Classification of voltage-dependent Ca2+ channels in trigeminal ganglion neurons from neonatal rats

We examined the subtypes and characteristics of the Ca(2+) channel in small (diameter < 30 microm) trigeminal ganglion (TG) neurons from neonatal rats by means of whole cell patch clamp techniques. There were two current components, low-voltage activated (LVA) and high-voltage activated (HVA) I(Ba), with different activation ranges and waveforms. LVA I(Ba) elicited from a depolarizing step pulse at a holding potential (HP) of -80 mV was inhibited by 0.25 mM amiloride (62%), which did not produce any significant inhibition of the peak amplitude of HVA I(Ba). The application of 0.5 mM amiloride inhibited 10% of the HVA I(Ba). The LVA I(Ba) was also reduced by changing the HP from -80 to -60 mV (61%), and under these conditions the peak amplitude of HVA I(Ba) did not change significantly. In addition, HVA I(Ba) and LVA I(Ba) showed marked differences in their inactivation properties. Experiments with several Ca(2+) channel blockers revealed that on average, 26% of the HVA I(Ba) was nifedipine (10 microM) sensitive, 55% was sensitive to omega-conotoxinGVIA (1 microM), 4% was blocked by omega-agatoxinIVA (1 microM), and the remainder of the current that was resistant to the co-application of all three Ca(2+) channel blockers was 15% of the total current. These results suggest that the application of amiloride and the alteration of the holding potential level can discriminate between HVA and LVA Ba(2+) currents in TG neurons, and that TG neurons expressed T-, L-, N-, P-/Q- and R-type Ca(2+) channels.     

Modulation by endogenously released ATP and opioids of chromaffin cell calcium channels in mouse adrenal slices

Modulation of high-threshold voltage-dependent calcium channels by neurotransmitters has been the subject of numerous studies in cultures of neurons and chromaffin cells. However, no studies on such modulation exist in chromaffin cells in their natural environment, the intact adrenal medullary tissue. Here we performed such a study in voltage-clamped chromaffin cells of freshly prepared mouse adrenal slices under the whole cell configuration of the patch-clamp technique. The subcomponents of the whole cell inward Ca(2+) current (I(Ca)) accounted for 49% for L-, 28% for N-, and 36% for P/Q-type channels. T-type Ca(2+) channels or residual R-type Ca(2+) currents were not seen. However, under the perforated-patch configuration, 20% of I(Ca) accounted for a toxin-resistant R-type Ca(2+) current. Exogenously applied ATP and methionine-enkephalin (Met-enk) inhibited I(Ca) by 33%. Stop-flow and Ca(2+) replacement by Ba(2+), which favored the release of endogenous ATP and opioids, also inhibited I(Ca), with no changes in activation or inactivation kinetics. This inhibition was partially voltage independent and insensitive to prepulse facilitation. Furthermore, in about half of the cells, suramin and naloxone augmented I(Ca) in the absence of exogenous application of ATP/Met-enk. No additional modulation of I(Ca) was obtained after bath application of exogenous ATP and opioids to these already inhibited cells. Augmentation of I(Ca) was also seen upon intracellular dialysis of guanosine 5'-[β-thio]diphosphate (GDPβS), indicating the existence in the intact slice of a tonic inhibition of I(Ca) in resting conditions. These results suggest that in the intact adrenal tissue a tonic inhibition of I(Ca) exists, mediated by purinergic and opiate receptors.     

R-Type Ca<Superscript>2+</Superscript>-channel Activity Is Associated with Chromogranin A Secretion in Human Neuroendocrine Tumor BON Cells

