Voltage-dependent inactivation of l-type ca2+ Currents in Guinea-Pig Ventricular Myocytes

The objective of this study was to describe the kinetics of voltage-dependent inactivation of native cardiac L-type Ca²⁺ currents. Whole-cell currents were recorded from guinea-pig isolated ventricular myocytes. Voltage-dependent inactivation was separated from Ca²⁺-dependent inactivation by replacing extracellular Ca²⁺ with Mg²⁺ and recording outward currents through Ca²⁺ channels. Voltage-dependent inactivation accelerated from slow monophasic decay at −30 mV to maximal rapid biphasic decay at +20 mV. Maximal voltage-dependent inactivation occurred with τ_f≈30 ms and τ_s≈300 ms, the fast component of decay accounted for 70 % of the current amplitude. In basal conditions Ca²⁺ current availability was sigmoid. Isoproterenol (isoprenaline) evoked a large increase in a time-independent component of the Ca²⁺ current which also increased with depolarisation. This was responsible for the apparent recovery of Ca²⁺ channel current availability at positive membrane potentials and thus a U-shaped availability-voltage ( A-V) relationship. It is concluded that β-adrenergic stimulation altered the reaction of native cardiac L-type Ca²⁺ channels to membrane voltage. In basal conditions, voltage accelerated inactivation. In isoproterenol, voltage could also reduce inactivation.     

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.     

Ca2+-activated K+ (BK) channel inactivation contributes to spike broadening during repetitive firing in the rat lateral amygdala

In many neurons, trains of action potentials show frequency-dependent broadening. This broadening results from the voltage-dependent inactivation of K⁺ currents that contribute to action potential repolarisation. In different neuronal cell types these K⁺ currents have been shown to be either slowly inactivating delayed rectifier type currents or rapidly inactivating A-type voltage-gated K⁺ currents. Recent findings show that inactivation of a Ca²⁺-dependent K⁺ current, mediated by large conductance BK-type channels, also contributes to spike broadening. Here, using whole-cell recordings in acute slices, we examine spike broadening in lateral amygdala projection neurons. Spike broadening is frequency dependent and is reversed by brief hyperpolarisations. This broadening is reduced by blockade of voltage-gated Ca²⁺ channels and BK channels. In contrast, broadening is not blocked by high concentrations of 4-aminopyridine (4-AP) or α-dendrotoxin. We conclude that while inactivation of BK-type Ca²⁺-activated K⁺ channels contributes to spike broadening in lateral amygdala neurons, inactivation of another as yet unidentified outward current also plays a role.     

Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current ( I _ti). I _ti is the result of three different [Ca]_i-sensitive currents: the Na⁺–Ca²⁺ exchange current, a Ca²⁺-activated chloride current and a Ca²⁺-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca²⁺-activated non-selective cation channel (NSC_Ca) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na⁺ and K⁺) and was slightly permeable to Ca²⁺ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K _d for [Ca²⁺]_i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC_Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca²⁺] overload and thus may be implicated in the genesis of arrhythmias.     

A Ca2+-activated Cl− conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity

Interstitial cells of Cajal (ICC) are unique cells that generate electrical pacemaker activity in gastrointestinal (GI) muscles. Many previous studies have attempted to characterize the conductances responsible for pacemaker current and slow waves in the GI tract, but the precise mechanism of electrical rhythmicity is still debated. We used a new transgenic mouse with a bright green fluorescent protein (copGFP) constitutively expressed in ICC to facilitate study of these cells in mixed cell dispersions. We found that ICC express a specialized 'slow wave' current. Reversal of tail current analysis showed this current was due to a Cl⁻ selective conductance. ICC express ANO1, a Ca²⁺-activated Cl⁻ channel. Slow wave currents are not voltage dependent, but a secondary voltage-dependent process underlies activation of these currents. Removal of extracellular Ca²⁺, replacement of Ca²⁺ with Ba²⁺, or extracellular Ni²⁺ (30 μ m ) blocked the slow wave current. Single Ca²⁺-activated Cl⁻ channels with a unitary conductance of 7.8 pS were resolved in excised patches of ICC. These are similar in conductance to ANO1 channels (8 pS) expressed in HEK293 cells. Slow wave current was blocked in a concentration-dependent manner by niflumic acid (IC₅₀= 4.8 μ m ). Slow wave currents are associated with transient depolarizations of ICC in current clamp, and these events were blocked by niflumic acid. These findings demonstrate a role for a Ca²⁺-activated Cl⁻ conductance in slow wave current in ICC and are consistent with the idea that ANO1 participates in pacemaker activity.     

