bluuebunny/crossref_metadata_2025 huggingface dataset:
Slow gating charge immobilization in the human potassium channel Kv1.5 and its prevention by 4‐aminopyridine.
Authors: Fedida D , Bouchard R , Chen F S
Published: December 2014

1. The relationship between ionic current inactivation and immobilization of ‘off'‐gating charge in human Kv1.5 channels expressed in human embryonic kidney (HEK293) cells was studied using 4‐aminopyridine (4‐AP) and tetraethylammonium chloride (TEA‐Cl). 2. The charge transferred during short (< 10 ms) depolarizations (Q(on)) was conserved on repolarization (Q(off)) although peak off‐gating current (off‐Ig) was reduced and the time course prolonged (tau decay increased from 0.4 to > 1.2 ms). For +80 mV pulses longer than 50 ms, Q(off) at 20 ms was less than Q(on) (Q(off)/Q(on) ratio was 0.26 +/‐ 0.06 at 450 ms). We attribute this to a relative ‘immobilization’ of gating charge during long depolarizations. 3. 4‐AP (0.1‐1 mM) prevented slowing of off‐Ig, allowing saturation of peak off‐Ig. 4‐AP also completely prevented immobilization of off‐Ig after long depolarizations. In 1 mM 4‐AP, off‐Ig waveforms decayed rapidly and the charge ratio Q(off)/Q(on) remained at 1.0. 4. In addition to its effects on Ig, 1 mM 4‐AP prevented the slow inactivation of ionic current seen during strong depolarizations. An initial block was caused by 4‐AP or 1 mM intracellular TEA internally applied. However, only 4‐AP prevented the slower, later development of C‐type inactivation. 5. We suggest that slow current inactivation is accompanied by a gating charge immobilization in Kv1.5. 4‐AP potently inhibits the changes in Q(off)/Q(on0, off‐Ig, and ionic currents that underlie slow inactivation. Some actions of 4‐AP appear independent of its properties as a blocker of open K+ channels, and are not mimicked by internal TEA.
Activity dependence and functional role of the apamin‐sensitive K+ current in rat supraoptic neurones in vitro.
Authors: Kirkpatrick K , Bourque C W
Published: December 2014

1. Intracellular recordings were obtained from seventy‐two magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus. The current underlying the after‐hyperpolarization (IAHP) following spike‐evoked trains of action potentials was characterized using the hybrid‐clamp technique. The activity‐dependent requirements for the genesis of the AHP were determined. The functional role of the conductance was investigated using saturating concentrations (50‐300 nM) of apamin, a selective blocker of the AHP in MNCs. 2. IAHP was reversibly abolished by the removal of extracellular Ca2+. The amplitude of IAHP varied linearly as a function of voltage and reversed at ‐100 +/‐ 3 mV in 3 mM external K+. Changes in the concentration of extracellular K+ resulted in shifts of the reversal potential consistent with Nernst equation predictions for a K+‐selective conductance. 3. Action potentials triggered by brief depolarizing pulses elicited an AHP during trains evoked at frequencies > 1 Hz. Onset of the AHP progressed exponentially, reaching a maximum after the first fifteen to twenty impulses. The steady‐state amplitude of the AHP increased logarithmically between 1 and 20 Hz. 4. Switching to voltage clamp during periods of continuous cell activity (firing rate > 4 Hz) confirmed the presence of an apamin‐sensitive Ca2(+)‐dependent K+ current. 5. Application of apamin produced a threefold increase in the mean firing rate of spontaneously active cells, but was without effect when applied to silent cells (firing rate < 0.5 Hz). 6. Apamin did not affect the ability of MNCs to fire in a phasic manner but caused a dramatic increase in the mean intraburst firing rate. Moreover, inhibition of IAHP by apamin strongly attenuated spike accommodation normally seen at the onset of phasic bursts. 7. While apamin did not enhance the amplitude of depolarizing after‐potentials following single spikes, post‐train plateau potentials and associated after‐discharges were enhanced. 8. The possible consequences of IAHP modulation are discussed in the context of the regulation of firing rate and pattern in MNCs.
Coupling between fast and slow inactivation revealed by analysis of a point mutation (F1304Q) in mu 1 rat skeletal muscle sodium channels.
Authors: Nuss H B , Balser J R , Orias D W , Lawrence J H , Tomaselli G F , Marban E
Published: December 2014

1. We sought to elucidate the mechanism of the defective inactivation that characterizes sodium channels containing mutations in the cytoplasmic loop between the third and fourth domains (the III‐IV linker). Specifically, we measured whole‐cell and single‐channel currents through wild‐type and F1304Q mutant mu 1 rat skeletal muscle Na+ channels expressed in Xenopus laevis oocytes. 2. In wild‐type channels, inactivation is complete and the faster of two decay components predominates. In F1304Q, inactivation is incomplete; the slow decay component is larger in amplitude and slower than in wild‐type. The fraction of non‐inactivating current is substantial (37 +/‐ 2% of peak current at ‐20 mV) in F1304Q. 3. Cell‐attached patch recordings confirmed the profound kinetic differences and indicated that permeation was not altered by the F1304Q mutation. The F1304Q phenotype must be conferred entirely by changes in gating properties and is not remedied by coexpression with the beta 1‐subunit. 4. Recovery from inactivation of F1304Q channels is faster than for wild‐type channels and three exponentials are required to describe recovery adequately following long (5 s) depolarizations. Thus, there are three inactivated states even in ‘inactivation‐deficient’ F1304Q channels. 5. The steady‐state voltage dependence of F1304Q inactivation is right‐shifted by 26 +/‐ 2 mV. 6. A gating model incorporating three inactivated states, all directly accessible from multiple closed states or the open state, was constrained to fit wild‐type and F1304Q inactivation (h infinitive) data and repriming data simultaneously. While it was necessary to alter the rate constants entering and exiting all three inactivated states, the model accounted for the F1304Q‐induced rightward shift in steady‐state inactivation without imposing voltage dependence on the inactivation rate constants. 7. We conclude that the F1304Q mutation in mu 1 sodium channels modifies several inactivation processes simultaneously. The fact that a single amino acid substitution profoundly alters both fast and slow inactivation indicates that these processes share physical determinants in Na+ channels.
The effects of continuous drainage of fetal fluids on salt and water balance in fetal sheep.
Authors: Gibson K J , Lumbers E R
Published: December 2014

1. In nine chronically catheterized fetuses in which all lung liquid was drained continuously from the time of surgery, the effects of continuous drainage of fetal urine for 1 week on fetal renal function, lung liquid production and salt and water balance were studied. Fetal wellbeing, as judged by fetal growth, urinary osmolality, blood gas status, arterial pressure and heart rate, was not adversely affected by urine drainage. The ewes, however, drank more water when fetal urine was drained. Thus. fetal plasma and urinary osmolalities declined (P < 0.25 and P < 0.05). 2. Fetal glomerular filtration rate fell from 75 +/‐ 4 ml kg‐1 h‐1 (+/‐ S.E.M., n = 9) before drainage to 54 +/‐ 7 ml kg‐1 h‐1 after drainage (n = 7; P < 0.005), and fetal renal sodium excretion also declined (P < 0.05). However, the excretion of sodium in lung liquid did not decrease and the fetal renin‐angiotensin system was not activated. Fetal extracellular volume (561 +/‐ 44 ml kg‐1, n = 7) and the calculated net sodium transfer (0.76 mmol kg‐1 h‐1, n = 6) and fluid transfer (15 +/‐ 2 ml kg‐1 h‐1, n = 8) to the fetus did not change. 3. It is concluded that overall fetal salt and water balance were maintained when all fetal urine and lung liquid were drained from fetal sheep in late gestation. Since drainage of urine and lung liquid considerably reduced the amniotic and allantoic fluids, transfer across the placenta and extraplacental membranes was able to compensate for the absence of these fluids. In response to the loss of sodium during drainage, fetal renal sodium conservation was about 11% of the total sodium conservation by the materno‐fetal unit.
Inhibition of nitric oxide synthase does not alter ocular dominance shifts in kitten visual cortex.
Authors: Reid S N , Daw N W , Czepita D , Flavin H J , Sessa W C
Published: December 2014

1. Since nitric oxide has been proposed as a feedback factor in plasticity in the hippocampus, we tested whether it might also be a feedback factor in sensory‐dependent plasticity in the cat visual cortex. 2. The effects of monocular deprivation were compared between eight hemispheres with infusion of a nitric oxide synthase inhibitor, and eight control hemispheres with either infusion of the inactive isomer, or no infusion. Although nitric oxide synthase activity was reduced significantly, the ocular dominance histograms were not substantially different in the two groups of animals. We conclude that the feedback factor for sensory‐dependent plasticity in the visual cortex is likely to be some factor other than nitric oxide.
Effect of postural changes on human lymphatic capillary pressure of the skin.
Authors: Franzeck U K , Fischer M , Costanzo U , Herrig I , Bollinger A
Published: December 2014

1. The influence of postural changes on cutaneous lymphatic capillary pressure and venous pressure was measured at the dorsum of the foot in twelve healthy volunteers. Measurements were performed in the supine and sitting positions. 2. Lymphatic skin capillaries were visualized by fluorescence microlymphography with fluorescein isothiocyanate (FITC)‐Dextran 150000. Subsequently a lymphatic capillary was punctured with a glass micropipette and pressure was measured using the servo‐nulling technique. Lymphatic capillary pressure, venous pressure, heart and respiration rates were recorded simultaneously. 3. Mean lymphatic capillary pressure was significantly higher (P = 0.0096) in the sitting (9.9 +/‐ 3.0 mmHg) than in the supine (3.9 +/‐ 4.2 mmHg) position. There was no significant difference (P = 0.09) between lymphatic capillary pressure and venous pressure (6.8 +/‐ 3.4 mmHg) in the supine position. During sitting mean lymphatic capillary pressure was significantly lower (P = 0.0022) than mean venous pressure (53.3 +/‐ 4.1 mmHg). The smaller increase in lymphatic capillary pressure may be caused by the discontinuous fluid column in the lymphatic system and enhanced orthostatic contractile activity of lymphatic collectors and precollectors. Spontaneous low frequency pressure fluctuations occurred in 89% of recordings during sitting, which was significantly (P = 0.02) higher than in the supine position (54%). 4. The present results support the suggestion of enhanced intrinsic contractile activity of lymph precollectors and collectors in the dependent position. This mechanism is primarily responsible for the propulsion of lymph from the periphery to the thoracic duct during quiet sitting, when extrinsic pumping by the calf muscles is not active.
Calcium gradients during excitation‐contraction coupling in cat atrial myocytes.
Authors: Hüser J , Lipsius S L , Blatter L A
Published: December 2014