This electrophysiological study was undertaken to investigate the role of voltage-operated Ca(2+) channels (VOCCs) in cultivated human neuroendocrine tumor (NET) cells. Patch-clamp techniques, measurements of intracellular Ca(2+) ([Ca(2+)](i)), and secretion analysis were performed using cultured human NET BON cells. Ba(2+) inward currents through R-type channels (Ca(V)2.3) were measured and identified by SNX-482 (10 n M), a novel voltage-sensitive R-type Ca(2+) channel antagonist. In the presence of nifedipine (5 micro M), omega-Conotoxin GVIA (100 n M) and omega-Agatoxin IVA (20 n M), R-type channel currents were also detectable. Release of Ca(2+) from intracellular Ca(2+) stores by intracellular application of inositol-1,4,5-trisphosphate (InsP(3); 10 micro M) via the patch pipette during whole-cell configuration as well as induction of capacitative Ca(2+) entry (CCE), a passive maneuver to release Ca(2+) from intracellular Ca(2+) stores, led to an increase in [Ca(2+)](i). This effect could be reduced by SNX-482 (20 n M). In addition, SNX-482 (25 n M) also decreased chromogranin A (CgA) secretion, whereas omega-Conotoxin GVIA (500 n M) and nifedipine (5 micro M) failed to reduce CgA secretion. We conclude that these data reveal neuronal R-type channel activity (Ca(V)2.3), for the first time associated with CgA secretion in BON cells. Influx of Ca(2+) by activation of R-type channels may lead to an increase of intracellular Ca(2+), which stimulates CgA secretion. Thus, R-type channels could play an important role in certain clinical characteristics of NETs, such as the hypersecretion syndrome.     

Tyrosine kinases act directly on the alpha1 subunit to modulate Ca(v)2.2 calcium channels.

Voltage-operated calcium channels are modulated by tyrosine kinases in different cell types. In this study, I(Ba) was measured by the whole cell voltage-clamp technique in single COS-7 cells overexpressing the Ca(v)2.2 calcium channels encoding N-type currents. Bath application of genistein, a nonselective PTK inhibitor (50-300 microM), concentration-dependently inhibited calcium channel currents, whereas the inactive structural analogue, daidzein, was without effect over the same concentration range. Similarly, PP1, a src family-selective tyrosine kinase inhibitor, inhibited I(Ba) in a concentration-dependent manner (500 nM-5 microM) over a range of test potentials. Expression of the Ca(v)2.2alpha1 (alpha(1B)) subunit alone gave rise to functional channels, and genistein (100 microM) also inhibited currents elicited by the alpha(1B) subunit alone. These results indicate that tyrosine kinase inhibitors are likely to inhibit Ca(v)2.2 calcium channels via an action on the pore-forming alpha(1) subunit and suggest that an endogenous member of the Src family may play a physiological role in modulating these channels.     

Functional coupling between 'R-type' Ca2+ channels and insulin secretion in the insulinoma cell line INS-1.

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.     

Differences in apparent pore sizes of low and high voltage-activated Ca2+ channels

Pore size is of considerable interest in voltage-gated Ca(2+) channels because they exemplify a fundamental ability of certain ion channels: to display large pore diameter, but also great selectivity for their ion of choice. We determined the pore size of several voltage-dependent Ca(2+) channels of known molecular composition with large organic cations as probes. T-type channels supported by the Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 subunits; L-type channels encoded by the Ca(V)1.2, beta(1), and alpha(2)delta(1) subunits; and R-type channels encoded by the Ca(V)2.3 and beta(3) subunits were each studied using a Xenopus oocyte expression system. The weak permeabilities to organic cations were resolved by looking at inward tails generated upon repolarization after a large depolarizing pulse. Large inward NH(4)(+) currents and sizable methylammonium and dimethylammonium currents were observed in all of the channels tested, whereas trimethylammonium permeated only through L- and R-type channels, and tetramethylammonium currents were observed only in L-type channels. Thus, our experiments revealed an unexpected heterogeneity in pore size among different Ca(2+) channels, with L-type channels having the largest pore (effective diameter = 6.2 A), T-type channels having the tiniest pore (effective diameter = 5.1 A), and R-type channels having a pore size intermediate between these extremes. These findings ran counter to first-order expectations for these channels based simply on their degree of selectivity among inorganic cations or on the bulkiness of their acidic side chains at the locus of selectivity.     

