Potentiated L-type Ca2+ channels rectify

Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type alpha1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-alpha1C) or repeat III (E3K-alpha1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-alpha1C and E3K-alpha1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (-)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-alpha1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-alpha1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels.     

Potentiation of recombinant L-type Ca channel currents by alpha1-adrenoceptors coexpressed in baby hamster kidney (BHK) cells.

In cardiac myocytes, the effect of alpha1-adrenergic stimulation on L-type Ca current remains to be clarified. We examined this issue by the transient coexpression of alpha1-adrenoceptors on BHKC12 cells, where recombinant Ca channels composed of cardiac alpha1 subunit and skeletal beta, gamma, alpha2/delta subunits were stably expressed. After transfection of plasmid DNA encoding bovine alpha1C-adrenoceptors, bath-applied phenylephrine potentiated the cloned Ca channel current during perforated-patch whole-cell recording by 26+/-6% in 6 out of 12 cells. The potentiation was elicited also by methoxamine, and was blocked by prazosin. Phenylephrine also increased the channel open probability during cell-attached single channel recording in 7 out of 15 cells. The ratio of successful modulation of Ca channels was in accordance with the ratio of successful expression of alpha1-adrenoceptors, as estimated by beta-galactosidase staining. These results suggest that the stimulation of alpha1C-adrenoceptors is linked to potentiation of cardiac L-type Ca current. BHK cells provide a valuable expression system to study the modulation of Ca channels evoked by a receptor stimulation.     

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.     

A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2+ channel and modulates its function.

In skeletal muscle, voltage-dependent potentiation of L-type Ca(2+) channel (Ca(V)1.1) activity requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A kinase-anchoring protein (AKAP15). However, the mechanism by which AKAP15 targets PKA to L-type Ca(2+) channels has not been elucidated. Here we report that AKAP15 directly interacts with the C-terminal domain of the alpha(1) subunit of Ca(V)1.1 via a leucine zipper (LZ) motif. Disruption of the LZ interaction effectively inhibits voltage-dependent potentiation of L-type Ca(2+) channels in skeletal muscle cells. Our results reveal a novel mechanism whereby anchoring of PKA to Ca(2+) channels via LZ interactions ensures rapid and efficient phosphorylation of Ca(2+) channels in response to local signals such as cAMP and depolarization.     

Anomalous L-type calcium channels of rat spinal motoneurons

Single channel patch-clamp recordings show that embryonic rat spinal motoneurons express anomalous L-type calcium channels, which reopen upon repolarization to resting potentials, displaying both short and long reopenings. The probability of reopening increases with increasing voltage of the preceding depolarization without any apparent correlation with inactivation during the depolarization. The probability of long with respect to short reopenings increases with increasing length of the depolarization, with little change in the total number of reopenings and in their delay. With less negative repolarization voltages, the delay increases, while the mean duration of both short and long reopenings decreases, remaining longer than that of the openings during the preceding depolarization. Open times decrease with increasing voltage in the range -60 to +40 mV. Closed times tend to increase at V > 20 mV. The open probability is low at all voltages and has an anomalous bell-shaped voltage dependence. We provide evidence that short and long reopenings of anomalous L-type channels correspond to two gating modes, whose relative probability depends on voltage. Positive voltages favor both the transition from a short-opening to a long-opening mode and the occupancy of a closed state outside the activation pathway within each mode from which the channel reopens upon repolarization. The voltage dependence of the probability of reopenings reflects the voltage dependence of the occupancy of these closed states, while the relative probability of long with respect to short reopenings reflects the voltage dependence of the equilibrium between modes. The anomalous gating persists after patch excision, and therefore our data rule out voltage-dependent block by diffusible ions as the basis for the anomalous gating and imply that a diffusible cytosolic factor is not necessary for voltage-dependent potentiation of anomalous L-type channels.     

