Regulation of Ca<Superscript>2+</Superscript> influx in proliferating and differentiating murine myoblasts with participation of L-type Ca<Superscript>2+</Superscript> channels

The basic mechanisms of regulation of Ca²⁺ influx have been studied in murine myoblasts proliferating and differentiating in culture. The presence of L-type Ca²⁺ channels in proliferating myoblasts is shown for the first time. It is also shown that the influx of Ca²⁺ through these channels is regulated by the adrenergic system. The influx of Ca²⁺ after activation of the adrenergic system by addition of adrenaline has been estimated in comparison with the contribution of reticular stocks exhausted by ATP in calcium-free medium. The Ca²⁺ influx in proliferating myoblasts is regulated by β-2 adrenergic receptors whose action is mediated by adenylate cyclase through L-type calcium channels. In differentiating myoblasts, the adrenaline-induced Ca²⁺ influx is substantially lower than in proliferating cells, and maximal influx of Ca²⁺ may be reached only upon exhaustion of reticular stocks.     

Ca2+ influx through L-type Ca2+ channels controls the trailing tail contraction in growth factor-induced fibroblast cell migration

Growth factor-induced cell migration underlies various physiological and pathological processes. The mechanisms by which growth factors regulate cell migration are not completely understood. Although intracellular elevation of Ca2+ is known to be critical in cell migration, the source of this Ca2+ elevation and the mechanism by which Ca2+ modulates this process in fibroblast cells are not well defined. Here we show that increase of cellular Ca2+ through Ca2+ influx, rather than Ca2+ release from intracellular stores, is essential for growth factor-induced fibroblast cell migration. Voltage-gated L-type Ca2+ channels, previously known to exist in excitable cells such as neurons and muscle cells, are shown here to be present in fibroblasts as well. Furthermore, these channels are responsible for the Ca2+ influx. L-type Ca2+ channel inhibitors block growth factor-induced Ca2+ influx and fibroblast cell migration. One mechanism by which Ca2+ signals control cell migration is to regulate the contraction of the trailing edge of migrating fibroblasts; this process is controlled by the small GTPase Rho in fast migrating cells such as leukocytes. Downstream of Ca2+, both calmodulin and myosin light chain kinase, but not calcineurin, are involved leading to phosphorylation of the myosin light chain at the trailing end. Thus, trailing edge contraction is critically regulated by Ca2+ influx through L-type Ca2+ channels in growth factor-induced fibroblast cell migration.     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

Functional coupling of voltage-dependent L-type Ca2+ current to Ca2+-activated K+ current in pituitary GH3 cells

Ca2+-activated K+ currents (I(K(Ca)) can contribute to action potential repolarization and after-hyperpolarization in GH3 cells. In this study, we examined how the activation of I(K(Ca) at the cellular level could be functionally coupled to Ca2+ influx through L-type Ca2+ channels. A 30-msec Ca2+ influx step to 0 mV was found to exhibit substantial contribution of Ca2+ influx through the activation of I(Ca,L) to the activation of I(K(Ca)). A bell-shaped relationship between the conditioning potentials and the integrated I(K(Ca)) was observed, suggesting that the magnitude of integrated I(Ca,L) correlates well with that of integrated I(K(Ca)) in the same cell. A linear relationship of integrated I(Ca,L) and integrated I(K(Ca)) was found with a coupling ratio of 69+/-7. The value of the coupling ratio was unaffected by the presence of Bay K 8644 or nimodipine, although these compounds could effectively affect the amplitudes of both I(K(Ca)) and I(Ca,L). However, tetrandrine could decrease the coupling ratio. Paxilline or intracellular Ca2+ buffer with EGTA decreased the coupling ratio, while apamin had no effect on it. Interestingly, phorbol 12-myristate 13-acetate also reduced the coupling ratio significantly, whereas thapsigargin increased this value. Thus, the present study indicates that the activation of I(K(Ca)) during brief Ca2+ influx, which is inhibited by paxilline, is coupled to Ca2+ influx primarily through the L-type channels. The selective modulation of I(K(Ca)) by second messengers or Ca2+ release from internal stores may affect the coupling efficiency and hence cellular excitability.     

