Modulation of Ca(v)3.2 T-type Ca2+ channels by protein kinase C

Although T-type Ca2+ channels have been implicated in numerous physiological functions, their regulations by protein kinases have been obscured by conflicting reports. We investigated the effects of protein kinase C (PKC) on Ca(v)3.2 T-type channels reconstituted in Xenopus oocytes. Phorbol-12-myristate-13-acetate (PMA) strongly enhanced the amplitude of Ca(v)3.2 channel currents (approximately 3-fold). The augmentation effects were not mimicked by 4alpha-PMA, an inactive stereoisomer of PMA, and abolished by preincubation with PKC inhibitors. Our findings suggest that PMA upregulates Ca(v)3.2 channel activity via activation of oocyte PKC.     

Angiotensin II type 1 receptor activation modulates L- and T-type calcium channel activity through distinct mechanisms in bovine adrenal glomerulosa cells

In adrenal zona glomerulosa cells, calcium entry is crucial for aldosterone production and secretion. This influx is stimulated by increases of extracellular potassium in the physiological range of concentrations and by angiotensin II (Ang II). The high threshold voltage-activated (L-type) calcium channels have been shown to be the major mediators for the rise in cytosolic free calcium concentration, [Ca2+]c, observed in response to a depolarisation by physiological potassium concentrations. Paradoxically, both T- and L-type calcium channels have been shown to be negatively modulated by Ang II after activation by a sustained depolarisation. While the modulation of T-type channels involves protein kinase C (PKC) activation, L-type channel inhibition requires a pertussis toxin-sensitive G protein. In order to investigate the possibility of additional modulatory mechanisms elicited by Ang II on L-type channels, we have studied the effect of PKC activation or tyrosine kinase inhibition. Neither genistein or MDHC, two strong inhibitors of tyrosine kinases, nor the phorbol ester PMA, a specific activator of PKC, affected the Ang II effect on the [Ca2+]c response and on the Ba2+ currents elicited by cell depolarisation with the patch-clamp method. We propose a model describing the mechanisms of the [Ca2+]c modulation by Ang II and potassium in bovine adrenal glomerulosa cells.     

Inhibition of recombinant human T-type calcium channels by Delta9-tetrahydrocannabinol and cannabidiol

Delta(9)-Tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most prevalent biologically active constituents of Cannabis sativa. THC is the prototypic cannabinoid CB1 receptor agonist and is psychoactive and analgesic. CBD is also analgesic, but it is not a CB1 receptor agonist. Low voltage-activated T-type calcium channels, encoded by the Ca(V)3 gene family, regulate the excitability of many cells, including neurons involved in nociceptive processing. We examined the effects of THC and CBD on human Ca(V)3 channels stably expressed in human embryonic kidney 293 cells and T-type channels in mouse sensory neurons using whole-cell, patch clamp recordings. At moderately hyperpolarized potentials, THC and CBD inhibited peak Ca(V)3.1 and Ca(V)3.2 currents with IC(50) values of approximately 1 mum but were less potent on Ca(V)3.3 channels. THC and CBD inhibited sensory neuron T-type channels by about 45% at 1 mum. However, in recordings made from a holding potential of -70 mV, 100 nm THC or CBD inhibited more than 50% of the peak Ca(V)3.1 current. THC and CBD produced a significant hyperpolarizing shift in the steady state inactivation potentials for each of the Ca(V)3 channels, which accounts for inhibition of channel currents. Additionally, THC caused a modest hyperpolarizing shift in the activation of Ca(V)3.1 and Ca(V)3.2. THC but not CBD slowed Ca(V)3.1 and Ca(V)3.2 deactivation and inactivation kinetics. Thus, THC and CBD inhibit Ca(V)3 channels at pharmacologically relevant concentrations. However, THC, but not CBD, may also increase the amount of calcium entry following T-type channel activation by stabilizing open states of the channel.     