A sequence in the carboxy-terminus of the alpha(1C) subunit important for targeting, conductance and open probability of L-type Ca(2+) channels

The role of the 80-amino acid motif 1572-1651 in the C-terminal tail of alpha(1C) Ca(2+) channel subunits was studied by comparing properties of the conventional alpha(1C,77) channel expressed in HEK-tsA201 cells to three isoforms carrying alterations in this motif. Replacement of amino acids 1572-1651 in alpha(1C,77) with 81 non-identical residues leading to alpha(1C,86) impaired membrane targeting and cluster formation of the channel. Similar to alpha(1C, 86), substitution of its 1572-1598 (alpha(1C,77L)) or 1595-1652 (alpha(1C,77K)) segments into the alpha(1C,77) channel yielded single-channel Ba(2+) currents with increased inactivation, reduced open probability and unitary conductance, when compared to the alpha(1C,77) channel. Thus, the C-terminal sequence 1572-1651 of the alpha(1C) subunit is important for membrane targeting, permeation and open probability of L-type Ca(2+) channels.     

Double-pulse facilitation of smooth muscle alpha 1-subunit Ca2+ channels expressed in CHO cells.

Frequent strong depolarizations facilitate Ca2+ channels in various cell types by shifting their gating behavior towards mode 2, which is characterized by long openings and high probability of being open. In cardiac cells, the same type of gating behavior is potentiated by beta-adrenoceptors presumably acting via phosphorylation of a protein identical to or associated with the channel. Voltage-dependent phosphorylation has also been reported to underlie Ca2+ channel facilitation in chromaffin adrenal medulla and in skeletal muscle cells. We studied a possible voltage-dependent facilitation of the principal channel forming alpha 1-subunit of the dihydropyridine-sensitive smooth muscle Ca2+ channel. Single channel and whole-cell Ca2+ currents were recorded in Chinese hamster ovary cells stably expressing the class Cb Ca2+ channel alpha 1-subunit. Strong depolarizing voltage-clamp steps preceding the test pulse resulted in a 2- to 3-fold increase of the single Ca2+ channel activity and induction of mode 2-like gating behavior. Accordingly we observed a significant potentiation of the whole-cell current by approximately 50%. In contrast to the previous suggestions we found no experimental evidence for involvement of channel phosphorylation by protein kinases (cAMP-dependent protein kinase, protein kinase C and other protein kinases utilizing ATP gamma S) in the control and facilitated current. The data demonstrate that the L-type Ca2+ channel alpha 1-subunit solely expressed in Chinese hamster ovary cells is subject to a voltage-dependent facilitation but not to phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Volume regulatory decrease in UMR-106.01 cells is mediated by specific α1 subunits of L-type calcium channels

An early cellular response of osteoblasts to swelling is plasma membrane depolarization, accompanied by a transient increase in intracellular calcium ([Ca2+]i), which initiates regulatory volume decrease (RVD). The authors have previously demonstrated a hypotonically induced depolarization of the osteoblast plasma membrane, sufficient to open L-type Ca channels and mediate Ca2+ influx. Herein is described the initiation of RVD in UMR-106.01 cells, mediated by hypotonically induced [Ca2+]i transients resulting from the activation of specific isoforms of L-type Ca channels. The authors further demonstrate that substrate interaction determines which specific alpha1 Ca channel subunit isoform predominates and mediates Ca2+ entry and RVD. Swelling-induced [Ca2+]i transients, and RVD in cells grown on a type I collagen matrix, are inhibited by removal of Ca from extracellular solutions, dihydropyridines, and antisense oligodeoxynucleotides directed exclusively to the alpha1C isoform of the L-type Ca channel. Ca2+ transients and RVD in cells grown on untreated glass cover slips were inhibited by similar maneuvers, but only by antisense oligodeoxynucleotides directed to the alpha1S isoform of the L-type Ca channel. This represents the first molecular identification of the Ca channels that transduce the initiation signal for RVD by osteoblastic cells.     

Functional expression and characterization of a voltage-gated CaV1.3 (alpha1D) calcium channel subunit from an insulin-secreting cell line.

