Christopher J. Partridge

Voltage-Gated Ion Channels in Human Pancreatic β-Cells: Electrophysiological Characterization and Role in Insulin Secretion

OBJECTIVE— To characterize the voltage-gated ion channels in human β-cells from nondiabetic donors and their role in glucose-stimulated insulin release. RESEARCH DESIGN AND METHODS— Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated β-cells. The ion channel complement was determined by quantitative PCR. RESULTS— Human β-cells express two types of voltage-gated K+ currents that flow through delayed rectifying (KV2.1/2.2) and large-conductance Ca2+-activated K+ (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of KV2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na+ currents (NaV1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na+ currents with TTX reduced glucose-stimulated (6–20 mmol/l) insulin secretion by 55–70%. Human β-cells are equipped with L- (CaV1.3), P/Q- (CaV2.1), and T- (CaV3.2), but not N- or R-type Ca2+ channels. Blockade of L-type channels abolished glucose-stimulated insulin release, while inhibition of T- and P/Q-type Ca2+ channels reduced glucose-induced (6 mmol/l) secretion by 60–70%. Membrane potential recordings suggest that L- and T-type Ca2+ channels participate in action potential generation. Blockade of P/Q-type Ca2+ channels suppressed exocytosis (measured as an increase in cell capacitance) by >80%, whereas inhibition of L-type Ca2+ channels only had a minor effect. CONCLUSIONS— Voltage-gated T-type and L-type Ca2+ channels as well as Na+ channels participate in glucose-stimulated electrical activity and insulin secretion. Ca2+-activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca2+ influx through P/Q-type Ca2+ channels.

A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse.

The neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25). The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic β-cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease.

R-type Ca 2+ -channel-evoked CICR regulates glucose-induced somatostatin secretion

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing β-cells and glucagon-secreting α-cells, the islets contain somatostatin-releasing δ-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K+-channels (KATP-channels) and a depolarization-evoked increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (≥10 mM) is unaffected by the KATP-channel activator diazoxide and proceeds normally in KATP-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca2+-induced Ca2+-release (CICR). This constitutes a novel mechanism for KATP-channel-independent metabolic control of pancreatic hormone secretion.

Cell coupling in mouse pancreatic beta-cells measured in intact islets of Langerhans.

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified β-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent β-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between β-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell (due to the firing of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of β-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to −55 mV) gave rise to a slow capacitive current indicative of coupling between β-cells, but not in non-β-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial β-cell in an islet is in electrical contact with six to seven other β-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell KATP channel conductance (GK,ATP) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC50 of approximately 4 mM. Theoretical considerations indicate that the coupling between β-cells within the islet is sufficient to allow propagation of [Ca2+]i waves to spread with a speed of approximately 80 μm s−1, similar to that observed experimentally in confocal [Ca2+]i imaging.

Impaired Insulin Exocytosis in Neural Cell Adhesion Molecule(-/-) Mice Due to Defective Reorganization of the Submembrane F-Actin Network

The neural cell adhesion molecule (NCAM) is required for cell type segregation during pancreatic islet organogenesis. We have investigated the functional consequences of ablating NCAM on pancreatic beta-cell function. In vivo, NCAM(-/-) mice exhibit impaired glucose tolerance and basal hyperinsulinemia. Insulin secretion from isolated NCAM(-/-) islets is enhanced at glucose concentrations below 15 mM but inhibited at higher concentrations. Glucagon secretion from pancreatic alpha-cells evoked by low glucose was also severely impaired in NCAM(-/-) islets. The diminution of insulin secretion is not attributable to defective glucose metabolism or glucose sensing (documented as glucose-induced changes in intracellular Ca(2+) and K(ATP)-channel activity). Resting K(ATP) conductance was lower in NCAM(-/-) beta-cells than wild-type cells, and this difference was abolished when F-actin was disrupted by cytochalasin D (1 muM). In wild-type beta-cells, the submembrane actin network disassembles within 10 min during glucose stimulation (30 mM), an effect not seen in NCAM(-/-) beta-cells. Cytochalasin D eliminated this difference and normalized insulin and glucagon secretion in NCAM(-/-) islets. Capacitance measurements of exocytosis indicate that replenishment of the readily releasable granule pool is suppressed in NCAM(-/-) alpha- and beta-cells. Our data suggest that remodeling of the submembrane actin network is critical to normal glucose regulation of both insulin and glucagon secretion.

Collagenase penetrates human pancreatic islets following standard intraductal administration.

Background. To optimize human islet isolation, it is important to improve our understanding of the collagenase digestion phase. Previous studies of collagenase action were mostly concerned with optimizing its composition, but the delivery and distribution of collagenase at the islet-exocrine interface is likely to be important for liberation of intact islets. The aim of this study was to characterize collagenase distribution in relation to islets in infused human pancreases. Methods. Human pancreases were retrieved from multiorgan donors with appropriate consent. Tissue samples were taken from the neck, body, and tail regions before and after collagenase infusion by manual syringe-loading (n=10) or recirculating perfusion (n=8), and snap frozen in liquid nitrogen. Frozen sections were immunolabeled for collagenase, insulin, CK19, collagen VI and CD31, then assessed by confocal microscopy. Results. Collagenase labeling was widespread throughout the pancreas, associated with collagen VI, and adjacent to CK19-labeled ducts. Collagenase was found within 67%±2% of islets (“intraislet”), associated with capillaries (CD31-positive). Intraislet collagenase was observed in 70%±3% of islets in the pancreatic tail, compared with 58%±2% and 53%±2% of islets in the body and neck, respectively (P<0.05 tail vs. neck), and was more prevalent in islets with diameters more than 150 &mgr;m (98%±1% of islets >150 &mgr;m vs. 52%±2% of islets <150 &mgr;m, P<0.05). There was no difference in intraislet collagenase labeling between perfused and syringe-loaded pancreases. Conclusions. Using current infusion techniques, collagenase penetrates the islet interior. This could cause islet fragmentation, and consequently, low islet yields. This study underlies the need to optimize collagenase delivery to preserve intact islets.

Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation.