Properties and molecular basis of the mouse urinary bladder voltage-gated K+ current

Potassium channels play an important role in controlling the excitability of urinary bladder smooth muscle (UBSM). Here we describe the biophysical, pharmacological and molecular properties of the mouse UBSM voltage-gated K⁺ current ( I _{K ( V)}). The I _{K ( V)} activated, deactivated and inactivated slowly with time constants of 29.9 ms at +30 mV, 131 ms at −40 mV and 3.4 s at +20 mV. The midpoints of steady-state activation and inactivation curves were 1.1 mV and −61.4 mV, respectively. These properties suggest that I _{K ( V)} plays a role in regulating the resting membrane potential and contributes to the repolarization and after-hyperpolarization phases of action potentials. The I _{K ( V)} was blocked by tetraethylammonium ions with an IC₅₀ of 5.2 m m and was unaffected by 1 m m 4-aminopyridine. RT-PCR for voltage-gated K⁺ channel (K_V) subunits revealed the expression of Kv2.1, Kv5.1, Kv6.1, Kv6.2 and Kv6.3 in isolated UBSM myocytes. A comparison of the biophysical properties of UBSM I _{K ( V)} with those reported for Kv2.1 and Kv5.1 and/or Kv6 heteromultimeric channels demonstrated a marked similarity. We propose that heteromultimeric channel complexes composed of Kv2.1 and Kv5.1 and/or Kv6 subunits form the molecular basis of the mouse UBSM I _{K ( V)}.     

Descending vasa recta pericytes express voltage operated Na+ conductance in the rat

We studied the properties of a voltage-operated Na⁺ conductance in descending vasa recta (DVR) pericytes isolated from the renal outer medulla. Whole-cell patch-clamp recordings revealed a depolarization-induced, rapidly activating and rapidly inactivating inward current that was abolished by removal of Na⁺ but not Ca⁺ from the extracellular buffer. The Na⁺ current ( I _Na) is highly sensitive to tetrodotoxin  (TTX, K _d= 2.2 n m )  . At high concentrations, mibefradil (10 μ m ) and Ni⁺ (1 m m ) blocked I _Na. I _Na was insensitive to nifedipine (10 μ m ). The L-type Ca⁺ channel activator FPL-64176 induced a slowly activating/inactivating inward current that was abolished by nifedipine. Depolarization to membrane potentials between 0 and 30 mV induced inactivation with a time constant of ∼1 ms. Repolarization to membrane potentials between −90 and −120 mV induced recovery from inactivation with a time constant of ∼11 ms. Half-maximal activation and inactivation occurred at −23.9 and −66.1 mV, respectively, with slope factors of 4.8 and 9.5 mV, respectively. The Na⁺ channel activator, veratridine (100 μ m ), reduced peak inward I _Na and prevented inactivation. We conclude that a TTX-sensitive voltage-operated Na⁺ conductance, with properties similar to that in other smooth muscle cells, is expressed by DVR pericytes.     

Ether-à-go-go-related gene K+ channels contribute to threshold excitability of mouse auditory brainstem neurons

The ionic basis of excitability requires identification and characterisation of expressed channels and their specific roles in native neurons. We have exploited principal neurons of the medial nucleus of the trapezoid body (MNTB) as a model system for examining voltage-gated K⁺ channels, because of their known function and simple morphology. Here we show that channels of the ether-à-go-go -related gene family (ERG, Kv11; encoded by kcnh ) complement Kv1 channels in regulating neuronal excitability around threshold voltages. Using whole-cell patch clamp from brainstem slices, the selective ERG antagonist E-4031 reduced action potential (AP) threshold and increased firing on depolarisation. In P12 mice, under voltage-clamp with elevated [K⁺]_o (20 m m ), a slowly deactivating current was blocked by E-4031 or terfenadine ( V _0.5,act=−58.4 ± 0.9 mV, V _0.5,inact=−76.1 ± 3.6 mV). Deactivation followed a double exponential time course (τ_slow= 113.8 ± 6.9 ms, τ_fast= 33.2 ± 3.8 ms at −110 mV, τ_fast 46% peak amplitude). In P25 mice, deactivation was best fitted by a single exponential (τ_fast= 46.8 ± 5.8 ms at −110 mV). Quantitative RT-PCR showed that ERG1 and ERG3 were the predominant mRNAs and immunohistochemistry showed expression as somatic plasma membrane puncta on principal neurons. We conclude that ERG currents complement Kv1 currents in limiting AP firing at around threshold; ERG may have a particular role during periods of high activity when [K⁺]_o is elevated. These ERG currents suggest a potential link between auditory hyperexcitability and acoustic startle triggering of cardiac events in familial LQT2.     

Temperature-sensitive TREK currents contribute to setting the resting membrane potential in embryonic atrial myocytes

TREK channels belong to the superfamily of two-pore-domain K⁺ channels and are activated by membrane stretch, arachidonic acid, volatile anaesthetics and heat. TREK-1 is highly expressed in the atrium of the adult heart. In this study, we investigated the role of TREK-1 and TREK-2 channels in regulating the resting membrane potential (RMP) of isolated chicken embryonic cardiac myocytes. At room temperature, the average RMP of embryonic day (ED) 11 atrial myocytes was −22 ± 2 mV. Raising the temperature to 35°C hyperpolarized the membrane to −69 ± 2 mV and activated a large outwardly rectifying K⁺ current that was relatively insensitive to conventional K⁺ channel inhibitors (TEA, 4-AP and Ba²⁺) but completely inhibited by tetracaine (200 μ m ), an inhibitor of TREK channels. The heat-induced hyperpolarization was mimicked by 10 μ m arachidonic acid, an agonist of TREK channels. There was little or no inwardly rectifying K⁺ current ( I _K1) in the ED11 atrial cells. In marked contrast, ED11 ventricular myocytes exhibited a normal RMP (−86.1 ± 3.4 mV) and substantial I _K1, but no temperature- or tetracaine-sensitive K⁺ currents. Both RT-PCR and real-time PCR further demonstrated that TREK-1 and TREK-2 are highly and almost equally expressed in ED11 atrium but much less expressed in ED11 ventricle. In addition, immunofluorescence demonstrated TREK-1 protein in the membrane of atrial myocytes. These data indicate the presence and function of TREK-1 and TREK-2 in the embryonic atrium. Moreover, we demonstrate that TREK-like currents have an essential role in determining membrane potential in embryonic atrial myocytes, where I _K1 is absent.     

A voltage-dependent K+ current contributes to membrane potential of acutely isolated canine articular chondrocytes

The electrophysiological properties of acutely isolated canine articular chondrocytes have been characterized using patch-clamp methods. The 'steady-state' current–voltage relationship ( I–V ) of single chondrocytes over the range of potentials from −100 to +40 mV was highly non-linear, showing strong outward rectification positive to the zero-current potential. Currents activated at membrane potentials negative to −50 mV were time independent, and the I–V from −100 to −60 mV was linear, corresponding to an apparent input resistance of 9.3 ± 1.4 GΩ ( n = 23). The outwardly rectifying current was sensitive to the K⁺ channel blocking ion tetraethylammonium (TEA), which had a 50% blocking concentration of 0.66 m m (at +50 mV). The 'TEA-sensitive' component of the outwardly rectifying current had time- and membrane potential-dependent properties, activated near −45 mV and was half-activated at −25 mV. The reversal potential of the 'TEA-sensitive' current with external K⁺ concentration of 5 m m and internal concentration of 145 m m , was −84 mV, indicating that the current was primarily carried by K⁺ ions. The resting membrane potential of isolated chondrocytes (−38.1 ± 1.4 mV; n = 19) was depolarized by 14.8 ± 0.9 mV by 25 m m TEA, which completely blocked the K⁺ current of these cells. These data suggest that this voltage-sensitive K⁺ channel has an important role in regulating the membrane potential of canine articular chondrocytes.     