Overexpressed Ca(v)beta3 inhibits N-type (Cav2.2) calcium channel currents through a hyperpolarizing shift of ultra-slow and closed-state inactivation

It has been shown that beta auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of beta3 subunit on macroscopic Ba(2+) currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at "physiological" holding potentials (HPs) of -60 and -80 mV. At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca(v)2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced current suppression was reversed at a HP of -120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba(2+) (40 mM) as a charge carrier also largely diminished the effect of beta3 at -80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and beta3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel beta3 effect. Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without beta3, and that beta3 caused an approximately 40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of beta3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV. Similar "ultra-slow" inactivation was observed for N-type channels without beta3. Thus, beta3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting "ultra-slow" and "closed-state" inactivation properties.     

Cu2+, Co2+, and Mn2+ modify the gating kinetics of high-voltage-activated Ca2+ channels in rat palaeocortical neurons

The effects of three divalent metal cations (Mn2+, Co2+, and Cu2+) on high-voltage-activated (HVA) Ca2+ currents were studied in acutely dissociated pyramidal neurons of rat piriform cortex using the patch-clamp technique. Cu2+, Mn2+, and Co2+ blocked HVA currents conducted by Ba2+ ( IBa) with IC50 of approximately 920 nM, approximately 58 micro M, and approximately 65 micro M, respectively. Additionally, after application of non-saturating concentrations of the three cations, residual currents activated with substantially slower kinetics than control IBa. As a consequence, the current fraction abolished by the blocking cations typically displayed, in its early phase, an unusually fast-decaying transient. The latter phenomenon turned out to be a subtraction artifact, since none of the pharmacological components (L-, N-, P/Q-, and R-type) that constitute the total HVA currents under study showed a similarly fast early decay: hence, the slow activation kinetics of residual currents was not due to the preferential inhibition of a fast-activating/inactivating component, but rather to a true slowing effect of the blocker cations. The percent IBa-amplitude inhibition caused by Mn2+, Co2+, and Cu2+ was voltage-independent over the whole potential range explored (up to +30 mV), hence the slowing of IBa activation kinetics was not due to a mechanism of voltage- and time-dependent relief from block. Moreover, Mn2+, Co2+, and Cu2+ significantly reduced I(Ba) deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism. The above results show that 1) Cu2+ is a particularly potent HVA Ca2+-channel blocker in rat palaeocortical neurons; and 2) Mn2+, Co2+, and Cu2+, besides exerting a blocking action on HVA Ca2+-channels, also modify Ca2+-current activation and deactivation kinetics, most probably by directly interfering with channel-state transitions.     

Expression of RGS2 alters the coupling of metabotropic glutamate receptor 1a to M-type K+ and N-type Ca2+ channels

Group I mGluRs heterologously expressed in sympathetic neurons inhibited calcium (I(Ca)) and M-type potassium (I(M)) currents. Treatment with pertussis toxin (PTX) revealed a voltage-dependent (VD), PTX-sensitive component of I(Ca) inhibition and a voltage-independent (VI), PTX-insensitive component. Coexpression of RGS2 occluded mGluR1a inhibition of I(M) and made I(Ca) inhibition VD in PTX-treated cells, presumably by blocking the effects of G alpha(q/11)-GTP. These data indicate that mGluR1a can couple to G(i/o) as well as G(q/11). In addition, VI I(Ca) inhibition proceeds through a G alpha(q/11)-GTP-mediated pathway, which can be occluded by expressing RGS2, leaving the VD, G betagamma-mediated inhibition active. These data may reveal a functional role for the upregulation of RGS2 expression in in vivo systems.     

Phoneutria nigriventer omega-phonetoxin IIA blocks the Cav2 family of calcium channels and interacts with omega-conotoxin-binding sites