Diverse intracellular signalling systems used by growth hormone-releasing hormone in regulating voltage-gated Ca2+ or K channels in pituitary somatotropes

Influx of Ca2+ via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+, are regulated by GH-releasing hormone (GHRH) through G-protein-coupled intracellular signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured ovine and human somatotropes were recorded. Growth hormone-releasing hormone (10 nmol/L) increased both L- and T-type voltage-gated Ca2+ currents. Inhibition of the cAMP/protein kinase A (PKA) pathway by either Rp-cAMP or H89 blocked this increase in both L- and T-type Ca2+ currents. Growth hormone-releasing hormone also decreased voltage-gated transient (IA) and delayed rectified (IK) K+ currents. Protein kinase C (PKC) inhibitors, such as calphostin C, chelerythrine or downregulation of PKC, blocked the effect of GHRH on K+ currents, whereas an acute activation of PKC by phorbol 12, 13-dibutyrate (1 micromol/L) mimicked the effect of GHRH. Intracellular dialysis of a specific PKC inhibitor (PKC19-36) also prevented the reduction in K+ currents by GHRH. It is therefore concluded that GHRH increases voltage-gated Ca2+ currents via cAMP/PKA, but decreases voltage-gated K+ currents via the PKC signalling system. The GHRH-induced alteration of Ca2+ and K+ currents augments the influx of Ca2+, leading to an increase in [Ca2+]i and the GH secretion.     

Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice Leydig cells

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)](i) transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH.     

Roles of molecular regions in determining differences between voltage dependence of activation of CaV3.1 and CaV1.2 calcium channels

Voltage-dependent calcium channels are classified into low voltage-activated and high voltage-activated channels. We have investigated the molecular basis for this difference in voltage dependence of activation by constructing chimeras between a low voltage-activated channel (Ca(V)3.1) and a high voltage-activated channel (Ca(V)1.2), focusing on steady-state activation properties. Wild type and chimeras were expressed in oocytes, and two-electrode voltage clamp recordings were made of calcium channel currents. Replacement of domains I, III, or IV of the Ca 3.1 channel with the corresponding domain of Ca(V)1.2 led (V)to high voltage-activated channels; for these constructs the current/voltage (I/V) curves were similar to those for Ca(V)1.2 wild type. However, replacement of domain II gave only a small shift to the right of the I/V curve and modulation of the activation kinetics but did not lead to a high voltage-activating channel with an I/V curve like Ca 1.2. We also investigated the role of the voltage sensor (V)S4 by replacing the S4 segment of Ca(V)3.1 with that of Ca 1.2. For domain I, there was no shift in the I/V curve (V)as compared with Ca(V)3.1, and there were relatively small shifts to the right for domains III and IV. Taken together, these results suggest that domains I, III, and IV (rather than domain II) are apparently critical for channel opening and, therefore, contribute strongly to the difference in voltage dependence of activation between Ca 3.1 and Ca(V)1.2. However, the S4 segments in domains I, (V)III, and IV did not account for this difference in voltage dependence.     

Dihydropyridine-sensitive calcium channels from skeletal muscle. II. Functional effects of differential phosphorylation of channel subunits.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are multisubunit proteins and are regulated by protein phosphorylation. The purpose of this study was to determine: 1) which subunits are the preferential targets of various protein kinases when the channels are phosphorylated in vitro in their native membrane-bound state and 2) the consequences of these phosphorylations in functional assays. Using as substrates channels present in purified transverse (T) tubule membranes, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM protein kinase) preferentially phosphorylated the 165-kDa alpha 1 subunit to an extent that was 2-5-fold greater than the 52-kDa beta subunit. A protein kinase endogenous to the skeletal muscle membranes preferentially phosphorylated the beta peptide and showed little activity toward the alpha 1 subunit; however, the extent of phosphorylation was low. Reconstitution of partially purified channels into liposomes was used to determine the functional consequences of phosphorylation by these kinases. Phosphorylation of channels by PKA or PKC resulted in an activation of the channels that was observed as increases in both the rate and extent of Ca2+ influx. However, phosphorylation of channels by either the CaM protein kinase or the endogenous kinase in T-tubule membranes was without effect. Phosphorylation did not affect the sensitivities of the channels toward the dihydropyridines. Taken together, the results demonstrate that the alpha 1 subunit is the preferred substrate of PKA, PKC, and CaM protein kinase when the channels are phosphorylated in the membrane-bound state and that phosphorylation of the channels by PKA and PKC, but not by CaM protein kinase or an endogenous T-tubule membrane protein kinase, results in activation of the dihydropyridine-sensitive Ca2+ channels from skeletal muscle.     

