . It was used in case the smaller amount of tissues collected from this species should prove to require a more sensitive technique for protein quantification , but this proved eventually not to be the case . -RRB- Both extraction media contained in addition a protease inhibitor cocktail , Control ® -LRB- Boehringer Mannheim Biochemical -RRB- , and 5mM phenylthiourea -LRB- PTU -RRB- . The midgut and , when present , the bursa copulatrix were discarded and the rest of the soft tissues were scraped out of the abdominal cuticle and added to the collected blood . Additional buffer with the protease inhibitor and PTU was added to bring the suspended tissues to a volume that would yield antigen concentrations convenient to measure by the method described below . For Cecropia the final volume was 6 ml per pupa , and 3 or 1.5 ml per adult female and male , respectively . For Monarch pupae the final volume was 3 ml and for adults 0.75 ml . Comparisons between stages were made possible by normalizing to the pupal volume ; e. g. , concentrations for adult Cecropia females were divided by two and for males by four in order to compare them with the contents of pupal extracts . The collected samples were stored frozen . For extraction , the tissue mixtures were thawed and transferred to a glass homogenizer . When chorionated eggs were present , they were crushed separately in a ceramic mortar and then combined with the rest of the tissues in the homogenizer . 4 . Measurement of Antigen Concentrations An immunodiffusion method was used to measure changes in extractable stores of the four proteins . Antigen concentrations were measured by layering the extracts over antisera that had been solidified in 3 mm -LRB- i. d. -RRB- glass tubes with 0.3 % agarose , PBS and 0.05 % sodium azide . The rate of penetration of an antigen-antibody precipitation front through the agarose is a function of the rate of diffusion of the antigen into the agarose layer and this in turn is a measure of antigen concentration in the upper reservoir . Procedures for setting up the tubes , for measuring rates of advance of precipitation fronts , and for constructing standard curves have already been described -LRB- Telfer et al. , 1983 ; Telfer and Pan , 1988 -RRB- . The method was developed for studies on human serum proteins -LRB- Oudin , 1948 -RRB- , and has been used many times to measure antigen concentrations in insect hemolymph and egg extracts . It requires larger volumes and higher antigen concentrations than rocket immunoelectrophoresis -LRB- Pan and Telfer , 1990 , 1992 -RRB- , but when these conditions are satisfied , as they proved to be for hexamerin measurements in Cecropia and Monarchs , it is convenient and reliable . The measurements are of relative concentrations and are expressed as percentages of the concentration in a standard solution . For the hexamerins , extracts of abdomens from female pupae of each species were used as the standards ; for VG , extracts of females containing chorionated eggs were used . The latter included newly eclosed adults in Cecropia and day 7 adults in long day Monarchs . 5 . SDS-PAGE Electrophoresis Insoluble residues that would escape detection by immunodiffusion were monitored by SDS-PAGE . For this purpose 0.1 ml of each of the homogenized samples from a chosen stage were vortexed and pooled . The pooled sample was centrifuged in a microfuge -LRB- Beckman ® -RRB- for 4 min . The lipid cap was removed , and the supernatant decanted . Pellet and supernatant were then separately dissolved in SDS-PAGE sample buffer so that equal volumes contained equal fractions of , respectively , the total pellet and supernatant material in the uncentrifuged pool . The samples were compared in neighboring lanes of pre-cast 4 -- 15 % gradient mini gels from Bio-Rad ® . Electrophoresis was conducted at constant voltage -LRB- 100 v -RRB- , and terminated after 1 hr and 12 min . Coomassie Brilliant Blue staining was used to reveal the banding patterns . Results 1 . Vitellogenin Cecropia produced Vg earlier than the Monarch -LRB- Figure 3 -RRB- . Like many other saturniids , Cecropia synthesizes part of its Vg , in this case about 30 mg per 6 g female , during the larval-pupal