1. Confocal microscopy in combination with the calcium‐sensitive fluorescent probe fluo‐3 was used to study spatial aspects of intracellular Ca2+ signals during excitation‐contraction coupling in isolated atrial myocytes from cat heart. 2. Imaging of [Ca2+]i transients evoked by electrical stimulation revealed that Ca2+ release started at the periphery and subsequently spread towards the centre of the myocyte. 3. Blocking sarcoplasmic reticulum (SR) Ca2+ release with 50 microM ryanodine unmasked spatial inhomogeneities in the [Ca2+]i was higher in the periphery than in central regions of the myocyte. 4. Positive (or negative) staircase or postrest potentiation of the ‘whole‐cell’ [Ca2+] transients were paralleled by characteristic changes in the spatial profile of the [Ca2+]i signal. With low SR Ca2+ load [Ca2+]i transients in the subsarcolemmal space were small and no Ca2+ release in the centre of the cell was observed. Loading of the SR increased subsarcolemmal [Ca2+]i transient amplitude and subsequently triggered further release in more central regions of the cell. 5. Spontaneous Ca2+ release from functional SR units, i.e. Ca2+ sparks, occurred at higher frequency in the subsarcolemmal space than in more central regions of the myocyte. 6. Visualization of the surface membrane using the membrane‐selective dye Di‐8‐ANEPPS demonstrated that transverse tubules (t‐tubules) were absent in atrial cells. 7. It is concluded that in atrial myocytes voltage‐dependent Ca2+ entry triggers Ca2+ release from peripheral coupling SR that subsequently induces further Ca2+ release from stores in more central regions of the myocyte. Spreading of Ca2+ release from the cell periphery to the centre accounts for [Ca2+]i gradients underlying the whole‐cell [Ca2+]i transient. The finding that cat atrial myocytes lack t‐tubules demonstrates the functional importance of Ca2+ release from extended junctional (corbular) SR in these cells.
Non‐ionic diffusion and carrier‐mediated transport drive extracellullar pH regulation of mouse colonic crypts.
Authors: Chu S , Montrose M H
Published: December 2014

1. Extracellular pH (pHo) regulation within mouse colonic crypt lumens is stimulated by transepithelial gradients of short‐chain fatty acids (SCFAs). Current work assesses underlying mechanisms contributing to pHo regulation. 2. Crypt luminal alkalinization was saturable by apical SCFA (substrate concentration activating half‐maximal transport (KT) of isobutyrate = 45 mM). However, saturation was consistent with either carrier‐mediated SCFA flux or non‐ionic diffusion, because the non‐ionized form was titrated by luminal alkalinization. Direct acidification of apical perfusates increased the magnitude of SCFA‐induced luminal alkalinization, roughly in the same proportion to the increased concentration of non‐ionized SCFA in the crypt lumen. 3. Transepithelial gradients of an alternative weak acid (CO2) produce pHo changes similar to SCFA. In contrast, a weak base (NH3) changes pHo with reverse dependence on the orientation of the transepithelial gradient compared with SCFA. Results implicate non‐ionic diffusion in pHo regulation, and suggest that pHo changes may underly SCFA‐stimulated bicarbonate secretion and ammonium absorption. 4. SCFA metabolism plays a minor role in extracellular pH regulation. An avidly metabolized SCFA (N‐butyrate) augments crypt luminal alkalinization only slightly (0.08 pH units) versus a poorly metabolized SCFA (isobutyrate). 5. Apical addition of 1 mM 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid (DIDS) partially inhibits luminal alkalinization caused by apical SCFA. DIDS has no effect on luminal alkalinization caused by transepithelial CO2 gradients. Probenecid (1 mM), alpha‐cyano‐4‐hydroxycinnamic acid (4 mM) or basolateral DIDS (1 mM) do not affect pHo regulation. Results suggest that DIDS‐sensitive, SCFA‐dependent transport in the colonocyte apical membrane contributes to pHo regulation.
Kinematic determinants of human locomotion.
Authors: Borghese N A , Bianchi L , Lacquaniti F
Published: December 2014

1. The aim of this study was to find kinematic patterns that are invariant across the normal range of locomotion speeds. Subjects walked at different, freely chosen speeds ranging from 0.9 to 2.1 m s‐1, while motion and ground reaction forces on the right side of the body were recorded in three‐dimensional space. 2. The time course of the anatomical angles of flexion‐extension at the hip and ankle was variable not only across subjects, but even from trial to trial in the same subject. By contrast, the time course of the changes in the angles of elevation of each limb segment (pelvis, thigh, shank and foot) relative to the vertical was stereotyped across subjects. 3. To compare the waveforms across speeds, data were scaled in time relative to gait cycle duration. The pattern of ground reaction forces was highly speed dependent. Several distinct families of curves could be recognized in the flexion‐extension angles at the hip and ankle. Instead, the waveforms of global length and elevation of the limb, elevation angles of all limb segments and flexion‐extension at the knee were invariant with speed. 4. When gait trajectories at all speeds are plotted in the position space defined by the elevation angles of the limb segments, they describe regular loops on a plane. The statistical characteristics of these angular covariations were quantified by means of principal component analysis. The first two principal components accounted together for > 99% of the total experimental variance, and were quantitatively comparable in all subjects. 5. This constraint of planar covariation of the elevation angles is closely reminiscent of that previously described for the control of posture. The existence of laws of intersegmental co‐ordination, common to the control of posture and locomotion, presumably assures the maintenance of dynamic equilibrium during forward progression, and the anticipatory adaptation to potentially destabilizing factors by means of co‐ordinated kinematic synergies of the whole body.
Effects of repetitive tetanic stimulation at long intervals on excitation‐contraction coupling in frog skeletal muscle.
Authors: Bruton J D , Lännergren J , Westerblad H
Published: December 2014