Plasma membrane expression of T-type calcium channel alpha(1) subunits is modulated by high voltage-activated auxiliary subunits

It has been suggested that the auxiliary subunits of high voltage-activated (HVA) calcium channels modulate T-type, low voltage-activated (LVA) calcium channels. Such a regulation has yet to be documented, especially because there has been no biochemical characterization of T-channels. To monitor total protein levels and plasma membrane expression of T-channels in living cells, external epitopes (hemagglutinin, FLAG) were introduced into human recombinant Ca(V)3 channels that were also N-terminally fused to green fluorescent protein. Utilizing Western blot techniques, fluorescence flow cytometry, immunofluorescence, luminometry, and electrophysiology, we describe here that beta(1b) and alpha(2)-delta(1) subunits enhance the level of Ca(V)3 proteins as well as their plasma membrane expression in various expression systems. We also report that, in both Xenopus oocytes and mammalian cells, the alpha(2)-delta(1) subunits increase by at least and beta(1b) 2-fold the current density of Ca(V)3 channels with no change in the electrophysiological properties. Altogether, these data indicate that HVA auxiliary subunits modulate Ca(V)3 channel surface expression, suggesting that the membrane targeting of HVA and LVA alpha(1) subunits is regulated dynamically through the expression of a common set of regulatory subunits.     

Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.     

Beta-adrenergic modulation of cardiac ion channels. Differential temperature sensitivity of potassium and calcium currents

beta-Adrenergic stimulation of ventricular heart cells results in the enhancement of two important ion currents that regulate the plateau phase of the action potential: the delayed rectifier potassium channel current (IK) and L-type calcium channel current (ICa). The temperature dependence of beta-adrenergic modulation of these two currents was examined in patch-clamped guinea pig ventricular myocytes at various steps in the beta-receptor/cyclic AMP-dependent protein kinase pathway. External applications of isoproterenol and forskolin were used to activate the beta-receptor and the enzyme adenylate cyclase, respectively. Internal dialysis of cyclic 3',5'-adenosine monophosphate (cAMP) or the catalytic subunit of cAMP-dependent protein kinase (CS), as well as the external addition of 8-chlorphenylthio cAMP (CPT-cAMP) was applied to increase intracellular levels of cAMP and CS. Isoproterenol-mediated increases in IK, but not ICa, were found to be very temperature dependent over the range of 20-37 degrees C. At room temperature (20-22 degrees C) isoproterenol produced a large (threefold) enhancement of ICa but had no effect on IK. In contrast, at warmer temperatures (30-37 degrees C) both currents increased in the presence of this agonist and the kinetics of IK were slowed at -30 mV. A similar temperature sensitivity also existed after exposure to forskolin, CPT-cAMP, cAMP, and CS, suggesting that this temperature sensitivity of IK may arise at the channel protein level. Modulation of IK during each of these interventions was accompanied by a slowing in IK kinetics. Thus, regulation of cardiac potassium channels but not calcium channels involves a temperature-dependent step that occurs after activation of the catalytic subunit of cAMP-dependent protein kinase.     

Chemical determinants involved in anandamide-induced inhibition of T-type calcium channels

Anandamide, originally described as an endocannabinoid, is the main representative molecule of a new class of signaling lipids including endocannabinoids and N-acyl-related molecules, eicosanoids, and fatty acids. Bioactive lipids regulate neuronal excitability by acting on G-protein-coupled receptors (such as CB1) but also directly modulate various ionic conductances including voltage-activated T-type calcium channels (T-channels). However, little is known about the properties and the specificity of this new class of molecules on their various targets. In this study, we have investigated the chemical determinants involved in anandamide-induced inhibition of the three cloned T-channels: Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3. We show that both the hydroxyl group and the alkyl chain of anandamide are key determinants of its effects on T-currents. As follows, T-currents are also inhibited by fatty acids. Inhibition of the three Ca(V)3 currents by anandamide and arachidonic acid does not involve enzymatic metabolism and occurs in cell-free inside-out patches. Inhibition of T-currents by fatty acids and N-acyl ethanolamides depends on the degree of unsaturation but not on the alkyl chain length and consequently is not restricted to eicosanoids. Inhibition increases for polyunsaturated fatty acids comprising 18-22 carbons when cis-double bonds are close to the carboxyl group. Therefore the major natural (food-supplied) and mammalian endogenous fatty acids including gamma-linolenic acid, mead acid, and arachidonic acid as well as the fully polyunsaturated omega3-fatty acids that are enriched in fish oil eicosapentaenoic and docosahexaenoic acids are potent inhibitors of T-currents, which possibly contribute to their physiological functions.     

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.     

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.     

1α,25-Dihydroxyvitamin D(3) signaling pathways on calcium uptake in 30-day-old rat Sertoli cells

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.     

Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli.