molt , and stores it in the hemolymph during the months of diapause -LRB- Telfer , 1954 -RRB- . Beginning 10 days before eclosion , when yolk formation begins , there is a second period of Vg synthesis -LRB- Pan , 1971 -RRB- -LRB- Figure 3 -RRB- that draws on precursors released from the hexamerins present at that time -LRB- Pan and Telfer , 1996 , 1999 -RRB- . In Monarchs , Vg was not detected in either pupae or short day adults . In females raised under long day conditions it was first detected 2 -- 3 days after eclosion -LRB- Figure 3 -RRB- and it continued to accumulate until day 7 when eggs began to be laid . In SDS-PAGE , Coomassie-stained Vg subunits were identified at 180 and 40 kDa by their presence in females and absence from males -LRB- Figure 4 , Vg -RRB- . They were found in pupal and adult extracts of Cecropia and in extracts of long day Monarch adults , the same samples in which Vg had been detected by immunodiffusion . Vg is stored as a solute in the hemolymph of female Cecropia pupae and accordingly centrifugal pellets of extracts at that stage did not produce detectable 180 and 40 kDa bands . In adults most Vg has been transferred to the yolk ; pellets at this stage produced weak Vg bands due , presumably , to small yolk spheres that are not readily disrupted by homogenization . 2 . Hexamerin utilization in females Cecropia All three Cecropia hexamerins were consumed during the first 13 days of somatic tissue metamorphosis , 55 % of the soluble MtH 's and 33 % of soluble ArH disappeared before VG began its second rise -LRB- Figure 5A -RRB- . The last eight days of adult development encompass late somatic tissue development as well as the second rise in Vg content , and during this period the three hexamerins fell to less than 2 % of pupal levels . Coomassie staining of SDS-PAGE gels of pupal extracts was concentrated in a cluster of circa 80 kDa bands -LRB- Figure 4A , asterisks -RRB- . Mini gels resolved the hexamerin subunit bands in the clusters poorly , but for present purposes that was not a problem . Low 80 kDa staining in centrifugal pellets of adult Cecropia extracts -LRB- Figure 4A , lanes A1 and A2 -RRB- showed that PBS-insoluble reserves , like the soluble reserves assayed by immunodiffusion , were essentially negligible after eclosion . Monarch Nearly all of the soluble ArH stored in Monarch pupae was consumed during metamorphosis , but substantial MtH reserves remained . This was first seen in individuals that had been collected in the field in October and then fed as captives for four weeks under short day conditions -LRB- Figure 6A -RRB- . At the end of the four weeks , short-day females still contained about 20 % as much M-MtH and 30 % as much V-MtH as female pupae . When captive females were held for two weeks under short day conditions and then for two additional weeks in the long days that promote egg formation , these figures were much lower -- 1.5 % for M-MtH -LRB- p < 0.01 -RRB- and 0.7 % for V-MtH -LRB- p < 0.02 -RRB- -LRB- Figure 6A -RRB- . These results were confirmed by SDS-PAGE , which detected a strong 80 kDa band in short day adults -LRB- Figure 4B , lanes Ast -RRB- and very little staining in long-day adults -LRB- lanes Alg -RRB- . The time-course of the decrease was examined in a set of females that had been raised from eggs in the laboratory under long day conditions -LRB- Figure 7 -RRB- . In this case 32 % of extractable pupal M-MtH and 47 % of V-MtH remained at the time of eclosion . During one week of feeding as adults under continuing long day conditions three quarters of the reserves present at eclosion were consumed . 