Mechanisms underlying the early phase of spike frequency adaptation in mouse spinal motoneurones

Spike frequency adaptation (SFA) is a fundamental property of repetitive firing in motoneurones (MNs). Early SFA (occurring over several hundred milliseconds) is thought to be important in the initiation of muscular contraction. To date the mechanisms underlying SFA in spinal MNs remain unclear. In the present study, we used both whole-cell patch-clamp recordings of MNs in lumbar spinal cord slices prepared from motor functionally mature mice and computer modelling of spinal MNs to investigate the mechanisms underlying SFA. Pharmacological blocking agents applied during whole-cell recordings in current-clamp mode demonstrated that the medium AHP conductance (apamin), BK-type Ca²⁺-dependent K⁺ channels (iberiotoxin), voltage-activated Ca²⁺ channels (CdCl₂), M-current (linopirdine) and persistent Na⁺ currents (riluzole) are all unnecessary for SFA. Measurements of Na⁺ channel availability including action potential amplitude, action potential threshold and maximum depolarization rate of the action potential were found to correlate with instantaneous firing frequency suggesting that the availability of fast, inactivating Na⁺ channels is involved in SFA. Characterization of this Na⁺ conductance in voltage-clamp mode demonstrated that it undergoes slow inactivation with a time course similar to that of SFA. When experimentally measured parameters for the fast, inactivating Na⁺ conductance (including slow inactivation) were incorporated into a MN model, SFA could be faithfully reproduced. The removal of slow inactivation from this model was sufficient to remove SFA. These data indicate that slow inactivation of the fast, inactivating Na⁺ conductance is likely to be the key mechanism underlying early SFA in spinal MNs.     

Small conductance Ca2+-activated K+ channels and calmodulin

Small conductance Ca²⁺-activated K⁺ channels (SK channels) contribute to the long lasting afterhyperpolarization (AHP) that follows an action potential in many central neurones. The biophysical and pharmacological attributes of cloned SK channels strongly suggest that one or more of them underlie the medium component of the AHP that regulates interspike interval and plays an important role in setting tonic firing frequency. The cloned SK channels comprise a distinct subfamily of K⁺ channels. Heterologously expressed SK channels recapitulate the biophysical and pharmacological hallmarks of native SK channels, being gated solely by intracellular Ca²⁺ ions with no voltage dependence to their gating, small unitary conductance values and sensitivity to the bee venom peptide toxin, apamin. Molecular, biochemical and electrophysiological studies have revealed that Ca²⁺ gating in SK channels is due to heteromeric assembly of the SK α pore-forming subunits with calmodulin (CaM). Ca²⁺ binding to the N-terminal E–F hands of CaM is responsible for SK channel gating. Crystallographic studies suggest that SK channels gate as a dimer-of-dimers, and that the physical gate of SK channels resides at or near the selectivity filter of the channels. In addition, Ca²⁺-independent interactions between the SK channel α subunits and CaM are necessary for proper membrane trafficking.     

Sodium and calcium currents shape action potentials in immature mouse inner hair cells

Before the onset of hearing at postnatal day 12, mouse inner hair cells (IHCs) produce spontaneous and evoked action potentials. These spikes are likely to induce neurotransmitter release onto auditory nerve fibres. Since immature IHCs express both α1D (Ca_v1.3) Ca²⁺ and Na⁺ currents that activate near the resting potential, we examined whether these two conductances are involved in shaping the action potentials. Both had extremely rapid activation kinetics, followed by fast and complete voltage-dependent inactivation for the Na⁺ current, and slower, partially Ca²⁺-dependent inactivation for the Ca²⁺ current. Only the Ca²⁺ current is necessary for spontaneous and induced action potentials, and 29 % of cells lacked a Na⁺ current. The Na⁺ current does, however, shorten the time to reach the action-potential threshold, whereas the Ca²⁺ current is mainly involved, together with the K⁺ currents, in determining the speed and size of the spikes. Both currents increased in size up to the end of the first postnatal week. After this, the Ca²⁺ current reduced to about 30 % of its maximum size and persisted in mature IHCs. The Na⁺ current was downregulated around the onset of hearing, when the spiking is also known to disappear. Although the Na⁺ current was observed as early as embryonic day 16.5, its role in action-potential generation was only evident from just after birth, when the resting membrane potential became sufficiently negative to remove a sizeable fraction of the inactivation (half inactivation was at −71 mV). The size of both currents was positively correlated with the developmental change in action-potential frequency.     

