N-and P͞Q-type Ca 2؉ channels regulate a number of critical physiological processes including synaptic transmission and hormone secretion. These Ca 2؉ channels are multisubunit proteins, consisting of a pore-forming ␣1, and accessory ␤ and ␣2␦ subunits each encoded by multiple genes and splice variants. ␤ subunits alter current amplitude and kinetics. The ␤2a subunit is associated with slowed inactivation, an effect that requires the palmitoylation of two N-terminal cysteine residues in ␤2a. In the current manuscript, we studied steady state inactivation properties of native Nand P͞Q-type Ca 2؉ channels and recombinant N-type Ca 2؉ channels. When bovine ␣1B and ␤2a and human ␣2␦ were coexpressed in tsA 201 cells, we observed significant variations in inactivation; some cells exhibited virtually no inactivation as the holding potential was altered whereas others exhibited significant inactivation. A similar variability in inactivation was observed in native channels from bovine chromaffin cells. In individual chromaffin cells, the amount of inactivation exhibited by N-type channels was correlated with the inactivation of P͞Q-type channels, suggesting a shared mechanism. Our results with recombinant channels with known ␤ subunit composition indicated that inactivation could be dynamically regulated, possibly by alterations in ␤ subunit palmitoylation. Tunicamycin, which inhibits palmitoylation, increased steady-state inactivation of Ca 2؉ channels in chromaffin cells. Cerulenin, another drug that inhibits palmitoylation, also increased inactivation. Tunicamycin produced a similar effect on recombinant N-type Ca 2؉ channels containing ␤2a but not ␤2b or ␤2a subunits mutated to be palmitoylation deficient. Our results suggest that Ca 2؉ channels containing ␤2a subunits may be regulated by dynamic palmitoylation. V oltage-dependent Ca 2ϩ channels are multimeric membrane proteins critical for a wide variety of cellular functions, including synaptic transmission, cellular growth, differentiation, and migration. Ca 2ϩ channels are classified based on their biophysical, pharmacological, and, more recently, molecular properties. Functionally, Ca 2ϩ channels have been classified as L-, N-, P͞Q-, R-and T-type; ten different ␣ 1 genes, with multiple splice variants, have been identified and called ␣ 1A -␣ 1I and ␣ 1S (1, 2). Both N-and P͞Q-type Ca 2ϩ channels have been directly linked to neurotransmitter release (3-6) and are associated with synaptic release proteins (7-11). In chromaffin cells, N-type Ca 2ϩ channels, when activated by themselves, trigger catecholamine release (12). L-and P͞Q-type channels can also initiate catecholamine release from chromaffin cells. Many chromaffin cells express an N-type current that exhibits little inactivation during long depolarizations and does not show reduced channel availability at depolarized holding potentials (13). A similar noninactivating N-type current has been described in chick ciliary ganglion (14). However, most neuronal N-type channels show robust inactivation (3), which...

Synaptotagmin (syt) I is a Ca2+-binding protein that is well accepted as a major sensor for Ca2+-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as Ca2+ sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference. Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting Ca2+ and stimulated Ca2+ influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca2+ sensor for regulated release of transmitter.

General anesthetics produce anesthesia by depressing central nervous system activity. Activation of inhibitory GABA(A) receptors plays a central role in the action of many clinically relevant general anesthetics. Even so, there is growing evidence that anesthetics can act at a presynaptic locus to inhibit neurotransmitter release. Our own data identified the neurotransmitter release machinery as a target for anesthetic action. In the present study, we sought to examine the site of anesthetic action more closely. Exocytosis was stimulated by directly elevating the intracellular Ca(2+) concentration at neurotransmitter release sites, thereby bypassing anesthetic effects on channels and receptors, allowing anesthetic effects on the neurotransmitter release machinery to be examined in isolation. Three different PC12 cell lines, which had the expression of different release machinery proteins stably suppressed by RNA interference, were used in these studies. Interestingly, there was still significant neurotransmitter release when these knockdown PC12 cells were stimulated. We have previously shown that etomidate, isoflurane, and propofol all inhibited the neurotransmitter release machinery in wild-type PC12 cells. In the present study, we show that knocking down synaptotagmin I completely prevented etomidate from inhibiting neurotransmitter release. Synaptotagmin I knockdown also diminished the inhibition produced by propofol and isoflurane, but the magnitude of the effect was not as large. Knockdown of SNAP-25 and SNAP-23 expression also changed the ability of these three anesthetics to inhibit neurotransmitter release. Our results suggest that general anesthetics inhibit the neurotransmitter release machinery by interacting with multiple SNARE and SNARE-associated proteins.