Molecular correlates of the M-current in cultured rat hippocampal neurons

M-type K⁺ currents ( I _K(M)) play a key role in regulating neuronal excitability. In sympathetic neurons, M-channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K⁺ channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of I _K(M) in hippocampal pyramidal neurons cultured from 5- to 7-day-old rats. RT-PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single-cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M-channel blocker, fully inhibited I _K(M) with an IC₅₀ of 3.6 ± 1.5 μM. In 70 % of these cells, TEA fully suppressed I _K(M) with an IC₅₀ of 0.7 ± 0.1 m m . In the remaining cells, TEA maximally reduced I _K(M) by only 59.7 ± 5.2 % with an IC₅₀ of 1.4 ± 0.3 m m ; residual I _K(M) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to I _K(M) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.     

Properties of single voltage-dependent K+ channels in dendrites of CA1 pyramidal neurones of rat hippocampus

Voltage-dependent K⁺ channels in the apical dendrites of CA1 pyramidal neurones play important roles in regulating dendritic excitability, synaptic integration, and synaptic plasticity. Using cell-attached, voltage-clamp recordings, we found a large variability in the waveforms of macroscopic K⁺ currents in the dendrites. With single-channel analysis, however, we were able to identify four types of voltage-dependent K⁺ channels and we categorized them as belonging to delayed-rectifier, M-, D-, or A-type K⁺ channels previously described from whole-cell recordings. Delayed-rectifier-type K⁺ channels had a single-channel conductance of 19 ± 0.5 pS, and made up the majority of the sustained K⁺ current uniformly distributed along the apical dendrites. The M-type K⁺ channels had a single-channel conductance of 11 ± 0.8 pS, did not inactivate with prolonged membrane depolarization, deactivated with slow kinetics (time constant 100 ± 6 ms at −40 mV), and were inhibited by bath-applied muscarinic agonist carbachol (10 μ m ). The D-type K⁺ channels had a single-channel conductance of around 18 pS, and inactivated with a time constant of 98 ± 4 ms at +54 mV. The A-type K⁺ channels had a single-channel conductance of 6 ± 0.6 pS, inactivated with a time constant of 23 ± 2 ms at +54 mV, and contributed to the majority of the transient K⁺ current previously described. These results suggest both functional and molecular complexity for K⁺ channels in dendrites of CA1 pyramidal neurones.     

Inhibition of HERG K+ Current and Prolongation of the Guinea-Pig Ventricular Action Potential by 4-Aminopyridine

4-Aminopyridine (4-AP) has been used extensively to study transient outward K⁺ current ( I _TO,1) in cardiac cells and tissues. We report here inhibition by 4-AP of HERG (the human ether-à-go-go -related gene) K⁺ channels expressed in a mammalian cell line, at concentrations relevant to those used to study I _TO,1. Under voltage clamp, whole cell HERG current ( I _HERG) tails following commands to +30 mV were blocked with an IC₅₀ of 4.4 ± 0.5 m m . Development of block was contingent upon HERG channel gating, with a preference for activated over inactivated channels. Treatment with 5 m m 4-AP inhibited peak I _HERG during an applied action potential clamp waveform by ∼59 %. It also significantly prolonged action potentials and inhibited resurgent I _K tails from guinea-pig isolated ventricular myocytes, which lack an I _TO,1. We conclude that by blocking the α-subunit of the I _Kr channel, millimolar concentrations of 4-AP can modulate ventricular repolarisation independently of any action on I _TO,1.     