omega-Phonetoxin IIA (omegaPtxIIA), a peptide from spider venom (Phoneutria nigriventer), inhibits high threshold voltage-dependent calcium currents in neurons. To define its pharmacological specificity, we have used patch-clamp methods in cell lines expressing recombinant Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 channels (P/Q-, N-, and R-type currents, respectively). Calcium currents generated by Ca(v)2.1 and Ca(v)2.2 were blocked almost irreversibly by 3 nm omegaPtxIIA, whereas Ca(v)2.3 showed partial and readily reversible inhibition. Binding assays with mono[(125)I]iodo-omegaPtxIIA indicated that membranes expressing recombinant Ca(v)2.1 or Ca(v)2.2 channels showed a single class of sites with similar affinity (K(D) approximately 50 pm), whereas low affinity interactions were detectable with Ca(v)2.3. Kinetic, saturation, and displacement assays demonstrated that rat brain synaptosomes displayed multiple classes of binding sites for (125)I-omegaPtxIIA. High affinity binding of (125)I-omegaPtxIIA was totally displaced by omegaPtxIIA (K(i) = 100 pm), but only partially by omega-conotoxin GVIA (25% inhibition) and omega-conotoxin MVIIC (50% inhibition at 0.3 microm). (125)I-omegaPtxIIA thus defines a unique high affinity binding site that is predominantly associated with Ca(v)2.1 or Ca(v)2.2 channels.     

Structural elements in domain IV that influence biophysical and pharmacological properties of human alpha1A-containing high-voltage-activated calcium channels

We have cloned two splice variants of the human homolog of the alpha1A subunit of voltage-gated Ca2+ channels. The sequences of human alpha1A-1 and alpha1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human alpha1A-2alpha2bdeltabeta1b Ca2+ channels expressed in HEK293 cells activate rapidly (tau+10mV = 2.2 ms), deactivate rapidly (tau-90mV = 148 micros), inactivate slowly (tau+10mV = 690 ms), and have peak currents at a potential of +10 mV with 15 mM Ba2+ as charge carrier. In HEK293 cells transient expression of Ca2+ channels containing alpha1A/B(f), an alpha1A subunit containing a 112 amino acid segment of alpha1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold larger compared to wild-type (wt) alpha1A-2-containing Ca2+ channels, and had inactivation kinetics similar to those of alpha1B-1-containing Ca2+ channels. Cells transiently transfected with alpha1A/B(f)alpha2bdeltabeta1b expressed higher levels of the alpha1, alpha2bdelta, and beta1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis we identified two locations in domain IV within the extracellular loops S3-S4 (N1655P1656) and S5-SS1 (E1740) that influence the biophysical properties of alpha1A. alpha1AE1740R resulted in a threefold increase in current magnitude, a -10 mV shift in steady-state inactivation, and an altered Ba2+ current inactivation, but did not affect ion selectivity. The deletion mutant alpha1ADeltaNP shifted steady-state inactivation by -20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by omega-Aga IVA was increased with alpha1ADeltaNP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of alpha1A-containing Ca2+ channels.     

Low-affinity Ca(2+) and Ba(2+) binding sites in the pore of alpha7 nicotinic acetylcholine receptors

alpha7 nicotinic receptors are highly permeable to Ca(2+) as well as monovalent cations. We extended the characterization of the Ca(2+) permeation of non-desensitizing chick alpha7 receptors (S240T/L247T alpha7 nAChRs) expressed in Xenopus oocytes by (1) measuring the concentration dependence of conductance under conditions in which Ca(2+) or Ba(2+) were the only permeant cations in the extracellular solution, and (2) measuring the concentration dependence of Ca(2+) block of K(+) currents through the receptors. The first set of experiments yielded an apparent affinity of 0.96 mM Ca(2+) activity (2.4 mM concentration) for Ca(2+) permeation and an apparent affinity of 0.65 mM Ba(2+) activity (1.7 mM concentration) for Ba(2+) permeation. The apparent affinity of Ca(2+) inhibition of K(+) currents was 0.49 mM activity (1.5 mM concentration). The similarity of these apparent affinities in the millimolar range suggests that the pore of alpha7 receptors has one or more low-affinity Ca(2+) binding sites and no high-affinity sites.     

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.     