Adaptor protein 3 (AP-3) is a vesicle-coat protein that forms a heterotetrameric complex. Two types of AP-3 subunits are found in mammalian cells. Ubiquitous AP-3 subunits are expressed in all tissues of the body, including the brain. In addition, there are neuronal AP-3 subunits that are thought to serve neuron-specific functions such as neurotransmitter release. In this study, we show that overexpression of neuronal AP-3 in mouse chromaffin cells results in a striking decrease in the neurotransmitter content of individual vesicles (quantal size), whereas deletion of all AP-3 produces a dramatic increase in quantal size; these changes were correlated with alterations in dense-core vesicle size. AP-3 appears to localize in the trans-Golgi network and possibly immature secretory vesicles, where it may be involved in the formation of neurosecretory vesicles.amperometry ͉ electron microscopy ͉ quantal content ͉ vesicle size ͉ chromaffin cells

Presynaptic N-type Ca 2؉ channels (Cav2.2, ␣1B) are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of the ␣1B subunit. Whether binding of syntaxin to the N-type Ca 2؉ channels is required for coupling Ca 2؉ ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, we deleted the synprint site from a recombinant ␣1B Ca 2؉ channel subunit and transiently transfected either the wild-type ␣1B or the synprint deletion mutant into mouse pheochromocytoma (MPC) cell line 9͞3L, a cell line that has the machinery required for rapid stimulated exocytosis but lacks endogenous voltage-dependent Ca 2؉ channels. Secretion was elicited by activation of exogenously transfected Ca 2؉ channel subunits. The current-voltage relationship was similar for the wild-type and mutant ␣1B-containing Ca 2؉ channels. Although total Ca 2؉ entry was slightly larger for the synprint deletion channel, compared with the wild-type channel, when Ca 2؉ entry was normalized to cell size and limited to cells with similar Ca 2؉ entry (Ϸ150 ؋ 10 6 Ca 2؉ ions͞pF cell size), total secretion and the rate of secretion, determined by capacitance measurements, were significantly reduced in cells expressing the synprint deletion mutant channels, compared with wild-type channels. Furthermore, the amount of endocytosis was significantly reduced in cells with the ␣1B synprint deletion mutant, compared with the wild-type subunit. These results suggest that the synprint site is necessary for efficient coupling of Ca 2؉ influx through ␣1B-containing Ca 2؉ channels to exocytosis. R elease of neurotransmitter from presynaptic vesicles is triggered by Ca 2ϩ influx through voltage-dependent Ca 2ϩ channels (1-3). Presynaptic vesicles are docked at release sites by binding of the vesicular protein synaptobrevin (also called VAMP) to two plasma membrane proteins, syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) (4-6). Collectively, these three proteins form the SNAP-25 receptor (SNARE) complex, which is an essential component of the exocytotic machinery (7). In addition to participating in SNARE complex formation, syntaxin and SNAP-25 bind to and modulate voltage-dependent Ca 2ϩ channels (8-10). The association of Ca 2ϩ channels with proteins of the SNARE complex was first demonstrated by coimmunoprecipitation of the N-type Ca 2ϩ channel subunits with syntaxin and synaptotagmin (11-13). Like other voltage-dependent Ca 2ϩ channels, the N-type channel is composed of at least three subunits: ␣ 1 , the pore-forming subunit, which is distinctive for each Ca 2ϩ channel subtype (␣ 1B for the N-type Ca 2ϩ channel), and two auxiliary subunits, ␣ 2 ␦ and ␤, which alter the voltage-dependent properties of the channels as well as surface expression of the channel complex (14,15 influx is efficiently coupled to secretion (9, 10). Consistent with this hypothesis, two recent papers by Mochida et al. (21,22) suggest...