1. Single skeletal muscle fibres of Xenopus frogs were used to investigate the possibility that excitation‐contraction (E‐C) coupling can be impaired under conditions of elevated intracellular free Ca2+ ([Ca2+]i). 2. Fibres were stimulated with a train of up to 200 tetani at 10 or 20s intervals; this long‐interval stimulation (LIS) scheme was chosen to minimize fatigue. After LIS, fibres were exposed to hypotonic Ringer solution for 5 min. At the end of LIS, force was about 90% of the original and the hypotonic challenge did not result in any force depression. 3. Caffeine, terbutaline and 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone increased both basal and tetanic [Ca2+]i. In ten out of thirteen fibres, the presence of any of these drugs during LIS resulted in a force reduction to about 10% of the control when fibres were returned to normal Ringer solution after the hypotonic challenge. Force production was severely depressed for at least 20 min and then recovered to control levels within 120 min. 4. Neither protease inhibitors nor a scavenger of reactive oxygen species prevented the impairment of E‐C coupling. 5. It is concluded that after a period of elevated [Ca2+]i, E‐C coupling in frog skeletal muscle becomes sensitive to the mechanical stress induced by exposure to hypotonic solution. The underlying molecular basis for this remains unclear.
Modulation of K+ conductances by Ca2+ and human chorionic gonadotrophin in Leydig cells from mature rat testis.
Authors: Desaphy J F , Rogier C , Joffre M
Published: December 2014

1. Although the control of steroidogenic activity of the Leydig cell by the peptides luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) is clearly mediated by cAMP, the extent to which Ca2+ controls the Leydig cell function is less well defined. In the present study, the whole‐cell configuration of the patch‐clamp technique was used to investigate the modulation of potassium conductances by calcium and hCG, in the Leydig cells from mature rat testis. 2. In symmetrical glutamate solutions, depolarizations elicited outwardly rectifying currents, which were mainly carried by potassium and were blocked by tetraethylammonium and 4‐aminopyridine. For values of [Ca2+]i below 10(‐8) M, transient currents of low amplitudes, insensitive to charybdotoxin (CTX) and iberiotoxin (IBTX), were activated above ‐40 mV. For [Ca2+]i values of 10(‐7) M and above, noisy currents with slow activation kinetics were activated above 0 mV. These currents were sustained and were sensitive to CTX and IBTX. 3. Both current types were modulated by intracellular calcium. Ionomycin and a [Ca2+]i elevation in the range from 10(‐9) to 10(‐7) M, both inhibited the CTX‐insensitive currents, whereas a rise in the calcium concentration above 10(‐7) M increased the amplitude and shifted the threshold of activation of the CTX‐sensitive currents to less positive levels. 4. hCG (1‐50 i.u. ml‐1), in conditions where the chloride currents were strongly inhibited by 4‐acetamido‐4'‐isothiocyanatostilbene‐2,2'‐disulphonic acid (SITS), induced a partial inhibition of the CTX‐insensitive currents but was unable to increase the CTX‐sensitive currents. 5. No voltage‐sensitive calcium current was recorded in control or hCG‐stimulated cells. 6. The results indicate that hCG inhibits one kind of Ca(2+)‐modulated channel, perhaps as a result of a moderate [Ca2+]i rise, but is unable to increase the intracellular Ca2+ concentration to the range in which large conductance Ca(2+)‐dependent channels are activated.
Central effects of 5‐HT on activity of respiratory and hypoglossally innervated muscles in newborn kittens.
Authors: Khater-Boidin J , Rose D , Duron B
Published: December 2014

1. In decerebrate kittens (n = 29), electrical activity was studied in the 3rd intercartilaginous (inspiratory), the 9th internal intercostal (expiratory) and the hypoglossally innervated muscles (geniohyoid m. and sternohyoid m.) evoked by the application of 5‐HT (n = 16) or related agents (5‐HT1A agonist, 8‐OH‐DPAT (n = 6) and 5‐HT2 agonist, DOI floor of the IVth ventricle. 2. The application of a control solution (n = 2) produced no significant changes either in minute inspiratory frequency (Fi) or in the electrical activity of the muscles studied. Except for these controls, only one trial with one dose of one drug was performed in a given kitten. 3. A dose‐related decrease in Fi was observed in response to 5‐HT. Low doses (50‐500 nmol, n1 = 8) induced a long‐lasting bradypnoea; high doses (5000‐10,000 nmol, n2 = 8) induced prolonged periods of apnoea. 4. The apnoeas observed in tracheotomized (n = 3) or non‐tracheotomized (n2 = 8) kittens were mainly of central origin and linked to the lengthening of expiratory time. The expiratory muscle activation came on with the reinforcement of the activity of hypoglossally innervated muscles. 5. Application of agonists showed that both the 5‐HT‐dependent modulation of Fi and the effects of 5‐HT on the activity of the muscles studied resulted predominantly from activation of 5‐HT2 receptors.
Neuropeptide Y‐like substances are released from the rostral brainstem of cats during the muscle pressor response.
Authors: Williams C A
Published: December 2014

1. In this study, the release of immunoreactive neuropeptide Y (NPY)‐like substances (irNPYs) was measured from the rostral brainstem in response to fatiguing isometric contractions of the hindlimb muscle of cats anaesthetized with alpha‐chloralose. 2. The irNPYs were measured using a solid phase autoradiographic immunoassay. NPY antibody‐coated glass microelectrodes were inserted stereotaxically into the rostral brainstem during rest and contractions. Control procedures such as passive leg flexing, stimulation of the central end of the cut tibial nerve, brief repeated carotid occlusions and carotid sinus nerve stimulations were performed to determine whether patterns of release of irNPYs were specific to the pathways activated during the muscle pressor response. 3. During isometric contractions, the mean arterial blood pressure (MABP) and heart rate (HR) increased by 60‐70 mmHg and 15‐20 beats min‐1, respectively. Only the brief carotid occlusions caused a similar increase in MABP and HR. 4. During isometric contractions, less irNPYs were released from sites in the lateral reticular nucleus, lateral tegmental field and vestibular nuclei at a level in the brainstem 4.0‐4.4 mm rostral to the obex. In contrast, irNPYs were released from sites near the lateral tegmental field and nucleus ambiguus at a more caudal level tested (1.3 mm anterior to the obex) in response to contractions. Brief carotid occlusions caused a release of irNPYs from the lateral reticular nucleus on the contralateral side of the brainstem (at 3.5 mm anterior to the obex). None of the other procedures resulted in a release of irNPYs from these sites in the brainstem. 5. These data indicate there are differences in the patterns of release of irNPYs in response to activation of reflex pathways which increase arterial pressure. Given the sites in the rostral brainstem where irNPYs were released, irNPYs may be involved with the central regulation of arterial pressure during the muscle pressor response.
Effects of photoreceptor metabolism on interstitial and glial cell pH in bee retina: evidence of a role for NH4+.
Authors: Coles J A , Marcaggi P , Véga C , Cotillon N
Published: December 2014