Effects of ginsenosides and their metabolites on voltage-dependent Ca(2+) channel subtypes

In previous reports we demonstrated that ginsenosides, active ingredients of Panax ginseng, affect some subsets of voltage-dependent Ca(2+) channels in neuronal cells expressed in Xenopus laevis oocytes. However, the major component(s) of ginseng that affect cloned Ca(2+) channel subtypes such as alpha(1C) (L)-, alpha(1B) (N)-, alpha(1A) (P/Q)-, a1E (R)- and a1G (T) have not been identified. Here, we used the two-microelectrode volt-age clamp technique to characterize the effects of ginsenosides and ginsenoside metabolites on Ba(2+) currents (IBa) in Xenopus oocytes expressing five different Ca(2+) channel subtypes. Exposure to ginseng total saponins (GTS) induced voltage-dependent, dose-dependent and reversible inhibition of the five channel subtypes, with particularly strong inhibition of the a1G-type. Of the various ginsenosides, Rb(1), Rc, Re, Rf, Rg(1), Rg(3), and Rh(2), ginsenoside Rg(3) also inhibited all five channel subtypes and ginsenoside Rh(2) had most effect on the a1C- and a1E-type Ca(2+) channels. Compound K (CK), a protopanaxadiol ginsenoside metabolite, strongly inhibited only the a(1G)-type of Ca(2+) channel, whereas M4, a protopanaxatriol ginsenoside metabolite, had almost no effect on any of the channels. Rg(3), Rh(2), and CK shifted the steady-state activation curves but not the inactivation curves in the depolarizing direction in the alpha(1B)- and alpha(1A)-types. These results reveal that Rg(3), Rh(2) and CK are the major inhibitors of Ca(2+) channels in Panax ginseng, and that they show some Ca(2+) channel selectivity.     

Mg(2+) block unmasks Ca(2+)/Ba(2+) selectivity of alpha1G T-type calcium channels

We have examined permeation by Ca(2+) and Ba(2+), and block by Mg(2+), using whole-cell recordings from alpha1G T-type calcium channels stably expressed in HEK 293 cells. Without Mg(o)(2+), inward currents were comparable with Ca(2+) and Ba(2+). Surprisingly, three other results indicate that alpha1G is actually selective for Ca(2+) over Ba(2+). 1) Mg(2+) block is approximately 7-fold more potent with Ba(2+) than with Ca(2+). With near-physiological (1 mM) Mg(o)(2+), inward currents were approximately 3-fold larger with 2 mM Ca(2+) than with 2 mM Ba(2+). The stronger competition between Ca(2+) and Mg(2+) implies that Ca(2+) binds more tightly than Ba(2+). 2) Outward currents (carried by Na(+)) are blocked more strongly by Ca(2+) than by Ba(2+). 3) The reversal potential is more positive with Ca(2+) than with Ba(2+), thus P(Ca) > P(Ba). We conclude that alpha1G can distinguish Ca(2+) from Ba(2+), despite the similar inward currents in the absence of Mg(o)(2+). Our results can be explained by a 2-site, 3-barrier model if Ca(2+) enters the pore 2-fold more easily than Ba(2+) but exits the pore at a 2-fold lower rate.     

Role of GABA<Subscript>B</Subscript> receptor in the regulation of excitatory synaptic transmission in trigeminal motoneurons

The aim of the present study was to determine if excitatory synaptic transmission onto trigeminal motoneurons is subject to a presynaptic modulation by gamma-aminobutyric acid (GABA) via GABA(B) receptor in this system. Whole cell recordings were made from trigeminal motoneurons in longitudinal brain stem slices taken from 8-day-old rats. Monosynaptic excitatory postsynaptic potential (EPSP) activity was evoked by placing bipolar stainless steel electrodes dorsal-caudal to the trigeminal motor nucleus. Bath application of the GABA(B) receptor agonist, baclofen, produced a marked reduction in the mean amplitude and variance of evoked EPSPs and also increased the portion of transmission failures. It also produced a decrease in the frequency, but not in the mean amplitude, of spontaneous miniature EPSPs. Bath application of GABA(B) receptor antagonists 6-hydroxy-saclofen and CGP35348 increased both the amplitude and frequency of miniature EPSP activity. Taken together the above results suggest that the excitatory synaptic inputs onto trigeminal motoneurons are controlled by tonic presynaptic modulation by GABA(B) receptor.     