Tyrosine kinase and phosphatase regulation of slow delayed-rectifier K+ current in guinea-pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the regulation of the cardiac slowly activating delayed-rectifier K⁺ current ( I _Ks) that is important for action potential repolarization. Constitutive I _Ks recorded from guinea-pig ventricular myocytes was suppressed by broad-spectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC₅₀, 4.1 ± 0.6 μ m ), tyrphostin A25 (IC₅₀, 12.1 ± 2.1 μ m ) and genistein (IC₅₀, 64 ± 4 μ m ), but was relatively insensitive to the inactive analogues tyrphostin A1, tyrphostin A63, daidzein and genistin. I _Ks was unaffected by AG1478 (10 μ m ), an inhibitor of epidermal growth factor receptor TK, and was strongly suppressed by the Src TK inhibitor PP2 (10 μ m ) but not by the inactive analogue PP3 (10 μ m ). The results of experiments with forskolin, H89 and bisindolylmaleimide I indicate that the suppression of I _Ks by TK inhibitors was not mediated via inhibition of ( I _Ks-stimulatory) protein kinases A and C. To evaluate whether the suppression was related to lowered tyrosine phosphorylation, myocytes were pretreated with TK inhibitors and then exposed to the phosphotyrosyl phosphatase inhibitor orthovanadate (1 m m ). Orthovanadate almost completely reversed the suppression of I _Ks induced by broad-spectrum TK inhibitors at concentrations around their IC₅₀ values. We conclude that basal I _Ks is strongly dependent on tyrosine phosphorylation of Ks channel (or channel-regulatory) protein.     

Mexiletine block of wild-type and inactivation-deficient human skeletal muscle hNav1.4 Na+ channels

Mexiletine is a class 1b antiarrhythmic drug used for ventricular arrhythmias but is also found to be effective for paramyotonia congenita, potassium-aggravated myotonia, long QT–3 syndrome, and neuropathic pain. This drug elicits tonic block of Na⁺ channels when cells are stimulated infrequently and produces additional use-dependent block during repetitive pulses. We examined the state-dependent block by mexiletine in human skeletal muscle hNav1.4 wild-type and inactivation-deficient mutant Na⁺ channels (hNav1.4-L443C/A444W) expressed in HEK293t cells with a β1 subunit. The 50% inhibitory concentrations (IC₅₀) for the inactivated-state block and the resting-state block of wild-type Na⁺ channels by mexiletine were measured as 67.8 ± 7.0 μ m and 431.2 ± 9.4 μ m , respectively ( n = 5). In contrast, the IC₅₀ for the block of open inactivation-deficient mutant channels at +30 mV by mexiletine was 3.3 ± 0.1 μ m ( n = 5), which was within the therapeutic plasma concentration range (2.8–11 μ m ). Estimated on- and off-rates for the open-state block by mexiletine at +30 mV were 10.4 μ m ⁻¹ s⁻¹ and 54.4 s⁻¹, respectively. Use-dependent block by mexiletine was greater in inactivation-deficient mutant channels than in wild-type channels during repetitive pulses. Furthermore, the IC₅₀ values for the block of persistent late hNav1.4 currents in chloramine-T-pretreated cells by mexiletine was 7.5 ± 0.8 μ m ( n = 5) at +30 mV. Our results together support the hypothesis that the in vivo efficacy of mexiletine is primarily due to the open-channel block of persistent late Na⁺ currents, which may arise during various pathological conditions.     

Small- and Intermediate-Conductance Calcium-Activated K+ Channels Provide Different Facets of Endothelium-Dependent Hyperpolarization in Rat Mesenteric Artery