3 . Sexual Differences . While males contained roughly as much soluble ArH as females in Cecropia pupae , their contents of V-MtH and M-MtH were significantly lower -LRB- Figure 5B -RRB- . A sexual difference was also seen during adult development : from days 13 to 21 , when vitellogenesis and chorion deposition occur , utilization of V-MtH and M-MtH was more rapid in females than in males -LRB- Figure 5A and B -RRB- . In males , as described above for females , immunodiffusion failed to detect more than 1 -- 2 % of the pupal levels of any of the three hexamerins at the time of eclosion -LRB- Figure 5B , Adult -RRB- . In confirmation of this , SDS-PAGE revealed only traces of 80 kDa material in centrifugal pellets -LRB- Figure 4A , stages A1 and A2 , lanes P -RRB- . Monarchs differed from the lepidopteran norm in showing no sexual differences in the amounts of soluble V - and M-MtH stored in pupae -LRB- Figure 6B , green bars -RRB- . In four week old adults raised under short day conditions , however , females retained larger reserves than males -LRB- Figure 6A and B , red bars -RRB- , but had consumed more of these two proteins under conditions permitting egg formation -LRB- Figure 6A and B. blue bars -RRB- . Discussion 1 . Egg formation versus metamorphosis The results are clear in showing that methionine-rich hexamerins in the absence of ArH support egg formation in Monarchs . Short-day adults , whose ovaries do not produce eggs , retained 20 -- 30 % of their pupal reserves of the MtH 's , but only 0.3 % of ArH -LRB- Figure 6A -RRB- . Reduction of V-and M-MtH to only 1 -- 2 % of pupal levels in long-day females that had begun to lay eggs confirms that adult MtH 's are in fact consumed during egg formation . The Monarch results also show that V - and M-MtH support somatic tissue development , as 50 % of pupal V-MtH and 70 % of M-MtH disappeared during adult development -LRB- Figure 7 -RRB- . The depletion of pupal V - and M-MtH in males -LRB- Figure 6B -RRB- also indicates functions other than the support of egg formation . ArH , too , is multi-functional . It is known to support larval molting in the absence of the MtH 's in Manduca sexta -LRB- Kramer et al. , 1980 ; Riddiford and Hice , 1985 ; Webb and Riddiford , 1988 -RRB- and Cecropia -LRB- Telfer et al. , 1983 -RRB- . This situation differs in the dictyopteran , Blatta orientalis , whose two hexamerins are both present in larval hemolymph and cycle together during molting -LRB- Duhamel and Kunkel , 1978 -RRB- . ArH can also support reproductive functions , for it was as effective as M-MtH in providing precursors for the synthesis of Vg and chorions in pharate adult Luna moths -LRB- Pan and Telfer , 1996 -RRB- . Selectivity in the consumption of the multiple hexamerins within a lepidopteran species is shown by these results to be related to availability : the MtH 's are not used in the larval molts because they are not synthesized until late in the last instar -LRB- Tojo et al. , 1978 ; Riddiford and Hice , 1985 ; Jones et al , 1993 ; Memmel et al , 1994 -RRB- ; and pupal reserves of ArH do not support egg formation in adult Monarchs because they are fully consumed during metamorphosis . When both are at hand , however , both contribute to whatever metamorphic and reproductive functions are under way . Two kinds of processes are apparent that affect availability . Most obvious is the early synthesis of ArH that makes it uniquely available for the support of larval molting . The second involves differences in mode of storage . In insects generally , storage hexamers are secreted into larval hemolymph , where they can attain extraordinary concentrations . When metamorphosis approaches they are reclaimed to varying degrees by the fat body , and packaged in cytoplasmic storage granules . In an earlier study over 99 % of injected M-MtH was cleared from the hemolymph of pharate pupae , compared with only 35 % of ArH -LRB- Pan and Telfer , 1992 -RRB- . A consequence of this in both Luna and Cecropia pupae is that the MtH 's are the most prominent proteins of fat body extracts while ArH is the most prominent protein of hemolymph -LRB- Figure 2 in Pan and Telfer , 1996 , and Figure 17 in Tojo et al , 1978 -RRB- . There can also be differences in the disposition of proteins within the fat body , for in Cecropia the hexamerin storage granules contain protein crystals embedded in an amorphous material -LRB- Tojo et al. , 1978 -RRB- . Differences in accessibility provide a plausible speculation on how V - and M-MtH , but not ArH , might survive metamorphosis in sufficient amounts to support post-eclosion egg formation . 