Human ClCa1 modulates anionic conduction of calcium-dependent chloride currents

Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca²⁺-dependent Cl⁻ currents. The present study examines the relationship between the hClCa1 protein and Ca²⁺-dependent Cl⁻ currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl⁻ currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca²⁺-dependent Cl⁻ currents and show that hClCa1 increases the amplitude of Ca²⁺-dependent Cl⁻ currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl⁻ conductance while decreasing the G _SCN−/ G _Cl− conductance ratio from ∼2–3 to ∼1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca²⁺-dependent Cl⁻ channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca²⁺-dependent Cl⁻ channels by lowering the energy barriers for ion translocation through the pore.     

Intracellular Na+ inhibits voltage-dependent N-type Ca2+ channels by a G protein βγ subunit-dependent mechanism

N-type  voltage-dependent  Ca²⁺ channels (N-VDCCs) play important roles in neurotransmitter release and certain postsynaptic phenomena. These channels are modulated by a number of intracellular factors, notably by Gβγ subunits of G proteins, which inhibit N-VDCCs in a voltage-dependent (VD) manner. Here we show that an increase in intracellular Na⁺ concentration inhibits N-VDCCs  in hippocampal pyramidal neurones and in Xenopus oocytes. In acutely dissociated hippocampal neurones, Ba²⁺ current via N-VDCCs was inhibited by Na⁺ influx caused by the activation of NMDA receptor channels. In Xenopus oocytes expressing N-VDCCs, Ba²⁺ currents were inhibited by Na⁺ influx and enhanced by depletion of Na⁺, after incubation in a Na⁺-free extracellular solution. The Na⁺-induced inhibition was accompanied by the development of  VD facilitation, a hallmark of a Gβγ-dependent process. Na⁺-induced regulation of N-VDCCs is Gβγ dependent, as suggested by the blocking of Na⁺ effects by Gβγ scavengers and by excess Gβγ, and may be mediated by the Na⁺-induced dissociation of Gαβγ heterotrimers. N-VDCCs may be novel effectors of Na⁺ion, regulated by the Na⁺ concentration via Gβγ.     

Hypoxia-induced secretion of serotonin from intact pulmonary neuroepithelial bodies in neonatal rabbit

We examined the effects of hypoxia on the release of serotonin (5-HT) from intact neuroepithelial body cells (NEB), presumed airway chemoreceptors, in rabbit lung slices, using amperometry with carbon fibre microelectrodes. Under normoxia ( P _O2∼155 mmHg; 1 mmHg ≈133 Pa), most NEB cells did not exhibit detectable secretory activity; however, hypoxia elicited a dose-dependent ( P _O2 range 95–18 mmHg), tetrodotoxin (TTX)-sensitive stimulation of spike-like exocytotic events, indicative of vesicular amine release. High extracellular K⁺ (50 m m ) induced a secretory response similar to that elicited by severe hypoxia. Exocytosis was stimulated in normoxic NEB cells after exposure to tetraethylammonium (20 m m ) or 4-aminopyridine (2 m m ). Hypoxia-induced secretion was abolished by the non-specific Ca²⁺ channel blocker Cd²⁺ (100 μ m ). Secretion was also largely inhibited by the L-type Ca²⁺ channel blocker nifedipine (2 μ m ), but not by the N-type Ca²⁺ channel blocker ω-conotoxin GVIA (1 μ m ). The 5-HT₃ receptor blocker ICS 205 930 also inhibited secretion from NEB cells under hypoxia. These results suggest that hypoxia stimulates 5-HT secretion from intact NEBs via inhibition of K⁺ channels, augmentation of Na⁺-dependent action potentials and calcium entry through L-type Ca²⁺ channels, as well as by positive feedback activation of 5-HT₃ autoreceptors.     

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.     

Dissociation of the store-operated calcium current ICRAC and the Mg-nucleotide-regulated metal ion current MagNuM

Rat basophilic leukaemia cells (RBL-2H3-M1) were used to study the characteristics of the store-operated Ca²⁺ release-activated Ca²⁺ current ( I _CRAC) and the magnesium-nucleotide-regulated metal cation current (MagNuM) (which is conducted by the LTRPC7 channel). Pipette solutions containing 10 m m BAPTA and no added ATP induced both currents in the same cell, but the time to half-maximal activation for MagNuM was about two to three times slower than that of I _CRAC. Differential suppression of I _CRAC was achieved by buffering free [Ca²⁺]_i to 90 n m and selective inhibition of MagNuM was accomplished by intracellular solutions containing 6 m m Mg.ATP, 1.2 m m free [Mg²⁺]_i or 100 μ m GTP-γ-S, allowing investigations on these currents in relative isolation. Removal of extracellular Ca²⁺ and Mg²⁺ caused both currents to be carried significantly by monovalent ions. In the absence or presence of free [Mg²⁺]_i, I _CRAC carried by monovalent ions inactivated more rapidly and more completely than MagNuM carried by monovalent ions. Since several studies have used divalent-free solutions on either side of the membrane to study selectivity and single-channel behaviour of I _CRAC, these experimental conditions would have favoured the contribution of MagNuM to monovalent conductance and call for caution in interpreting results where both I _CRAC and MagNuM are activated.     