Inhibitory effect of C-type natriuretic peptide on L-type calcium channel currents in gastric antral myocytes of guinea pigs

The role of C-type natriuretic peptide (CNP) in the gastrointestinal tract is still unclear. This study was designed to investigate the effect of CNP on barium current (I(Ba)) through the L-type calcium channel in gastric antral myocytes of guinea pigs. The whole-cell patch clamp technique was performed in gastric antral myocytes isolated by collagenase in guinea pigs. CNP significantly inhibited I(Ba) in a dose-dependent manner at the concentrations of 0.001, 0.01, and 0.1 micromol/l, CNP inhibited I(Ba) to 81.56 +/- 2.48 %, 73.64 +/- 3.65 %, and 57.77 +/- 4.93 % of control at 0 mV, respectively. The values of steady-state half-inactivation voltage (33.6 +/- 2.6 mV and 33.8 +/- 3.4 mV, in control and CNP groups, respectively) or the half-activation voltage (-12.6 +/- 2.2 mV and 12.4 +/- 1.8 mV) of I(Ba) were not significantly changed (p > 0.05, n = 6). 8-br-cGMP (1 mmol/l) mimicked the effect of CNP on I(Ba), and the peak current of I(Ba) was inhibited from -403.84 +/- 61.87 pA to 318.94 +/- 67.17 pA (p < 0.05, n = 5). In the presence of LY83583 (0.1 micromol/l), a nonspecific inhibitor of guanylate cyclase, CNP (0.1 micromol/l)-induced inhibition of I(Ba) was partially blocked (n = 13, p < 0.05 ). However, when the cell was pretreated with zaprinast (0.1 micromol/l), an inhibitor of cyclic guanosine monophosphate (cGMP) sensitive phosphoesterase, the inhibitory effect of CNP on I(Ba) was significantly potentiated (n = 11, p < 0.05). KT5823 (1 micromol/l), a cGMP-dependent protein kinase (PKG) inhibitor, almost completely blocked CNP-induced inhibition of I(Ba). The results suggested that CNP can inhibit L-type calcium channel currents, and the inhibitory effect is mediated by pGC-cGMP-PKG-dependent signal pathway in gastric antral myocytes of guinea pigs.     

S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca(2+) channels

Modulation of smooth muscle, L-type Ca(2+) channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba(2+) currents through channels derived either by expression of alpha1C-b or by coexpression of alpha1C-b plus beta2a and alpha2-delta. The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba(2+) currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha1C-b channel function nor their modulation by GSNO. Analysis of single-channel behavior revealed that GSNO inhibited Ca(2+) channels mainly by reducing open probability. The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca(2+) channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca(2+) channel gating and conductance.     

Calcium currents in hair cells isolated from semicircular canals of the frog

L-type and R-type Ca(2+) currents were detected in frog semicircular canal hair cells. The former was noninactivating and nifedipine-sensitive (5 microM); the latter, partially inactivated, was resistant to omega-conotoxin GVIA (5 microM), omega-conotoxin MVIIC (5 microM), and omega-agatoxin IVA (0.4 microM), but was sensitive to mibefradil (10 microM). Both currents were sensitive to Ni(2+) and Cd(2+) (>10 microM). In some cells the L-type current amplitude increased almost twofold upon repetitive stimulation, whereas the R-type current remained unaffected. Eventually, run-down occurred for both currents, but was prevented by the protease inhibitor calpastatin. The R-type current peak component ran down first, without changing its plateau, suggesting that two channel types generate the R-type current. This peak component appeared at -40 mV, reached a maximal value at -30 mV, and became undetectable for voltages > or =0 mV, suggestive of a novel transient current: its inactivation was indeed reversibly removed when Ba(2+) was the charge carrier. The L-type current and the R-type current plateau were appreciable at -60 mV and peaked at -20 mV: the former current did not reverse for voltages up to +60 mV, the latter reversed between +30 and +60 mV due to an outward Cs(+) current flowing through the same Ca(2+) channel. The physiological role of these currents on hair cell function is discussed.