1. Measurements were made with pH microelectrodes in superfused slices of the retina of the honey‐bee drone. In the dark, the mean +/‐ S.E.M. pH values in the three compartments of the tissue were: neurones (photoreceptors), 6.99 +/‐ 0.04; glial cells (outer pigment cells), 7.31 +/‐ 0.03; extracellular space, 6.60 +/‐ 0.03. 2. Stimulation of the photoreceptors with light caused transient pH changes: a decrease in the photoreceptors (pHn) and in the glial cells (pHg), and an increase in the interstitial clefts (pHo). 3. The effects of inhibition and activation of aerobic metabolism showed that part, perhaps all, of the light‐induced delta pHo resulted from the increased aerobic metabolism in the photoreceptors. 4. Addition of 2 mM NH4+ to the superfusate produced changes in pHo and pHg of the same sign as and similar amplitude to those caused by light stimulation. Manipulation of transmembrane pH gradients had similar effects on changes in pHo induced by light or by exogenous NH4+. 5. Measurements with NH(4+)‐sensitive microelectrodes showed that stimulation of aerobic metabolism in the photoreceptors increased [NH4+]o and also that exogenous NH4+/NH3 was taken up by cells, presumably the glial cells. 6. We conclude that within seconds of an increase in the aerobic metabolism in the photoreceptors, they release an increased amount of NH4+/NH3 which affects pHo and enters glial cells. Other evidence suggests that in drone retina the glial cells supply the neurones with amino acids as substrates of energy metabolism; the present results suggest that fixed nitrogen is returned to the glial cells as NH4+/NH3.
Distinct modes of channel gating underlie inactivation of somatic K+ current in rat hippocampal pyramidal cells in vitro.
Authors: Bossu J L , Gähwiler B H
Published: December 2014

1. We have used the cell‐attached configuration of the patch‐clamp recording method to characterize the biophysical properties of the voltage‐gated K+ channel underlying a 4‐aminopyridine (4‐AP)‐ and tetraethylammonium (TEA)‐sensitive K+ current (IK(AT)) in pyramidal cells of hippocampal slice cultures. 2. The unitary conductance of channels carrying IK(AT) current (KAT channels) was 19.1 +/‐ 5.1 pS with a physiological K+ gradient (2.7 mM external K+) and 39.0 +/‐ 3.6 pS with high external K+ (140 mM). The reversal potential changed with the external K+ concentration as expected for a channel with a dominant K+ selectivity. Channel activity was blocked under both conditions by either external application of 4‐AP at 100 microM or by including 20 mM TEA in the pipette solution. 3. An analysis of kinetic behaviour showed that open times were distributed as a single exponential. The mean open time (+/‐ S.D.) was 4.4 +/‐ 1.4 ms at a voltage 30 mV positive to resting potential and increased with further depolarization to reach a value of 16.2 +/‐ 7.4 ms at 70 mV positive to the resting potential. At this depolarized potential, we observed bursts of channel openings with a mean burst duration around 100 ms. 4. With repeated depolarizing pulses, response failures of the KAT channel occurred in a non‐random manner and were grouped (referred to as mode 0). This mode was associated with a voltage‐dependent inactivation process of the channel and was favoured when the opening probability of the channel was reduced by increasing steady‐state inactivation or by bath application of 4‐AP. This is consistent with the localization of the binding site for 4‐AP at or near the inactivation gate of the channel. 5. When KAT channel openings were elicited by 500 ms depolarizing steps, activity was either transient or it persisted throughout the duration of the pulse. These two modes of activity alternated in a random manner or occurred in groups giving rise to transient (time constant, 20‐100 ms) or sustained ensemble currents. In the presence of low concentrations of 4‐AP (20‐40 microM), the transient pattern of activity was more frequently observed. 6. In addition to mode 0, we propose the existence of at least two further gating modes for KAT channels: mode T (transient current) and mode S (sustained current) that underlie the three decaying components of the IK(AT) ensemble current. These gating modes are probably under the control of intracellular factors that remain to be identified.
Developmental adaptation of withdrawal reflexes to early alteration of peripheral innervation in the rat.
Authors: Holmberg H , Schouenborg J
Published: December 2014