Electrophysiological properties of two axonal sodium channels, Nav1.2 and Nav1.6, expressed in mouse spinal sensory neurones

Sodium channels Na_v1.2 and Na_v1.6 are both normally expressed along premyelinated and myelinated axons at different stages of maturation and are also expressed in a subset of demyelinated axons, where coexpression of Na_v1.6 together with the Na⁺/Ca²⁺ exchanger is associated with axonal injury. It has been difficult to distinguish the currents produced by Na_v1.2 and Na_v1.6 in native neurones, and previous studies have not compared these channels within neuronal expression systems. In this study, we have characterized and directly compared Na_v1.2 and Na_v1.6 in a mammalian neuronal cell background and demonstrate differences in their properties that may affect neuronal behaviour. The Na_v1.2 channel displays more depolarized activation and availability properties that may permit conduction of action potentials, even with depolarization. However, Na_v1.2 channels show a greater accumulation of inactivation at higher frequencies of stimulation (20–100 Hz) than Na_v1.6 and thus are likely to generate lower frequencies of firing. Na_v1.6 channels produce a larger persistent current that may play a role in triggering reverse Na⁺/Ca²⁺ exchange, which can injure demyelinated axons where Na_v1.6 and the Na⁺/Ca²⁺ exchanger are colocalized, while selective expression of Na_v1.2 may support action potential electrogenesis, at least at lower frequencies, while producing a smaller persistent current.     

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.     

A Ca2+-inhibited non-selective cation conductance contributes to pacemaker currents in mouse interstitial cell of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity in some smooth muscles. The nature of the pacemaker conductance is unclear, but studies suggest that pacemaker activity is due to a voltage-independent, Ca²⁺-regulated, non-selective cation conductance. We investigated Ca²⁺-regulated conductances in murine intestinal ICC and found that reducing cytoplasmic Ca²⁺ activates whole-cell inward currents and single-channel currents. Both the whole-cell currents and single-channel currents reversed at 0 mV when the equilibrium potentials of all ions present were far from 0 mV. Recordings from on-cell patches revealed oscillations in unitary currents at the frequency of pacemaker currents in ICC. Voltage-clamping cells to −60 mV did not change the oscillatory activity of channels in on-cell patches. Depolarizing cells with high external K⁺ caused loss of resolvable single-channel currents, but the oscillatory single-channel currents were restored when the patches were stepped to negative potentials. Unitary currents were also resolved in excised patches. The single-channel conductance was 13 pS, and currents reversed at 0 mV. The channels responsible were strongly activated by 10⁻⁷ m Ca²⁺, and 10⁻⁶ m Ca²⁺ reduced activity. The 13 pS channels were strongly activated by the calmodulin inhibitors calmidazolium and W-7 in on-cell and excised patches. Calmidazolium and W-7 also activated a persistent inward current under whole-cell conditions. Murine ICC express Ca²⁺-inhibited, non-selective cation channels that are periodically activated at the same frequency as pacemaker currents. This conductance may contribute to the pacemaker current and generation of electrical slow waves in GI muscles.     

Selectivity and interactions of Ba2+ and Cs+ with wild-type and mutant TASK1 K+ channels expressed in Xenopus oocytes

The acid-sensitive K⁺ channel, TASK1 is a member of the K⁺-selective tandem-pore domain (K2P) channel family. Like many of the K2P channels, TASK1 is relatively insensitive to conventional channel blockers such as Ba²⁺. In this paper we report the impact of mutating the pore-neighbouring histidine residues, which are involved in pH sensing, on the sensitivity to blockade by Ba²⁺ and Cs⁺; additionally we compare the selectivity of these channels to extracellular K⁺, Na⁺ and Rb⁺. H98D and H98N mutants showed reduced selectivity for K⁺ over both Na⁺ and Rb⁺, and significant permeation of Rb⁺. This enhanced permeability must reflect changes in the structure or flexibility of the selectivity filter. Blockade by Ba²⁺ and Cs⁺ was voltage-dependent, indicating that both ions block within the pore. In 100 m m K⁺, the K _D at 0 mV for Ba²⁺ was 36 ± 10 m m  ( n = 6)  , whilst for Cs⁺ it was 20 ± 6.0 m m  ( n = 5)  . H98D was more sensitive to Ba²⁺ than the wild-type (WT); in addition, the site at which Ba²⁺ appears to bind was altered (WT: δ, 0.64 ± 0.16, n = 6; H98D: δ, 0.16 ± 0.03, n = 5, statistically different from WT; H98N: δ, 0.58 ± 0.09, not statistically different from WT). Thus, the pore-neighbouring residue H98 contributes not only to the pH sensitivity of TASK1, but also to the structure of the conduction pathway.     

