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.

Although N-and P-type Ca 2؉ channels predominant in fast-secreting systems, Lc-type Ca 2؉ channels (C-class) can play a similar role in certain secretory cells and synapses. For example, in retinal bipolar cells, Ca 2؉ entry through the Lc channels triggers ultrafast exocytosis, and in pancreatic ␤-cells, evoked secretion is highly sensitive to Ca 2؉ . These findings suggest that a rapidly release pool of vesicles colocalizes with the Ca 2؉ channels to allow high Ca 2؉ concentration and a tight coupling of the Lc channels at the release site. In binding studies, we show that the Lc channel is physically associated with synaptotagmin (p65) and the soluble N-ethylmaleimide-sensitive attachment proteins receptors: syntaxin and synaptosomal-associated protein of 25 kDa. Soluble N-ethylmaleimide-sensitive attachent proteins receptors coexpressed in Xenopus oocytes along with the Lc channel modify the kinetic properties of the channel. The modulatory action of syntaxin can be overcome by coexpressing p65, where at a certain ratio of p65͞syntaxin, the channel regains its unaltered kinetic parameters. The cytosolic region of the channel, Lc 753-893 , separating repeats II-III of its ␣1C subunit, interacts with p65 and ''pulls'' down native p65 from rat brain membranes. Lc 753-893 injected into single insulinsecreting ␤-cell, inhibits secretion in response to channel opening, but not in response to photolysis of caged Ca 2؉ , nor does it affect Ca 2؉ current. These results suggest that Lc 753-893 competes with the endogenous channel for the synaptic proteins and disrupts the spatial coupling with the secretory apparatus. The molecular organization of the Lc channel and the secretory machinery into a multiprotein complex (named excitosome) appears to be essential for an effective depolarization evoked exocytosis.Regulated secretion in synapses occurs at a fast speed from vesicles preassembled with N-and P-type voltage sensitive Ca 2ϩ channels (1). In contrast, in many neuroendocrine cells exocytosis triggered by Ca 2ϩ entry through Lc channel, is slower and persists for tens of milliseconds after Ca 2ϩ influx has stopped, implying that the vesicles are localized at a distance from the source of Ca 2ϩ entry (2-4). Although exocytosis is slow in various endocrine cells in which secretion is mediated by Lc channel (C-class), there are reports suggesting a close association of Lc channels with the exocytotic machinery (5-8). For example a combined study of amperometry and laser imaging in chromaffin cells have shown that the sites of Ca 2ϩ entry and catecholamine release are close (5, 6). Similarly, in mouse pancreatic ␤-cells, Lc channels have been shown to colocalize with insulin-containing secretory granules (7). Previously, we showed that the expression of syntaxin, synaptosomal-associated protein of 25 kDa (SNAP-25), and p65 along with the L-and N-type channel modify the kinetic properties of the channels (8-10). The N-type Ca 2ϩ channel binds syntaxin and SNAP-25 (11-14) at a site in the cytoplasmic domain of ␣1...

We have used an electrophysiological assay to investigate the functional interaction of syntaxin 1A and SNAP‐25 with the class C, L‐type, and the class B, N‐type, voltage‐sensitive calcium channels. Co‐expression of syntaxin 1A with the pore‐forming subunits of the L‐ and N‐type channels in Xenopus oocytes generates a dramatic inhibition of inward currents (>60%) and modifies the rate of inactivation (tau) and steady‐state voltage dependence of inactivation. Syntaxin 1–267, which lacks the transmembrane region (TMR), and syntaxin 2 do not modify channel properties, suggesting that the syntaxin 1A interaction site resides predominantly in the TMR. Co‐expression of SNAP‐25 significantly modifies the gating properties of L‐ and N‐type channels and displays modest inhibition of current amplitude. Syntaxin 1A and SNAP‐25 combined restore the syntaxin‐inhibited N‐type inward current but not the reduced rate of inactivation. Hence, a distinct interaction of a putative syntaxin 1A‐SNAP‐25 complex with the channel is apparent, consistent with the formation of a synaptosomal SNAP receptors (SNAREs) complex. The in vivo functional reconstitution: (i) establishes the proximity of the SNAREs to calcium channels; (ii) provides new insight into a potential regulatory role for the two SNAREs in controlling calcium influx through N‐ and L‐type channels; and (iii) may suggest a pivotal role for calcium channels in the secretion process.

No abstract

The secretion of neurotransmitters is a rapid Ca 21 -regulated process that brings about vesicle fusion with the plasma membrane. This rapid process (, 100 ms) involves multiple proteins located at the plasma and vesicular membranes. In recent years, multiple specialized proteins that mediate this tightly controlled process have been identi®ed, although their precise role in the process is still unclear (see reviews: Burgoyne et al. 1993;Bennett 1997;Goda 1997;Hanson et al. 1997;Stanley 1997;Bajjalieh 1999;Fossier et al. 1999). Three membrane proteins, syntaxin, synaptosomeassociated protein of 25 kDa (SNAP-25) and synaptobrevin (VAMP), also known as soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs) were shown to be key players in the fusion process. These proteins assemble with equal molar stoichiometry into a tight stable ternary complex. Three models have been postulated for the role of the SNARE proteins in secretion: the ternary complex together with a-SNAP and NSF form a 20S complex. This complex dissociates upon ATP hydrolysis and leads to membrane fusion via a mechanism that is still not fully understood (So Èllner et al. 1993;Pevsner et al. 1994;Hanson et al. 1997;Lin and Scheller 1997). This model was modi®ed and it now seems that after SNARE proteins form a complex and fusion takes place, dissociation of the complex through recruitment of a-SNAP and NSF, is needed as means of priming the SNAREs for another round of fusion (Hanson et al. 1997;Fasshauer et al. 1998). A more recent model suggests that Ca 21, through binding to a low-af®nity site, initiates pairing of SNARE proteins on apposing membranes, and the generated high-af®nity trans-SNARE complex leads to fusion of bilayers (Chen et al. 1999). This complex is stabilized by the interactions of the 972 q