The lipidic polymer, poly-3-hydroxybutyrate (PHB), is found in the plasma membranes of Escherichia col complexed to calcium polyphosphate (CaPPi). The composition, location, and putative structure of the polymer salt complexes led Reusch and Sadoff (1988) to propose that the complexes function as Ca2+ channels. Here we use bilayer patch-clamp techniques to demonstrate that voltage-activated Ca2+ channels composed of PHB and CaPPi are in the plasma membranes of E. coli. Single channel calcium currents were observed in vesicles of plasma membranes incorporated into planar bilayers of synthetic 1-palmitoyl, 2-oleoyl phosphatidylcholine. The channels were extracted from cells and incorporated into bilayers, where they displayed many of the signal characteristics of protein Ca2+ channels: voltage-activated selective for divalent over monovalent cations, permeant to Ca2+, manner by La3+, Co2+, Cd2+, and Mg2+, in that order. The channel-active extract, purified by size exclusion chromatography, was found to contain only PHB and CaPPi. This composition was confirmed by the observation of comparable single channel currents with complexes reconstituted from synthetic CaPPi and PHB, isolated from E. coli. This is the first report of a biological non-proteinaceous calcium channel. We suggest that poly-3-hydroxybutyrate/calcium polyphosphate complexes are evolutionary antecedents of protein Ca2+ channels.     

Modulation of cardiac Ca2+ channels in Xenopus oocytes by protein kinase C.

L-Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Ca2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4 beta-phorbol 12-myristate 13-acetate) were studied. Currents through channels composed of the main (alpha 1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the alpha 1 subunit was expressed in combination with alpha 2/delta, beta and/or gamma subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. beta subunit moderate the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L-type Ca2+ currents by PKC are mediated via an effect on the alpha 1 subunit, while the beta subunit may play a mild modulatory role.     

A Ca(v)3.2/syntaxin-1A signaling complex controls T-type channel activity and low-threshold exocytosis

T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.     

Regulation of one type of Ca2+ current in smooth muscle cells by diacylglycerol and acetylcholine

Electrophysiological recordings from freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus revealed two types of Ca2+ currents. One has a low threshold of activation and inactivates rapidly; the other has a high threshold of activation and inactivates more slowly. Acetylcholine (ACh) increased the high-threshold current but not the low-threshold current. The synthetic diacylglycerol analog sn-1,2-dioctanoylglycerol, an activator of protein kinase C (PKC), mimicked these effects of ACh on Ca2+ currents. However, another diacylglycerol analog, 1,2-dioctanoyl-3-thioglycerol, which has a closely related structure but does not activate PKC, failed to increase the Ca2+ current. The same was true of 1,2-dioctanoyl-3-chloropropanediol, an analog that even at high concentrations only minimally activates PKC. These results suggest that diacylglycerol may be the second messenger mediating the effects of ACh on one type of voltage-activated Ca2+ channel, possibly by activating PKC.     

Cch1 mediates calcium entry in Cryptococcus neoformans and is essential in low-calcium environments

The ability of Cryptococcus neoformans to grow at the mammalian body temperature (37 degrees C to 39 degrees C) is a well-established virulence factor. Growth of C. neoformans at this physiological temperature requires calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase. When cytosolic calcium concentrations are low ( approximately 50 to 100 nM), calcineurin is inactive and becomes active only when cytosolic calcium concentrations rise ( approximately 1 to 10 microM) through the activation of calcium channels. In this study we analyzed the function of Cch1 in C. neoformans and found that Cch1 is a Ca(2+)-permeable channel that mediates calcium entry in C. neoformans. Analysis of the Cch1 protein sequence revealed differences in the voltage sensor (S4 regions), suggesting that Cch1 may have diminished voltage sensitivity or possibly an alternative gating mechanism. The inability of the cch1 mutant to grow under conditions of limited extracellular calcium concentrations ([Ca(2+)](extracellular), approximately 100 nM) suggested that Cch1 was required for calcium uptake in low-calcium environments. These results are consistent with the role of ScCch1 in mediating high-affinity calcium uptake in Saccharomyces cerevisiae. Although the growth defect of the cch1 mutant under conditions of limited [Ca(2+)](extracellular) ( approximately 100 nM) became more severe with increasing temperature (25 degrees C to 38.5 degrees ), this temperature sensitivity was not observed when the cch1 mutant was grown on rich medium ([Ca(2+)](extracellular), approximately 0.140 mM). Accordingly, the cch1 mutant strain displayed only attenuated virulence when tested in the mouse inhalation model of cryptococcosis, further suggesting that C. neoformans may have a limited requirement for Cch1 and that this requirement appears to include ion stress tolerance.