Subpopulation of store-operated Ca2+ channels regulate Ca2+-induced Ca2+ release in non-excitable cells

Ca2+-induced Ca2+ release (CICR) is a well characterized activity in skeletal and cardiac muscles mediated by the ryanodine receptors. The present study demonstrates CICR in the non-excitable parotid acinar cells, which resembles the mechanism described in cardiac myocytes. Partial depletion of internal Ca2+ stores leads to a minimal activation of Ca2+ influx. Ca2+ influx through this pathway results in an explosive mobilization of Ca2+ from the majority of the stores by CICR. Thus, stimulation of parotid acinar cells in Ca2+ -free medium with 0.5 microm carbachol releases approximately 5% of the Ca2+ mobilizable by 1 mm carbachol. Addition of external Ca2+ induced the same Ca2+ release observed in maximally stimulated cells. Similar results were obtained by a short treatment with 2.5-10 microm cyclopiazonic acid, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase pump. The Ca2+ release induced by the addition of external Ca2+ was largely independent of IP(3)Rs because it was reduced by only approximately 30% by the inhibition of the inositol 1,4,5-trisphosphate receptors with caffeine or heparin. Measurements of Ca2+ -activated outward current and [Ca2+](i) suggested that most CICR triggered by Ca2+ influx occurred away from the plasma membrane. Measurement of the response to several concentrations of cyclopiazonic acid revealed that Ca2+ influx that regulates CICR is associated with a selective portion of the internal Ca2+ pool. The minimal activation of Ca2+ influx by partial store depletion was confirmed by the measurement of Mn2+ influx. Inhibition of Ca2+ influx with SKF96365 or 2-aminoethoxydiphenyl borate prevented activation of CICR observed on addition of external Ca2+. These findings provide evidence for activation of CICR by Ca2+ influx in non-excitable cells, demonstrate a previously unrecognized role for Ca2+ influx in triggering CICR, and indicate that CICR in non-excitable cells resembles CICR in cardiac myocytes with the exception that in cardiac cells Ca2+ influx is mediated by voltage-regulated Ca2+ channels whereas in non-excitable cells Ca2+ influx is mediated by store-operated channels.     

Store-Operated Ca2+ Influx and Voltage-Gated Ca2+ Channels Coupled to Exocytosis in Pheochromocytoma (PC12) Cells

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+-evoked exocytosis was entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K+-evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+-free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca2+ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca2+ channels, but less than that caused by Ca2+ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage-gated Ca2+ channels.     

Ca2+ current activation rate correlates with alpha 1 subunit density.

We report here that L-type Ca2+ channels activate rapidly in myotubes expressing current at high density and slowly in myotubes expressing current at low density. Partial block of the current in individual cells does not slow activation, indicating that Ca2+ influx does not link activation rate to current density. Activation rate is positively correlated with the density of gating charge (Qmax) associated with the L-type Ca2+ channels. The range of values for Qmax, and the relationship between activation rate and Qmax, are similar for myotubes expressing native or recombinant L-type Ca2+ channels, whereas peak Ca2+ current density is approximately 3-fold higher for native channels. Taken together, these results suggest that Ca2+ channel density can govern activation kinetics. Our findings have important important implications for studies of ion channel function because they suggest that biophysical properties can be significantly influenced by channel density, both in heterologous expression systems and in native tissues.     

Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.     

Characterization of Ca2+ influx induced by dimethylphytosphingosine and lysophosphatidylcholine in U937 monocytes

Calcium is a ubiquitous second messenger controlling a broad range of cellular functions. We previously observed that N,N-dimethyl-D-ribo-phytosphingosine (DMPH) and lysophosphatidylcholine (LPC) induced Ca2+ influx across the plasma membrane in U937 monocytes. In this study, we characterized the Ca2+ influx induced by DMPH and LPC. L-type voltage-gated Ca2+ channel blockers, verapamil and nifedipine, significantly reduced LPC-induced Ca2+ influx, but not DMPH-induced one. On the other hand, non-specific Ca2+ channel blockers, Ga3+ and La3+, considerably reduced DMPH- and LPC-induced Ca2+ influx. Preincubation of the cells with forskolin enhanced DMPH-induced Ca2+ influx, however, LPC-induced Ca2+ influx was not affected by the treatment. The enhancement by forskolin was blocked by KT5720, a PKA inhibitor. We also confirmed the presence of TRPM7 and absence of TRPM3 in U937 cells. Therefore, our characterization of Ca2+ influx in U937 human monocytes shows the presence of two different types of Ca2+ channels modulated by lysolipid molecules, DMPH and LPC. LPC may induce Ca2+ influx via L-type Ca2+ channels and DMPH seems to induce Ca2+ influx through TRPM7 in U937 human monocytes.     