L-type calcium channels mediate depolarization-induced calcium influx in insulin-secreting cells and are thought to be modulated by G protein-coupled receptors (GPCRs). The major fraction of L-type alpha1-subunits in pancreatic beta-cells is of the neuroendocrine subtype (CaV1.3 or alpha1D). Here we studied the biophysical properties and receptor regulation of a CaV1.3 subunit previously cloned from HIT-T15 cells. In doing so, we compared this neuroendocrine CaV1.3 channel with the cardiac L-type channel CaV1.2a (or alpha1C-a) after expression together with alpha2delta- and beta3-subunits in Xenopus oocytes. Both the current voltage relation and voltage dependence of inactivation for the neuroendocrine CaV1.3 channel were shifted to more negative potentials compared with the cardiac CaV1.2 channel. In addition, the CaV1.3 channel activated and inactivated more rapidly than the CaV1.2a channel. Both subtypes showed a similar sensitivity to the dihydropyridine (+)isradipine. More interestingly, the CaV1.3 channels were found to be stimulated by ligand-bound G(i)/G(o)-coupled GPCRs whereas a neuronal CaV2.2 (or alpha1B) channel was inhibited. The observed receptor-induced stimulation of CaV1.3 channels could be mimicked by phorbol-12-myristate-13-acetate and was sensitive to inhibitors of protein kinases, but not to the phosphoinositol-3-kinase-inhibitor wortmannin, pointing to serine/threonine kinase-dependent regulation. Taken together, we describe a neuroendocrine L-type CaV1.3 calcium channel that is stimulated by G(i)/G(o)-coupled GPCRs and differs significantly in distinct biophysical characteristics from the cardiac subtype (CaV1.2a), suggesting that the channels have different roles in native cells.     

Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by c-Src and protein kinase A pathways

L-type, voltage-gated Ca2+ channels (CaL) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle CaL channel isoforms are acutely potentiated following alpha5beta1 integrin activation. Only the alpha1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by alpha5beta1 integrin requires phosphorylation of alpha1C C-terminal residues Ser1901 and Tyr2122. These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following alpha5beta1 integrin activation, native CaL channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of CaL channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.     

Modulation of L-type Ca2+ channels by gbeta gamma and calmodulin via interactions with N and C termini of alpha 1C

Neuronal voltage-dependent Ca(2+) channels of the N (alpha(1B)) and P/Q (alpha(1A)) type are inhibited by neurotransmitters that activate G(i/o) G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of Gbetagamma subunit of G proteins to the channel. Since cardiac and neuronal L-type (alpha(1C)) voltage-dependent Ca(2+) channels are not modulated in this way, they are presumed to lack interaction with Gbetagamma. However, here we demonstrate that both Gbetagamma and calmodulin directly bind to cytosolic N and C termini of the alpha(1C) subunit. Coexpression of Gbetagamma reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca(2+), and is eliminated by EGTA. The N and C termini of alpha(1C) appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of Gbetagamma. Deletion of the N terminus profoundly impairs the Ca(2+)/calmodulin-dependent inactivation. We propose that Gbetagamma and calmodulin regulate the L-type Ca(2+) channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of alpha(1C).     

Mechanisms of p-methoxycinnamic acid-induced increase in insulin secretion

p-Methoxycinnamic acid (p-MCA) is a cinnamic acid derivative that shows various pharmacologic actions such as hepatoprotective and antihyperglycemic activities. The present study was to elucidate the mechanisms by which p-MCA increases [Ca2?]i and insulin secretion in INS-1 cells. p-MCA (100 μM) increased [Ca2?]i in INS-1 cells. The p-MCA-induced insulin secretion and rise in [Ca2?]i were markedly inhibited in the absence of extracellular Ca2? or in the presence of an L-type Ca2? channel blocker nimodipine. These results suggested that p-MCA increased Ca2? influx via the L-type Ca2? channels. Diazoxide, an ATP-sensitive K? channel opener, did not alter p-MCA-induced insulin secretion, nor [Ca2?]i response. In addition, p-MCA enhanced glucose-, glibenclamide-induced insulin secretion whereas it also potentiated the increase in insulin secretion induced by arginine, and Bay K 8644, an L-type Ca2? channel agonist. Taken together, our results suggest that p-MCA stimulated insulin secretion from pancreatic β-cells by increasing Ca2? influx via the L-type Ca2? channels, but not through the closure of ATP-sensitive K? channels.     

Regulation of L-type calcium channels in pituitary GH(4)C(1) cells by depolarization.