2 . How hexamerins support egg formation Hexamerins presumably support egg formation primarily by providing precursors for protein synthesis , for the synthesis of yolk proteins by fat body and of chorion proteins by the follicle cells both entail transcription and the incorporation of free amino acids -LRB- Wyatt , 1991 ; Paul and Kafatos , 1975 ; Nadel and Kafatos , 1980 -RRB- . Cases of intact hexamerin incorporation by ovaries have been described , but these are idiosyncratic rather than general . Antigenic epitopes of all four lepidopteran hexamerins are present among yolk proteins of saturniid moths , but they are only 0.2 % as concentrated as those of Vg -LRB- Telfer and Pan , 1988 -RRB- . In Hyphantria cunea antigenic activity of an M-MtH-like protein was detected in intercellular spaces and yolk during vitellogenesis , but was absent from mature eggs -LRB- Seo et al. , 1998 -RRB- . The authors speculated that this protein provides an internal source of amino acids for protein synthesis in the maturing egg . A more striking exception occurs in Riptortus clavatus , a hemipteran whose yolk contains approximately equal amounts of Vg and a 500 kDa , biliverdin-binding hexamerin -LRB- Chinzei et al. , 1990 ; Miura et al. , 1998 -RRB- . The yolk form of this otherwise conventional storage protein is secreted by the fat body of adult females in synchrony with Vg and selectively endocytosed by the vitellogenic oocyte . In another unusual case , the principal hemolymph storage proteins of last instar larvae and pupae of Bombyx mori are not hexamerins but a family of 30 kDa proteins that persist into the adult stage and are accumulated during vitellogenesis as major constituents of yolk -LRB- Zhu et al. , 1986 ; Chen and Yamashita , 1990 -RRB- . But immunodiffusion and SDS-PAGE revealed nothing comparable to either the Riptortus or Bombyx storage proteins in egg-filled abdomens of long-day Monarchs . 3 . Hexamerin synthesis in adults ? There are other cases of the resumption of hexamerin synthesis in eclosed adults -LRB- e.g. , Kunkel and Pan , 1976 ; Wyatt et al. , 1992 -RRB- , but these occur in males as well as females and are thus not necessarily related to egg formation . Post-eclosion synthesis is not ruled out as a source of V - and M-MtH in adult Monarchs , but if it occurs it is not sufficient to prevent the depletion that accompanies egg formation . It would not , in any case , affect the argument that the consumption of residual pupal V - and M-MtH , rather than ArH , is correlated with post-ecdysial egg formation . 4 . V-MtH versus M-MtH The two methionine-rich hexamerins are electrophoretically distinct and show no detectable similarities in precipitation by polyclonal rabbit antibodies . Yet they behaved here identically in the timing of their disappearance during adult development and in the fraction of their pupal reserves that survived metamorphosis . Cladistic analyses of amino acid sequences deduced from cDNA clones suggest that they are descended from products of a gene duplication occurring early in lepidopteran evolution -LRB- Burmester et al. , 1998 -RRB- . But we are as yet left with no clue as to why natural selection has favored a pair of diverging MtH 's over a single protein that might have averaged their amino acid compositions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


SYD1/444 : Assessing the Readership of the UK National Database of Telemedicine Introduction The National Database of Telemedicine -LRB- NDTM -RRB- is a Web site containing information resources on UK telemedicine projects and related work . Its purpose is to provide a source of information to anyone researching the field or proposing a trial or a larger scale implementation of Telemedicine . The NDTM Web site was launched on 27th October 1998 and publicised extensively . Before and after its launch , the Web site has been kept up to date with information about new projects and modifications to the details kept about existing ones . It is clearly important for such a resource to keep track of who is reading it . We may need to tailor information to a particular audience , or bring the Web site to the attention of people who may not yet have found it . This paper describes a mechanism for tracking usage of the Web site that provides more information than conventional approaches .

O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O