Activation of both small-conductance (SK_Ca) and intermediate-conductance (IK_Ca) Ca²⁺-activated K⁺ channels in endothelial cells leads to vascular smooth muscle hyperpolarization and relaxation in rat mesenteric arteries. The contribution that each endothelial K⁺ channel type makes to the smooth muscle hyperpolarization is unknown. In the presence of a nitric oxide (NO) synthase inhibitor, ACh evoked endothelium and concentration-dependent smooth muscle hyperpolarization, increasing the resting potential (approx. −53 mV) by around 20 mV at 3 μ m . Similar hyperpolarization was evoked with cyclopiazonic acid (10 μ m , an inhibitor of sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA)) while 1-EBIO (300 μ m , an IK_Ca activator) only increased the potential by a few millivolts. Hyperpolarization in response to either ACh or CPA was abolished with apamin (50 n m , an SK_Ca blocker) but was unaltered by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (1 μ m TRAM-34, an IK_Ca blocker). During depolarization and contraction in response to phenylephrine (PE), ACh still increased the membrane potential to around −70 mV, but with apamin present the membrane potential only increased just beyond the original resting potential ( circa −58 mV). TRAM-34 alone did not affect hyperpolarization to ACh but, in combination with apamin, ACh-evoked hyperpolarization was completely abolished. These data suggest that true endothelium-dependent hyperpolarization of smooth muscle cells in response to ACh is attributable to SK_Ca channels, whereas IK_Ca channels play an important role during the ACh-mediated repolarization phase only observed following depolarization.     

Biophysical and pharmacological properties of the voltage-gated potassium current of human pancreatic β-cells

Voltage-gated potassium (Kv) currents of human pancreatic islet cells were studied by whole-cell patch clamp recording. On average, 75% of the cells tested were identified as β-cells by single cell, post-recording RT-PCR for insulin mRNA. In most cells, the dominant Kv current was a delayed rectifier. The delayed rectifier activated at potentials above −20 mV and had a V _½ for activation of −5.3 mV. Onset of inactivation was slow for a major component (τ= 3.2 s at +20 mV) observed in all cells; a smaller component (τ= 0.30 s) with an amplitude of ∼25% was seen in some cells. Recovery from inactivation had a τ of 2.5 s at −80 mV and steady-state inactivation had a V _½ of −39 mV. In 12% of cells (21/182) a low-threshold, transient Kv current (A-current) was present. The A-current activated at membrane potentials above −40 mV, inactivated with a time constant of 18.5 ms at −20 mV, and had a V _½ for steady-state inactivation of −52 mV. TEA inhibited total Kv current with an  IC₅₀= 0.54 m m   and PAC, a disubstituted cyclohexyl Kv channel inhibitor, inhibited with an  IC₅₀= 0.57 μ m   . The total Kv current was insensitive to margatoxin (100 n m ), agitoxin-2 (50 n m ), kaliotoxin (50 n m ) and ShK (50 n m ). Hanatoxin (100 n m ) inhibited total Kv current by 65% at +20 mV. Taken together, these data provide evidence of at least two distinct types of Kv channels in human pancreatic β-cells and suggest that more than one type of Kv channel may be involved in the regulation of glucose-dependent insulin secretion.     

An amiloride-sensitive H+-gated Na+ channel in Caenorhabditis elegans body wall muscle cells

About 30 genes are predicted to encode degenerin/epithelial sodium channels (DEG/ENaCs) in Caenorhabditis elegans but the gating mode of these channels has not been determined. Using the whole-cell configuration of the patch-clamp technique in acutely dissected C. elegans , we investigated the effects of H⁺ as a potential activating factor of DEG/ENaCs on electrical properties of body wall muscle cells. Under current-clamp conditions, decreasing external pH from 7.2 to 6.1 led to a reversible depolarization of muscle cells associated with a decrease in input resistance which was partially inhibited by amiloride. Under voltage-clamp conditions, extracellular acidification activated an inward desensitizing current at −60 mV. In the absence of external Ca²⁺, H⁺-gated channels were found to be slightly more permeable to Na⁺ than to K⁺ and were blocked by amiloride with a K _0.5 of 31 μ m at −60 mV. An inward current could be also activated by protons in a GABA receptor null mutant in the presence of d -tubocurare and in an unc-105 null mutant. These results demonstrate that ion channels sharing common properties with mammalian acid-sensing ion channels (ASICs) are functional in C. elegans muscle which should prove useful for understanding proton sensing in animals.     