Role of Ca2+ and Ca2+-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L6. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca2+ transport into myoblasts. We have also demonstrated that a Ca2+-activated protease, CAF (mM), appears to relocate in response to the Ca2+ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca2+ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.     

Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization

The mechanism by which extracellular ATP stimulates insulin secretion was investigated in RINm5F cells. ATP depolarized the cells as demonstrated both by using the patch-clamp technique and a fluorescent probe. The depolarization is due to closure of ATP-sensitive K+ channels as shown directly in outside-out membrane patches. ATP also raised cytosolic Ca2+ [( Ca2+]i). At the single cell level the latency of the [Ca2+]i response was inversely related to ATP concentration. The [Ca2+]i rise is due both to inositol trisphosphate mediated Ca2+ mobilization and to Ca2+ influx. The former component, as well as inositol trisphosphate generation, were inhibited by phorbol myristate acetate which uncouples agonist receptors from phospholipase C. This manoeuvre did not block Ca2+ influx or membrane depolarization. Diazoxide, which opens ATP-sensitive K+ channels, attenuated membrane depolarization and part of the Ca2+ influx stimulated by ATP. However, the main Ca2+ influx component was unaffected by L-type channel blockers, suggesting the activation of other Ca2+ conductance pathways. ATP increased the rate of insulin secretion by more than 12-fold but the effect was transient. Prolonged exposure to EGTA dissociated the [Ca2+]i rise from ATP-induced insulin secretion, since the former was abolished and the latter only decreased by about 60%. In contrast, vasopressin-evoked insulin secretion was more sensitive to Ca2+ removal than the accompanying [Ca2+]i rise. Inhibition of phospholipase C stimulation by phorbol myristate acetate abrogated vasopressin but only reduced ATP-induced insulin secretion by 34%. These results suggest that ATP stimulates insulin release by both phospholipase C dependent and distinct mechanisms. The Ca2+)-independent component of insulin secretion points to a direct triggering of exocytosis by ATP.     

Rapid modulation of Ca2+ uptake in human jejunal enterocytes

The active metabolite of D vitamin, 1,25(OH)2D3, has been suggested to promote acute uptake of calcium through the intestinal lining in cell lines and murine models. In this study, the effects of D vitamin on the cytoplasmic Ca2+ of single human jejunal enterocytes, obtained with LOC-I-GUT technique, was analyzed in vivo in a fluorometric system using fura-2 as the Ca2+-sensing probe. Vitamin-promoted acute Ca2+ influx exhibited dual kinetics, indicating initial release from intracellular Ca2+ pools and fast entry from the extracellular space. Furthermore, providing a chemical clamp of membrane potential close to 0 mV did not activate voltage-sensitive calcium channels in the cellular membrane, neither was the hormone-induced Ca2+ influx affected by verapamil. This advocates that voltage-operated channels like L-type Ca2+ channels do not participate in the process of Ca2+ uptake. In fact, the existence of calcium-release-activated-calcium channels (I(CRAC)) was implied by the findings that irreversible depletion of intracellular Ca2+ stores by thapsigargin promoted Ca2+ entry. In the thapsigargin-treated enterocytes, D vitamin lost its ability to promote calcium entry indicating an important role for intracellular store-operated Ca2+ stores in the acute effects of 1,25(OH)2D3.     

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.     