The neurosecretory anterior pituitary GH(4)C(1) cells exhibit the high voltage-activated dihydropyridine-sensitive L-type and the low voltage-activated T-type calcium currents. The activity of L-type calcium channels is tightly coupled to secretion of prolactin and other hormones in these cells. Depolarization induced by elevated extracellular K(+) reduces the dihydropyridine (+)-[(3)H]PN200-110 binding site density and (45)Ca(2+) uptake in these cells (). This study presents a functional analysis by electrophysiological techniques of short term regulation of L-type Ca(2+) channels in GH(4)C(1) cells by membrane depolarization. Depolarization of GH(4)C(1) cells by 50 mm K(+) rapidly reduced the barium currents through L-type calcium channels by approximately 70% and shifted the voltage dependence of activation by 10 mV to more depolarized potentials. Down-regulation depended on the strength of the depolarizing stimuli and was reversible. The currents recovered to near control levels on repolarization. Down-regulation of the calcium channel currents was calcium-dependent but may not have been due to excessive accumulation of intracellular calcium. Membrane depolarization by voltage clamping and by veratridine also produced a down-regulation of calcium channel currents. The down-regulation of the currents had an autocrine component. This study reveals a calcium-dependent down-regulation of the L-type calcium channel currents by depolarization.     

Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death

Sustained, mild K+ depolarization caused bovine chromaffin cell death through a Ca(2+)-dependent mechanism. During depolarization, Ca(2+) entered preferentially through L-channels to induce necrotic or apoptotic cell death, depending on the duration of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) signal, as proven by the following. (i) The L-type Ca(2+) channel activators Bay K 8644 and FPL64176, more than doubled the cytotoxic effects of 30 mm K+; (ii) the L-type Ca(2+) channel blocker nimodipine suppressed the cytotoxic effects of K+ alone or K+ plus FPL64176; (iii) the potentiation by FPL64176 of the K+ -evoked [Ca(2+)](c) elevation was totally suppressed by nimodipine. Cell exposure to K+ plus the L-type calcium channel agonist FPL64176 caused an initial peak rise followed by a sustained elevation of the [Ca(2+)](c) that, in turn, increased [Ca(2+)](m) and caused mitochondrial membrane depolarization. Cyclosporin A, a blocker of the mitochondrial transition pore, and superoxide dismutase prevented the apoptotic cell death induced by Ca(2+) overload through L-channels. These results suggest that Ca(2+) entry through L-channels causes both calcium overload and mitochondrial disruption that will lead to the release of mediators responsible for the activation of the apoptotic cascade and cell death. This predominant role of L-type Ca(2+) channels is not shared by other subtypes of high threshold voltage-dependent neuronal Ca(2+) channels (i.e. N, P/Q) expressed by bovine chromaffin cells.     

Galanin Reduces Carbachol Stimulation of Phosphoinositide Turnover in Rat Ventral Hippocampus by Lowering Ca2+ Influx Through Voltage-Sensitive Ca2+ Channels

The 29-amino-acid peptide galanin (GAL) caused concentration-dependent inhibition of the accumulation of 3H-inositol phosphates (3H-InsPs) induced by the muscarinic agonist carbachol (CARB; 10(-3)-10(-5) M) in the presence of 5 mM lithium, specifically in tissue miniprisms from rat ventral hippocampus. The inhibitory effect of GAL involved the mono-, bis-, tris-, and tetrakisphosphates formed during activation for 2 min of phospholipase C by CARB (1 mM) in the absence of lithium. GAL (1 microM) did not affect alpha-adrenergic or serotonergic type 2 receptor-mediated phosphoinositide (PI) breakdown in the same tissue. GAL by itself neither acted on basal levels of 3H-InsPs nor affected muscarinic receptors in binding studies. Blockade of the T-, N-, and L-types of voltage-sensitive calcium channel (VSCC) with 200 microM Cd2+ reduced muscarinic receptor-mediated PI breakdown by 50% and abolished the inhibitory effect of GAL (1 microM). Reduction of the extracellular Ca2+ concentration from 1.3 mM to 0.49 microM abolished the GAL inhibition of CARB-stimulated PI hydrolysis. Ca2+ influx promoted by 18 mM K+ depolarization or by 1 microM Bay K 8644, a selective agonist of the L-type VSCC, prevented the inhibitory effect of GAL. Blockade of the L-type VSCC with nifedipine (1 microM) potentiated the inhibitory effects of GAL without affecting muscarinic stimulation of PI breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)     

CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated integrator of Ca2+ signals for facilitation

Ca2+-dependent facilitation (CDF) of voltage-gated calcium current is a powerful mechanism for up-regulation of Ca2+ influx during repeated membrane depolarization. CDF of L-type Ca2+ channels (Ca(v)1.2) contributes to the positive force-frequency effect in the heart and is believed to involve the activation of Ca2+/calmodulin-dependent kinase II (CaMKII). How CaMKII is activated and what its substrates are have not yet been determined. We show that the pore-forming subunit alpha(1C) (Ca(v)alpha1.2) is a CaMKII substrate and that CaMKII interaction with the COOH terminus of alpha1C is essential for CDF of L-type channels. Ca2+ influx triggers distinct features of CaMKII targeting and activity. After Ca2+-induced targeting to alpha1C, CaMKII becomes tightly tethered to the channel, even after calcium returns to normal levels. In contrast, activity of the tethered CaMKII remains fully Ca2+/CaM dependent, explaining its ability to operate as a calcium spike frequency detector. These findings clarify the molecular basis of CDF and demonstrate a novel enzymatic mechanism by which ion channel gating can be modulated by activity.     

Multiple structural domains contribute to voltage-dependent inactivation of rat brain alpha(1E) calcium channels.

We have investigated the molecular determinants that mediate the differences in voltage-dependent inactivation properties between rapidly inactivating (R-type) alpha(1E) and noninactivating (L-type) alpha(1C) calcium channels. When coexpressed in human embryonic kidney cells with ancillary beta(1b) and alpha(2)-delta subunits, the wild type channels exhibit dramatically different inactivation properties; the half-inactivation potential of alpha(1E) is 45 mV more negative than that observed with alpha(1C), and during a 150-ms test depolarization, alpha(1E) undergoes 65% inactivation compared with only about 15% for alpha(1C). To define the structural determinants that govern these intrinsic differences, we have created a series of chimeric calcium channel alpha(1) subunits that combine the major structural domains of the two wild type channels, and we investigated their voltage-dependent inactivation properties. Each of the four transmembrane domains significantly affected the half-inactivation potential, with domains II and III being most critical. In particular, substitution of alpha(1C) sequence in domains II or III with that of alpha(1E) resulted in 25-mV negative shifts in half-inactivation potential. Similarly, the differences in inactivation rate were predominantly governed by transmembrane domains II and III and to some extent by domain IV. Thus, voltage-dependent inactivation of alpha(1E) channels is a complex process that involves multiple structural domains and possibly a global conformational change in the channel protein.     

Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells.

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.     

Blockade of GABAA receptor channels by niflumic acid prevents agonist dissociation

The modulation by the nonsteroidal anti-inflammatory drug niflumic acid (NFA) of the GABAA receptor-mediated currents was studied in acutely isolated cerebellar Purkinje cells using the whole-cell recording and fast drug application system. At concentrations of 3–300 μM NFA potentiated GABA (2 μM)-activated currents, and at concentrations of 1–3 mM NFA blocked these responses. The NFA-induced block was strongly voltage-dependent. Analysis of the voltage dependence of the block suggests that the blocking action of NFA is a result of NFA binding at the site located within GABAA channel pore. The termination of GABA and NFA application was followed by a transient increase of the inward current — “tail” current. These data suggest that NFA acts as a sequential open channel blocker, which prevents dissociation of agonist while the channel is blocked. The tail current develops because, prior to dissociation of agonist, the channels that are in the blocked state must return to the close state via the open state. The tail currents were compared in the presence and absence of gabazine, a competitive antagonist that also allosterically inhibits GABA_A receptors. Application of gabazine only during development of tail current did not change neither amplitude nor time course of this current, while noncompetitive antagonists picrotoxin and penicillin blocked it. Protection of tail current from gabazine block indicates that GABA cannot dissociate from the open-blocked state and the agonist was trapped on the receptor while the channel was open. Trapping was specific for the agonist, because the positive allosteric modulator zolpidem (benzodiazepine site agonist) was able to potentiate the tail current in the absence of GABA in the external solution. Our observations provide a model-independent functional support of the hypothesis that open channel block of GABAA channels by NFA prevents an escape of the agonist from its binding sites.