1. In adult decerebrate spinal rats whose plantar nerves (PLN) had been transected at either postnatal day 1 (P1) or P21 the nociceptive withdrawal reflexes (NWR) of musculi extensor digitorum longus (EDL), peroneus longus (PER) and semitendinosus (ST) were characterized with respect to receptive field (RF) organization, magnitude and time course, using electromyography. Thermal (short CO2 laser pulses) and mechanical (calibrated pinch) stimulation were used. The innervation patterns in normal and lesioned adult rats were assessed by acute nerve lesions. 2. The spatial organization of the mean mechano‐ and thermonociceptive RFs of all the muscles studied was similar to normal in both P1‐ and P21‐lesioned rats, although in some P21‐lesioned rats atypical EDL RFs were encountered. 3. In P1‐lesioned rats thermo‐NWR of PER and EDL had normal magnitudes, while mechano‐NWR were reduced. In P21‐lesioned rats both thermo‐ and mechano‐NWR of these muscles had reduced magnitudes. Except for thermo‐NWR of ST in P1‐lesioned rats, which were increased, NWR of ST had normal magnitudes in both P1‐ and P21‐lesioned rats. The time course of thermonociceptive NWR of the muscles studied were near normal in both P1‐ and P21‐lesioned rats. 4. Acute nerve lesions in adult P1‐lesioned rats revealed an essentially abolished contribution to NWR from the PLN. Instead, the contribution to NWR from other hindpaw nerves, such as the superficial and deep peroneal nerves, was dramatically increased. By contrast, in P21‐lesioned rats, the regenerated PLN contributed significantly to the NWR. 5. It is concluded that despite profound alterations of plantar hindpaw innervation induced by early PLN transection the cutaneous nociceptive input to NWR attained an essentially normal spatial organization. An experience‐dependent mechanism is suggested to be instrumental in adapting the reflex connectivity to the peripheral innervation.
The chloride current induced by expression of the protein pICln in Xenopus oocytes differs from the endogenous volume‐sensitive chloride current.
Authors: Voets T , Buyse G , Tytgat J , Droogmans G , Eggermont J , Nilius B
Published: December 2014

1. Phenotypical similarities between ICl,swell, the cell‐swelling‐induced chloride current and ICln, the nucleotide‐sensitive chloride current induced by expression of mammalian pICln in Xenopus oocytes, have led to models which identify pICln either as the volume‐sensitive chloride channel or as a cytosolic regulator thereof. 2. To investigate critically the relationship between ICl,swell and pICln two‐microelectrode voltage clamp experiments were performed on Xenopus oocytes in which either human pICln was expressed or endogenous ICl,swell was activated. 3. Several criteria that clearly differentiated ICln from ICl,swell were detected. Outward rectification and the discrimination between NO3‐ and Cl‐ were more pronounced for ICln. Cyclamate blocked ICln but not ICl,swell. In contrast to ICl,swell, inactivation kinetics of ICln were pH independent and extracellular cAMP blocked only the outward ICln component. Finally, ICln was readily expressed in collagenase‐defolliculated oocytes and was not modulated by extracellular hypotonicity, whereas ICl,swell could only be triggered in follicle‐enclosed or manually defolliculated oocytes. 4. We therefore conclude that ICln and ICl,swell are two different chloride currents. Consequently, any model which invokes a crucial role for pICln in ICl,swell should be critically reviewed.
The ionic dependence of the histamine‐induced depolarization of vasopressin neurones in the rat supraoptic nucleus.
Authors: Smith B N , Armstrong W E
Published: December 2014

1. The ionic basis of the histamine‐induced depolarization of immunohistochemically identified neurones in the supraoptic nucleus (SON) was investigated in the hypothalamo‐neurohypophysial explant of male rats. Histamine (0.1‐100 microM) caused an H1 receptor‐mediated, dose‐dependent depolarization of fifty of sixty‐two vasopressin neurones in the SON. In contrast, twenty‐three oxytocin neurones were either depolarized (n = 6), hyperpolarized (n = 4), or unaffected (n = 13) by histamine. Due to the low percentage of responding cells, oxytocin neurones were not further investigated. 2. Chelation of intracellular Ca2+ with 1,2‐bis(2‐aminophenoxy)ethane N,N,N',N'‐tetraacetic acid (BAPTA; 100‐500 mM) blocked the depolarization, whereas blocking Ca2+ influx and synaptic transmission with equimolar Co2+ or elevated (5‐20 mM) Mg2+ in nominally Ca(2+)‐free solutions was without effect. 3. The amplitude of the histamine‐induced depolarization was relatively independent of membrane potential. The input resistance was unaltered by histamine in nine neurones, but in nine other neurones it was decreased and in two neurones it was increased by more than 5%. Neither elevating extracellular K+ nor addition of the K+ channel blockers, apamin, d‐tubocurarine, tetraethylammonium (TEA), or intracellular Cs+ decreased the histamine effect. Indeed, broadly blocking K+ currents with TEA and Cs+ significantly increased the depolarization to histamine. 4. Tetrodotoxin (2‐3 microM) did not inhibit the histamine‐induced depolarization. However, equimolar replacement of approximately 50% of extracellular Na+ with Tris+ or N‐methyl‐D‐glucamine reduced or eliminated the response. 5. The depolarization of vasopressin neurones by histamine thus requires extracellular Na+ and intracellular Ca2+. Activation of a Ca(2+)‐activated non‐specific cation current or a Ca(2+)‐Na+ pump are possible mechanisms for this effect.
Inwardly rectifying currents in hair cells and supporting cells in the goldfish sacculus.
Authors: Sugihara I , Furukawa T
Published: December 2014

1. Inwardly rectifying ionic currents were studied using patch‐clamp recording methods in oscillatory‐type and spike‐type hair cells and supporting cells dissociated from the goldfish sacculus. These cells had different types of inwardly rectifying currents. The biophysical properties of these currents were investigated. 2. A unique potassium current (Isc) was the sole ionic current recognized in supporting cells. Isc was active throughout the membrane potential range between +30 and ‐170 mV, but showed weak inward rectification and no inactivation. 3. In spike‐type hair cells, inwardly rectifying current (Ik1) was selectively permeable to K+ (K+:Na+ permeability ratio, 1:0.0021). Ik1 could underlie the high negative resting potential of these hair cells because it is partially active at this potential. The strong inward rectification of Ik1 contributed to the low negative plateau potential seen in spike‐type hair cells. 4. In oscillatory‐type hair cells, hyperpolarization‐activated potassium‐sodium current (Ih), which had properties similar to that in photoreceptor and other neurons, was present instead of inwardly rectifying K+ current. 5. In the cell‐attached and inside‐out modes with 125 microM external K+ ([K+]o), IK1 channel had a unitary conductance of 27 pS and showed inactivation with increasing hyperpolarization. Putative Ih and Iso single channels had unitary conductances of 7 and 61 pS, respectively, in the cell‐attached mode with 125 microM Ko+.
Protein kinase C inhibition of cloned inward rectifier (HRK1/KIR2.3) K+ channels expressed in Xenopus oocytes.
Authors: Henry P , Pearson W L , Nichols C G
Published: December 2014