Proton modulation of ion channels in isolated horizontal cells of the goldfish retina

Transient changes in extracellular pH (pH_o) occur in the retina and may have profound effects on neurotransmission and visual processing due to the pH sensitivity of ion channels. The present study characterized the effects of acidification on the activity of membrane ion channels in isolated horizontal cells (HCs) of the goldfish retina using whole-cell patch-clamp recording. Currents recorded from HCs were characterized by prominent inward rectification at potentials negative to −80 mV, a negative slope conductance between −70 and −40 mV, a sustained inward current, and outward rectification positive to 40 mV. Inward currents were identified as those of inward rectifier K⁺ (Kir) channels and Ca²⁺ channels by their sensitivity to 10 m m Cs⁺ or 20 μ m Cd²⁺, respectively. Both of these currents were reduced when pH_o decreased from 7.8 to 6.8. Glutamate (1 m m )-activated currents were also identified, as were hemichannel currents that were enhanced by removal of extracellular Ca²⁺ and application of 1 m m quinidine. Both glutamate-activated and hemichannel currents were suppressed by a similar reduction of pH_o. When all of these H⁺-inhibited currents were blocked, a small, sustained inward current at −60 mV increased following a decrease in pH_o from 7.8 to 6.8. In addition, slope conductance between −70 and −20 mV increased during this acidification. Suppression of this H⁺-activated current by removal of extracellular Na⁺, and an extrapolated E _rev near E _Na, indicated that this current was carried predominantly by Na⁺ ions.     

Actions of noradrenaline on substantia gelatinosa neurones in the rat spinal cord revealed by in vivo patch recording

To elucidate the mechanisms of antinociception mediated by the descending noradrenergic pathway in the spinal cord, the effects of noradrenaline (NA) on noxious synaptic responses of substantia gelatinosa (SG) neurones, and postsynaptic actions of NA were investigated in rats using an in vivo whole-cell patch-clamp technique. Under urethane anaesthesia, the rat was fixed in a stereotaxic apparatus after the lumbar spinal cord was exposed. In the current-clamp mode, pinch stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSPs, some of which initiated an action potential. Perfusion with NA onto the surface of the spinal cord hyperpolarized the membrane (5.0–9.5 mV) and suppressed the action potentials. In the voltage-clamp mode ( V _H, −70 mV), the application of NA produced an outward current that was blocked by Cs⁺ and GDP-β-S added to the pipette solution and reduced the amplitude of EPSCs evoked by noxious stimuli. Under the blockade of postsynaptic actions of NA, a reduction of the evoked and spontaneous EPSCs of SG neurones was still observed, thus suggesting both pre- and postsynaptic actions of NA. The NA-induced outward currents showed a clear dose dependency (EC₅₀, 20 μ m ), and the reversal potential was −88 mV. The outward current was mimicked by an α₂-adrenoceptor agonist, clonidine, and suppressed by an α₂-adrenoceptor antagonist, yohimbine, but not by α₁- and β-antagonists. These findings suggest that NA acts on presynaptic sites to reduce noxious stimuli-induced EPSCs, and on postsynaptic SG neurones to induce an outward current by G-protein-mediated activation of K⁺ channels through α₂-adrenoceptors, thereby producing an antinociceptive effect.     

Initial segment Kv2.2 channels mediate a slow delayed rectifier and maintain high frequency action potential firing in medial nucleus of the trapezoid body neurons