Muscarinic acetylcholine receptors activate TRPC6 channels in PC12D cells via Ca2+ store-independent mechanisms

In this paper we report that stimulation of mAChRs in PC12D cells activates Ca2+ channels that are regulated independently of intracellular Ca2+ stores. In nominally Ca2+-free medium, exposure of PC12D cells to carbachol stimulates a robust influx of Ba2+, a Ca2+ substitute. This influx is blocked by atropine, but not by inhibitors of the nicotinic acetylcholine receptor or L-, N-, or T-type voltage-regulated Ca2+ channels. By contrast, depletion of intracellular Ca2+ stores with thapsigargin only weakly stimulates Ba2+ influx. Unlike store-operated Ca2+ channels (SOCCs), which close only after intracellular Ca2+ stores refill, channels mediating carbachol-stimulated Ba2+ influx rapidly close following the inactivation of mAChRs with atropine. Ba2+ influx is inhibited by extracellular Ca2+, by the Ca2+ channel blocker SKF-96365, and by activation of protein kinase C (PKC). Exogenous expression of antisense RNA encoding the rat canonical-transient receptor potential Ca2+ channel subtype 6 (TRPC6) or the N-terminal domain of TRPC6 blocks carbachol-stimulated Ba2+ influx in PC12D cells. Expression of TRPC6 antisense RNA or the TRPC6 N-terminal domain also blocks Ba2+ influx stimulated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a diacylglycerol analog previously shown to activate exogenously expressed TRPC6 channels. These data show that mAChRs in PC12D cells activate endogenous Ca2+ channels that are regulated independently of Ca2+ stores and require the expression of TRPC6.     

Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.     

Noradrenaline, vasopressin and angiotensin increase Ca2+ influx by opening a common pool of Ca2+ channels in isolated rat liver cells.

The effects of the Ca2+-mobilizing hormones noradrenaline, vasopressin and angiotensin on the unidirectional influx of Ca2+ were investigated in isolated rat liver cells by measuring the initial rate of 45Ca2+ uptake. The three hormones increased Ca2+ influx, with EC50 values (concentrations giving half-maximal effect) of 0.15 microM, 0.44 nM and 0.8 nM for noradrenaline, vasopressin and angiotensin respectively. The actions of noradrenaline and angiotensin were evident within seconds after their addition to the cells, whereas the increase in Ca2+ influx initiated by vasopressin was slightly delayed (by 5-15s). The activation of Ca2+ influx was maintained as long as the receptor was occupied by the hormone. The measurement of the resting and hormone-stimulated Ca2+ influxes at different external Ca2+ concentrations revealed Michaelis-Menten-type kinetics compatible with a saturable channel model. Noradrenaline, vasopressin and angiotensin increased both Km and Vmax. of Ca2+ influx. It is proposed that the hormones increase the rate of translocation of Ca2+ through a common pool of Ca2+ channels without changing the number of available channels or their affinity for Ca2+.     

The L-type voltage-gated Ca2+ channel is the Ca2+ sensor protein of stimulus-secretion coupling in pancreatic beta cells

L-type voltage-gated Ca2+ channels (Cav1.2) mediate a major part of insulin secretion from pancreatic beta-cells. Cav1.2, like other voltage-gated Ca2+ channels, is functionally and physically coupled to synaptic proteins. The tight temporal coupling between channel activation and secretion leads to the prediction that rearrangements within the channel can be directly transmitted to the synaptic proteins, subsequently triggering release. La3+, which binds to the polyglutamate motif (EEEE) comprising the selectivity filter, is excluded from entry into the cells and has been previously shown to support depolarization-evoked catecholamine release from chromaffin and PC12 cells. Hence, voltage-dependent trigger of release relies on Ca2+ ions bound at the EEEE motif and not on cytosolic Ca2+ elevation. We show that glucose-induced insulin release in rat pancreatic islets and ATP release in INS-1E cells are supported by La3+ in nominally Ca2+-free solution. The release is inhibited by nifedipine. Fura 2 imaging of dispersed islet cells exposed to high glucose and La3+ in Ca2+-free solution detected no change in fluorescence; thus, La3+ is excluded from entry, and Ca2+ is not significantly released from intracellular stores. La3+ by interacting extracellularlly with the EEEE motif is sufficient to support glucose-induced insulin secretion. Voltage-driven conformational changes that engage the ion/EEEE interface are relayed to the exocytotic machinery prior to ion influx, allowing for a fast and tightly regulated process of release. These results confirm that the Ca2+ channel is a constituent of the exocytotic complex [Wiser et al. (1999) PNAS 96, 248-253] and the putative Ca2+-sensor protein of release.