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...

The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial glucagon‐secreting α‐cells in intact mouse pancreatic islets. α‐cells were distinguished from the β‐ and δ‐cells by the presence of a large TTX‐blockable Na+ current, a TEA‐resistant transient K+ current sensitive to 4‐AP (A‐current) and the presence of two kinetically separable Ca2+ current components corresponding to low‐ (T‐type) and high‐threshold (L‐type) Ca2+ channels. The T‐type Ca2+, Na+ and A‐currents were subject to steady‐state voltage‐dependent inactivation, which was half‐maximal at −45, −47 and −68 mV, respectively. Pancreatic α‐cells were equipped with tolbutamide‐sensitive, ATP‐regulated K+ (KATP) channels. Addition of tolbutamide (0·1 mm) evoked a brief period of electrical activity followed by a depolarisation to a plateau of −30 mV with no regenerative electrical activity. Glucagon secretion in the absence of glucose was strongly inhibited by TTX, nifedipine and tolbutamide. When diazoxide was added in the presence of 10 mm glucose, concentrations up to 2 μm stimulated glucagon secretion to the same extent as removal of glucose. We conclude that electrical activity and secretion in the α‐cells is dependent on the generation of Na+‐dependent action potentials. Glucagon secretion depends on low activity of KATP channels to keep the membrane potential sufficiently negative to prevent voltage‐dependent inactivation of voltage‐gated membrane currents. Glucose may inhibit glucagon release by depolarising the α‐cell with resultant inactivation of the ion channels participating in action potential generation.

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS–insensitive) component correlated with a rapid increase in membrane capacitance of ∼80 fF that plateaued within ∼200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K d values were 6 and 29 μM for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1−/− mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1−/− mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl− into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane KATP-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca2+-induced exocytosis.

The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer) 4 (FKBP12) 4 and (RyR2 protomer) 4 (FKBP12.6) 4 , respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35 S-labeled FKBP12 and 35 S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12.6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBPstripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.FK506 is a potent immunosuppressive drug that binds to a family of related intracellular receptors termed FK506-binding proteins, varying in size from 12 to 54 kDa. Among these FKBPs, 1 FKBP12 is the most abundant and is involved in mediating the immunosuppressive action of FK506 in T lymphocytes. All of the known FKBP family members display cis-trans peptidyl-prolyl isomerase (PPIase) activity that is inhibited by FK506 and a structurally related compound, rapamycin (1). However, neither the immunosuppressive nor toxic side effects (including severe neuro-and nephrotoxicity) associated with FK506 therapy result from the inhibition of PPIase activity. Rather, the action of FK506 results from the specific inhibition of calcineurin by the FKBP12⅐drug complex. Calcineurin is a calcium-dependent protein phosphatase involved in the activation of T-lymphocytes (2). FKBP12 is a mere bystander protein during T-cell activation that becomes involved in blocking activation after it binds FK506. To date, the only physiological function directly associated with FKBP12 is its tight binding to (3, 4) and modulation of the ryanodine receptor (RyR-1) or calcium release channel of skeletal muscle SR (5-10). The purified SkM CRC 2 isolated from the CHAPS-solubilized TC is a hetero-oligomeric complex, with the structural formula of (RyR-1 protomer) 4 (FKBP12) 4 (5). Both FK506 and rapamycin bind to and dissociate FKBP12 from the SkM CRC (5). Both the native and FKBP-stripped CRCs have similar unitary conductance and sensitivity to ruthenium red (6). However, in co...

␣-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of ␣-cells correlated with a small cell size measured as the average cell diameter (10 µm) and whole-cell capacitance (<4 pF). The ␣-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. ␣-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na + current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca 2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through -conotoxin GVIA (25%)-and nifedipine-sensitive (50%) Ca G lucagon is a major catabolic and hyperglycemic hormone of 29 amino acids and is secreted from the ␣-cells of the islets of Langerhans (1). Its main biological effect is the regulation of glucose metabolism by enhancing synthesis and mobilization of glucose in the liver. Normally, secretion of the hormone is stimulated by low blood glucose (2), amino acids (3), and a variety of hormones and neurotransmitters, such as adrenaline, glucose-dependent insulinotropic polypeptide, and glucagon-like peptide-1 (4,5). Hyperglycemia and fatty acids are the main inhibitors (6), but the islet hormones insulin and somatostatin (4) also appear to reduce glucagon secretion, possibly by a paracrine mechanism (7). Whereas insulin levels are inadequately low in hyperglycemic diabetic subjects, glucagon levels are actually elevated, and this increase exacerbates the disease (8). The reason for this abnormality is unknown, and studies on ␣-cells are complicated by the scarcity of islet tissue and the low occurrence of ␣-cells compared with ␤-cells. Therefore, how glucose physiologically regulates secretion in the ␣-cell remains unknown. Electrical activity in glucagon-secreting cells has been observed using several experimental approaches (5,9,10), and it is, at least in part, attributable to voltage-gated Ca 2+ channels. Available evidence also suggests that glucagon release is a Ca 2+ -dependent process. Indeed, capacitance measurements on single rat ␣-cells revealed a close relationship between N-type Ca 2+ channels and the secretory granules under basal conditions, whereas L-type Ca 2+ channels appeared more important when secretion was stimulated with adrenaline (5). The finding that glucagon secretion is Ca 2+ -dependent, coupled with the fact that glucagon release is suppressed by glucose, suggests that the ␣-cells must be electrically silent at elevated glucose concentrations, contrary to the situation in the ␤-cell. Thus, it is surprising that ATP-sensitive potassium channels (i.e...

A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain approximately 9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10 min stimulation with high K+. This stimulus depolarized the cell to -10 mV, elevated cytosolic [Ca2+] ([Ca2+](i)) from a basal concentration of 130 nM to a peak of 1.3 microM and released 0.5 ng insulin/islet, corresponding to 200-300 granules/cell. The Ca2+ transient decayed towards the prestimulatory concentration within approximately 200 s, presumably reflecting Ca2+ channel inactivation. Renewed stimulation with high K+ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca2+. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.

The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin‐secreting B‐cells and 65 % non‐B‐cells (A‐ and D‐cells). Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mm glucose and had the electrophysiological characteristics of isolated B‐cells maintained in tissue culture. The Ca2+ current recorded from B‐cells in situ was 80 % larger than that of isolated B‐cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. Voltage‐dependent K+ currents were observed during depolarisations to membrane potentials above −20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between −90 and −30 mV. The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP‐regulated K+ (KATP) channels, amounted to ≈4 nS. Glucose produced a concentration‐dependent reduction of KATP channel conductance with half‐maximal inhibition observed with 5 mm glucose. Combining voltage‐ and current‐clamp recording allowed the estimation of the gap junction conductance between different B‐cells. These experiments indicated that the input conductance of the B‐cell at stimulatory glucose concentrations (≈1 nS) is almost entirely accounted for by coupling to neighbouring B‐cells.