1. The effect of protein kinase activators on cloned inward rectifier channels expressed in Xenopus oocytes was examined using a two‐electrode voltage clamp. PKA activators caused no change in KIR1.1, KIR2.1, or KIR2.3 current. The PKC activators phorbol 12‐myristate 14‐acetate (PMA) and phorbol 12, 13‐dibutyrate (PDBu) inhibited KIR2.3 currents, but not KIR2.1 or KIR1.1 current. This inhibition was blocked by staurosporine. An inactive phorbol ester, 4 alpha‐phorbol 12, 13‐didecanoate (4 alpha‐PDD), had no effect on KIR2.3. 2. Upon changing solution from 2 to 98 microM K+, KIR2.3 but not KIR1.1 or KIR2.1 currents typically ‘ran down’ over 5 min to 60‐80% of maximum amplitude. Rundown occurred even if PMA was applied before changing to high [K+] solution, indicating that rundown was independent of PKC activity. Rundown was evoked by substituting NMG+ for Na+, showing that it results from low [Na+] and not from high [K+]. 3. These results suggest that KIR2.3, but not KIR1.1 or KIR2.1, is subject to regulation, both by PKC activation and as a consequence of low [Na+]o. The difference in secondary regulation may account for specific responses to PKC stimulation of tissues expressing otherwise nearly identical KIR channels.
Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues.
Authors: Burnashev N , Villarroel A , Sakmann B
Published: December 2014

1. Recombinant alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate receptor (AMPAR) subunits (GluR‐A or GluR‐B) and kainate receptor (KAR) subunit (GluR‐6) in their unedited (Q)‐ and edited (R)‐forms were expressed in HEK 293 cells. To estimate the dimensions of the narrow portion of these channels, biionic reversal potentials for organic cations of different mean diameters were determined with Cs+ as the internal reference ion. 2. Homomeric channels assembled from Q‐form subunits were cation selective. The relation between the relative permeability and the mean size of different organic cations suggests that the diameter of the narrow portion of Q‐form channels is approximately 0.78 nm for AMPAR and 0.75 nm for KAR channels. 3. Homomeric channels assembled from R‐form subunits were permeant for anions and cations. When probed with CsC1 gradients the relative chloride permeability (PC1/PCs) was estimated as 0.14 for GluR‐B(R) and 0.74 for GluR‐6(R)‐subunit channels. The permeability versus mean size relation for large cations measured with the weakly permeant F‐ as anion, indicates that for the R‐form KAR channels the apparent pore diameter is close to 0.76 nm. 4. Heteromeric AMPAR and KAR channels co‐assembled from Q‐ and R‐form subunits were cation selective. The diameter of the narrow portion of these channels is estimated to be in the range between 0.70 and 0.74 nm. 5. The results indicated that the diameters of the narrow portion of AMPAR and KAR channels of different subunit composition and of widely different ion selectivity are comparable. Therefore, the differences in the anion versus cation selectivity, in Ca2+ permeability and in channel conductance are likely to be determined by the difference in charge density of the channel.
Force‐dependent and force‐independent heat production in single slow‐ and fast‐twitch muscle fibres from Xenopus laevis.
Authors: Buschman H P , van der Laarse W J , Stienen G J , Elzinga G
Published: December 2014

1. The origin of labile heat production, i.e. a heat component which rapidly decays after the onset of stimulation, and of stable (maintenance) heat production was investigated in intact single fast‐twitch (type 1) and slow‐twitch (type 3) iliofibularis muscle fibres from Xenopus laevis, at 20 degrees C, by varying stimulation frequency and by varying sarcomere length and the concentration of 2,3‐butanedione 2‐monoxime (BDM) added. 2. The labile heat produced consisted of a force‐independent and a force‐dependent part. The average parvalbumin (PA) content found in type 1 fibre bundles (0.84 +/‐ 0.08 mM; mean +/‐ S.E.M.; n = 5) and in type 3 fibre bundles (0.12 +/‐ 0.02 mM; n = 5) indicates that the force‐independent labile heat is explained by Ca(2+)‐Mg2+ exchange on PA, and amounts to a molar enthalpy change of ‐78 kJ (molPA)‐1. 3. Force‐dependent labile heat during fused contractions was similar to the calculated heat production resulting from the formation of force‐generating cross‐bridges, assuming an enthalpy change associated with cross‐bridge formation of ‐30 kJ mol‐1. 4. Activation heat, i.e. the part of the total stable heat that is not related to the contractile apparatus, and of which the calcium sequestration by the sarcoplasmic reticulum is the most important contributor, determined by varying sarcomere length or BDM concentration, was identical. For fused contractions the fraction activation heat of the stable maintenance rate of heat production was 34 +/‐ 4% (mean +/‐ S.E.M.; n = 13) in type 1 fibres, and 52 +/‐ 4% (n = 15) in type 3 fibres. In unfused contractions this was 48 +/‐ 5% (n = 13) in type 1 fibres, and 35 +/‐ 2% (n = 11) in type 3 fibres. 5. From the force‐dependent stable rate of heat production the economy of cross‐bridge cycling, expressed as the force‐time integral for a single myosin head per ATP molecule hydrolysed, was calculated. It followed that cross‐bridge interaction in type 3 fibres is more economical than in type 1 fibres, and that fused contractions are more economical than unfused contractions.
Differential expression of outer hair cell potassium currents in the isolated cochlea of the guinea‐pig.
Authors: Mammano F , Ashmore J F
Published: December 2014