The medial nucleus of the trapezoid body (MNTB) is specialized for high frequency firing by expression of Kv3 channels, which minimize action potential (AP) duration, and Kv1 channels, which suppress multiple AP firing, during each calyceal giant EPSC. However, the outward K⁺ current in MNTB neurons is dominated by another unidentified delayed rectifier. It has slow kinetics and a peak conductance of ∼37 nS; it is half-activated at −9.2 ± 2.1 mV and half-inactivated at −35.9 ± 1.5 mV. It is blocked by several non-specific potassium channel antagonists including quinine (100 μ m ) and high concentrations of extracellular tetraethylammonium (TEA; IC₅₀= 11.8 m m ), but no specific antagonists were found. These characteristics are similar to recombinant Kv2-mediated currents. Quantitative RT-PCR showed that Kv2.2 mRNA was much more prevalent than Kv2.1 in the MNTB. A Kv2.2 antibody showed specific staining and Western blots confirmed that it recognized a protein ∼110 kDa which was absent in brainstem tissue from a Kv2.2 knockout mouse. Confocal imaging showed that Kv2.2 was highly expressed in axon initial segments of MNTB neurons. In the absence of a specific antagonist, Hodgkin–Huxley modelling of voltage-gated conductances showed that Kv2.2 has a minor role during single APs (due to its slow activation) but assists recovery of voltage-gated sodium channels (Nav) from inactivation by hyperpolarizing interspike potentials during repetitive AP firing. Current-clamp recordings during high frequency firing and characterization of Nav inactivation confirmed this hypothesis. We conclude that Kv2.2-containing channels have a distinctive initial segment location and crucial function in maintaining AP amplitude by regulating the interspike potential during high frequency firing.     

KCNQ/M-currents contribute to the resting membrane potential in rat visceral sensory neurons

The M-current is a slowly activating, non-inactivating potassium current that has been shown to be present in numerous cell types. In this study, KCNQ2, Q3 and Q5, the molecular correlates of M-current in neurons, were identified in the visceral sensory neurons of the nodose ganglia from rats through immunocytochemical studies. All neurons showed expression of each of the three proteins. In voltage clamp studies, the cognition-enhancing drug linopirdine (1–50 μ m ) and its analogue, XE991 (10 μ m ), quickly and irreversibly blocked a small, slowly activating current that had kinetic properties similar to KCNQ/M-currents. This current activated between −60 and −55 mV, had a voltage-dependent activation time constant of 208 ± 12 ms at −20 mV, a deactivation time constant of 165 ± 24 ms at −50 mV and V _1/2 of −24 ± 2 mV, values which are consistent with previous reports for endogenous M-currents. In current clamp studies, these drugs also led to a depolarization of the resting membrane potential at values as negative as −60 mV. Flupirtine (10–20 μ m ), an M-current activator, caused a 3–14 mV leftward shift in the current–voltage relationship and also led to a hyperpolarization of resting membrane potential. These data indicate that the M-current is present in nodose neurons, is activated at resting membrane potential and that it is physiologically important in regulating excitability by maintaining cells at negative voltages.     

A novel osmosensitive voltage gated cation current in rat supraoptic neurones

The magnocellular neurosecretory cells of the hypothalamus (MNCs) regulate water balance by releasing vasopressin and oxytocin as a function of plasma osmolality. Release is determined largely by the rate and pattern of action potentials generated in the MNC somata. Changes in firing are mediated in part by a stretch-inactivated non-selective cation current that causes the cells to depolarize when increased osmolality leads to cell shrinkage. We have obtained evidence for a new current that may regulate MNC firing during changes in external osmolality, using whole-cell patch clamp of acutely isolated rat MNC somata. In internal and external solutions lacking K⁺, with high concentrations of TEA, and with Na⁺ as the only likely permeant cation, the current appears as a slow inward current during depolarizations and yields a large tail current upon return to the holding potential of −80 mV. Approximately 60% of the MNCs tested (79 out of 134 cells) displayed a large increase in tail current density (from 5.2 ± 0.9 to 10.5 ± 1.4 pA pF⁻¹; P < 0.001) following an increase in external osmolality from 295 to 325 mosmol kg⁻¹. The current is activated by depolarization to potentials above −60 mV and does not appear to depend on changes in internal Ca²⁺. The current is carried by Na⁺ under these conditions, but is blocked by Cs⁺ and Ba²⁺ and by internal K⁺, which suggests that the current could be a K⁺ current under physiological conditions. This current could play an important role in regulating the response of MNCs to osmolality.