Physiological modulation of inactivation in L-type Ca2+ channels: one switch

The relative contributions of voltage- and Ca²⁺-dependent mechanisms of inactivation to the decay of L-type Ca²⁺ channel currents ( I _CaL) is an old story to which recent results have given an unexpected twist. In cardiac myocytes voltage-dependent inactivation (VDI) was thought to be slow and Ca²⁺-dependent inactivation (CDI) resulting from Ca²⁺ influx and Ca²⁺-induced Ca²⁺-release (CICR) from the sarcoplasmic reticulum provided an automatic negative feedback mechanism to limit Ca²⁺ entry and the contribution of I _CaL to the cardiac action potential. Physiological modulation of I _CaL by β-adrenergic and muscarinic agonists then involved essentially more or less of the same by enhancing or reducing Ca²⁺ channel activity, Ca²⁺ influx, sarcoplasmic reticulum load and thus CDI. Recent results on the other hand place VDI at the centre of the regulation of I _CaL. Under basal conditions it has been found that depolarization increases the probability that an ion channel will show rapid VDI. This is prevented by β-adrenergic stimulation. Evidence also suggests that a channel which shows rapid VDI inactivates before CDI can become effective. Therefore the contributions of VDI and CDI to the decay of I _CaL are determined by the turning on, by depolarization, and the turning off, by phosphorylation, of the mechanism of rapid VDI. The physiological implications of these ideas are that under basal conditions the contribution of I _CaL to the action potential will be determined largely by voltage and by Ca²⁺ following β-adrenergic stimulation.     

Modulation of Ca2+-activated K+ currents and Ca2+-dependent action potentials by exocytosis in goldfish bipolar cell terminals

Retinal bipolar cells convey light-evoked potentials from photoreceptors to ganglion cells and mediate the initial stages of visual signal processing. They do not fire Na⁺-dependent action potentials (APs) but the Mb1 class of goldfish bipolar cell exhibits Ca²⁺-dependent APs and regenerative potentials that originate in the axon terminal. I have examined the properties of Ca²⁺-dependent APs in isolated bipolar-cell terminals in goldfish retinal slices. All recorded terminals fired spontaneous or evoked APs at frequencies of up to 15 Hz. When an AP waveform was used as a voltage stimulus, exocytosis was evoked by single APs, maintained throughout AP trains and modulated by AP frequency. Furthermore, feedback inhibition of the Ca²⁺ current ( I _Ca) by released vesicular protons reduced depression of exocytosis during AP trains. In the absence of K⁺ current inhibition, step depolarizations and AP waveforms evoked a rapidly activated outward current that was dependent on Ca²⁺ influx ( I _K(Ca)). I therefore investigated whether proton-mediated feedback inhibition of I _Ca affected the activation of I _K(Ca). A transient inhibition of I _K(Ca) was observed that was dependent on exocytosis, blocked by high-pH extracellular buffer, of similar magnitude to inhibition of I _Ca but occurred with a delay of 2.7 ms. In addition, the amplitude of APs evoked under current clamp was inhibited by the action of vesicular protons released by the APs. Protons released via exocytosis may therefore be a significant modulator of Ca²⁺-dependent currents and regenerative potentials in bipolar-cell terminals.     

Ca2+-regulated, neurosecretory granule channel involved in release from neurohypophysial terminals

Ion channels from bovine neurohypophysial secretory granules (NSG) were incorporated into artificial lipid bilayers. Specific antibodies against identified synaptic vesicle proteins were tested on such incorporated channel activity and on peptide release from rat permeabilized neurohypophysial terminals. Both the NSG cation channel and Ca²⁺-dependent release were inhibited by only SY-38, a monoclonal antibody directed against the C-terminus of synaptophysin. SY-38 and Ca²⁺ altered both the gating and conductance of the NSG cation channel, but in opposite ways. The close correlation between SY-38 effects on Ca²⁺-dependent channel activity and release leads us to conclude that this synaptophysin-like NSG channel is directly involved in peptide secretion from these central nervous system terminals.