1. Whole‐cell currents were recorded from outer hair cells (OHCs) in undissociated tissues from the organ of Corti. The experiments allowed ionic currents to be measured in cells with precise localization on the three most apical cochlear turns. 2. Two major potassium currents were expressed in the cells. One current, named IK, was half‐activated at ‐24 mV and was most prominent in the most apical turn, turn 4. A second, named IK.n, was half‐activated at ‐92 mV and was the major contributor to the current‐voltage (I‐V) curve of cells from the more basal turns, turns 3 and 2, of the cochlea. 3. IK was specifically blocked by 100 microM 4‐aminopyridine (4‐AP). In contrast, IK.n was reduced by 5 mM external barium. Superfusion with zero calcium produced no effect on currents in the range from ‐60 to 0 mV, but reduced currents by a maximum of 15% outside this range. 4. The cell input conductance increased systematically from 3.4 nS in turn 4 to 40 nS in turn 2 measured at a holding potential of ‐70 mV. 5. The mean leak conductance, measured from the slope of the I‐V curve at ‐110 mV, decreased systematically from 5.2 nS in turn 2, to 2.9 nS in turn 3 and 2.2 nS in turn 4. 6. These data show that hair cell properties can be determined in undissociated cells and are likely to provide a good estimate of the properties of the cells in the intact cochlea. Differences with the properties of isolated OHCs are discussed.
Movement illusions evoked by ensemble cutaneous input from the dorsum of the human hand.
Authors: Collins D F , Prochazka A
Published: December 2014

1. In this study we tested the hypothesis that ensemble activity in human cutaneous sensory afferents evoked by the stretching of skin over and around the finger joints contributes to the conscious perception of movement of the fingers. 2. In nineteen normal adults, ensembles of cutaneous afferents were activated either by electrical stimulation, delivered through an array of electrodes on the dorsum of the hand and fingers, or by mechanical stretching of the skin over and around the joints. The stretching was applied through an array of threads stuck to the skin, in such a way as to avoid or minimize moving the underlying joints and to avoid applying pressure to underlying tendons and ligaments. Perceived movements were mimicked by voluntary movements of the fingers of the contralateral hand. 3. By way of comparison, kinaesthetic illusions were also evoked by activation of muscle receptors by vibration. 4. Illusions of movement were elicited with each type of stimulus. Electrical stimulation of skin afferents caused clear illusory movements in six out of seventeen subjects (35%), and borderline movement illusions in three out of the same seventeen subjects (total 9/17, 53%). Various other localized skin sensations were also reported. Skin stretch evoked movement illusions in eleven out of nineteen of subjects (58%). In all subjects who received both cutaneous stimuli, twelve out of seventeen (71%) reported some movement sensations with one or other of the stimulation techniques. Vibration tended to be the most reliable stimulus modality, eliciting illusory movements in fourteen out of sixteen subjects (88%). 5. Although the skin stretching technique did cause minute movements of nearby joints in several cases, these were monitored and shown in separate control experiments to be below perceptual threshold, and so the movement illusions could be safely attributed to the cutaneous afferent input evoked by skin stretch. 6. The results support the hypothesis that input from skin stretched during finger movement contributes to the conscious perception of the movement. Vibration‐evoked muscle afferent input tended to be more reliable than the skin input in producing kinaesthetic illusions, though comparisons of the relative efficacy of the three techniques must be made with caution.
Asymmetric interactions between phosphorylation pathways regulating ciliary beat frequency in human nasal respiratory epithelium in vitro.
Authors: Smith R P , Shellard R , Dhillon D P , Winter J , Mehta A
Published: December 2014

1. The effects of the sequential stimulation of ciliary beat frequency (CBF) via two different phosphorylation cascades (dependent on protein kinase A (PKA) and calmodulin, respectively) were determined using video microscopy applied to a perfused preparation of human nasal respiratory epithelium in vitro. Dibutyryl cyclic AMP (db‐cAMP) (10(‐3) M) was used to stimulate PKA and the calcium ionophore 4‐Br‐A23187 (10(‐5) M) was used to stimulate calmodulin‐dependent phosphorylation. 2. Perfusion with db‐cAMP (10(‐3) M) alone showed an early rise in CBF (15.0 +/‐ 4%, mean +/‐ S.E.M., P < 0.05) by 10 min which remained elevated for 35 min; in contrast, the highest CBF response to 4‐Br‐A23187 (10(‐5) M) alone was not achieved until 35 min (16.1 +/‐ 1.8%, P < 0.05). 3. When a db‐cAMP stimulus was applied to cells which had been pre‐incubated with 4‐Br‐A23187 for 30 min, a further rise in CBF (maximal at 20 min, 14.3 +/‐ 2%, P < 0.05) was observed. Reversing the sequence of perfusions, cells pre‐incubated with db‐cAMP showed no further rise in response to stimulation with 4‐Br‐A23187. 4. We hypothesized that PKA inhibited the response to the 4‐Br‐A23187. This notion was supported by the restoration of the CBF response (22.8 +/‐ 4%, P < 0.05) to 4‐Br‐A23187 when the cells were pre‐incubated with the protein kinase inhibitor 1‐(5‐isoquinolinyl‐sulphonyl)‐2‐methylpiperazine (10(‐3) M), before the sequential perfusions with db‐cAMP and 4‐Br‐A23187. We conclude that the A23187‐dependent pathway, which regulates intrinsic CBF, is inhibited by db‐cAMP but not vice versa.