Kevin D. Gillis

Protein Kinase C Enhances Exocytosis from Chromaffin Cells by Increasing the Size of the Readily Releasable Pool of Secretory Granules

We have used membrane capacitance measurements to assay Ca2+-triggered exocytosis in single bovine adrenal chromatin cells. Brief application of phorbol ester (PMA) enhances depolarization-evoked exocytosis severalfold while actually decreasing the Ca2+ current. Ca2+ metabolism is unchanged. Three different protocols were used to show that PMA increases the size of the readily releasable pool of secretory granules. PMA treatment leads to a large increase in amplitude, but little change in the time course of the exocytic burst that results from rapid elevation of [Ca2+]i upon photolysis of DMI-Nitrophen. Thus, PKC appears to affect a late step in secretion but not the Ca2+ sensitivity of the final step.

Techniques for Membrane Capacitance Measurements

Understanding the process whereby cells transduce an external signal to a secretory response (“stimulus—secretion coupling”: Douglas, 1968) has been an important topic of research for many years. The understanding of early events in the cascade in excitable cells, whereby an external signal evokes an electrical response mediated by ion channels, has certainly been revolutionized by the development of the patch-clamp technique. Extensions of the technique, however, have also provided surprising flexibility in reporting events late in the cascade whereby intracellular Ca2+ and other second messengers lead to exocytosis. In 1982, Neher and Marty reported that the patch-clamp technique together with basic impedance analysis could be used to monitor membrane (electrical) capacitance as a single-cell assay of exocytosis and endocytosis. Since exocytosis involves the fusion of secretory granule membrane with the plasma membrane and a corresponding increase in surface area, an increase in membrane capacitance is observed. The excess membrane is reclaimed in the process of endocytosis, which leads to a corresponding decrease in capacitance. Present techniques can detect changes in capacitance on the order of a femtofarad, allowing the fusion of single secretory granules with diameters greater that about 200 nm to be resolved. The temporal resolution possible is on the order of milliseconds (e.g., Breckenridge and Almers, 1987); therefore, capacitance-recording techniques can almost achieve the resolution of synaptic preparations, where the electrical response of a postsynaptic cell serves as a reporter of secretion. Resolution limits of membrane capacitance estimation techniques are discussed in greater detail in Section 5.

Electrophysiology of Stimulus-Secretion Coupling in Human β-Cells

Herein, we review the applicability to human β-cells of an electrophysiologically based hypothesis of the coupling of glucose metabolism to insulin secretion. According to this hypothesis, glucose metabolism leads to the generation of intracellular intermediates (including ATP), which leads to closure of ATP-sensitive K+ channels. Channel closure results in membrane depolarization, the onset of electrical activity, and voltage-dependent Ca2+ entry. The resultant rise in cytosolic Ca2+ leads to Ca2+-dependent exocytosis of insulin granules. We found that most of the published experimental evidence for human β-cells supports this hypothesis. In addition, we present three other emerging lines of evidence in support of this hypothesis for human islet β-cells: 1) the effects of pH1-altering maneuvers on insulin secretion and electrical activity; 2) preliminary identification of LVA and HVA single Ca2+ channel currents; and 3) validation of the feasibility of Cm measurements to track insulin granule exocytosis. On the basis of this last new line of evidence, we suggest that combinations of Cm measurements and electrical activity/membrane current measurements may help define the roles of diverse electrical activity patterns, displayed by human β-cells, in stimulus-induced insulin secretion.

A Highly Ca2+-sensitive Pool of Granules Is Regulated by Glucose and Protein Kinases in Insulin-secreting INS-1 Cells

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca2+ to directly measure the Ca2+ sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca2+ sensitivity of release in that a small proportion of granules makes up a highly Ca2+-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca2+. A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca2+ are not located close to Ca2+ channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca2+-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca2+, suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca2+ sensor that is poised to respond to modest, global elevations of [Ca2+]i.

A highly Ca2+-sensitive pool of vesicles is regulated by protein kinase C in adrenal chromaffin cells

We have used flash photolysis of caged Ca2+ and membrane capacitance measurements to probe exocytosis in chromaffin cells at low concentrations of intracellular Ca2+ ([Ca2+]i) (<10 μM). We observed a small pool of granules that is more sensitive to [Ca2+]i than the previously described “readily releasable pool.” Upon activation of PKC, this “highly Ca2+-sensitive pool” is enhanced in size to a greater extent than the readily releasable pool but is eliminated upon expression of a C-terminal deletion mutant (Δ9) of synaptosome-associated protein of 25 kDa (SNAP-25). Thus, in chromaffin cells, PKC enhances exocytosis both by increasing the number of readily releasable vesicles and by shifting vesicles to a highly Ca2+-sensitive state, enabling exocytosis at sites relatively distant from Ca2+ channels.

Design of a CMOS Potentiostat Circuit for Electrochemical Detector Arrays

High-throughput electrode arrays are required for advancing devices for testing the effect of drugs on cellular function. In this paper, we present design criteria for a potentiostat circuit that is capable of measuring transient amperometric oxidation currents at the surface of an electrode with submillisecond time resolution and picoampere current resolution. The potentiostat is a regulated cascode stage in which a high-gain amplifier maintains the electrode voltage through a negative feedback loop. The potentiostat uses a new shared amplifier structure in which all of the amplifiers in a given row of detectors share a common half circuit permitting us to use fewer transistors per detector. We also present measurements from a test chip that was fabricated in a 0.5-mum, 5-V CMOS process through MOSIS. Each detector occupied a layout area of 35 mumtimes15 mum and contained eight transistors and a 50-fF integrating capacitor. The rms current noise at 2-kHz bandwidth is ap110 fA. The maximum charge storage capacity at 2 kHz is 1.26times106 electrons

The Origin of Quantal Size Variation: Vesicular Glutamate Concentration Plays a Significant Role

Fusion of a single vesicle induces a quantal response, which is critical in determining synaptic strength. Quantal size varies at most synapses. Its underlying mechanisms are not well understood. Here, we examined five sources of variation: vesicular glutamate concentration ([Glu]v), vesicle volume, ultrafast fusion pore closure, the postsynaptic receptor, and the location between release and the postsynaptic receptor cluster at glutamatergic, calyx of Held synapses. By averaging 2.66 million fusion events from 459 synapses, we resolved the capacitance jump evoked by single vesicle fusion. This capacitance jump, an indicator of vesicle volume, was independent of the amplitude of the miniature EPSC (mEPSC) recorded simultaneously at the same synapses. Thus, vesicle volume is not the main source of mEPSC variation. The capacitance jump was not followed by submillisecond endocytosis, excluding ultrafast endocytosis as a source of variation. Larger mEPSCs were increased to a lesser extent by presynaptic glutamate dialysis, and reduced to a lesser extent by γ-DGG (γ-d-glutamylglycine), a competitive AMPA receptor blocker, suggesting that a higher glutamate concentration in the synaptic cleft contributes to the large size of mEPSCs. Larger mEPSCs were not accompanied by briefer rise times, inconsistent with the prediction by, and thus arguing against, the scenario that larger mEPSCs are caused by a shorter distance between the release site and the postsynaptic receptor cluster. In summary, the different amplitudes of mEPSCs were mainly attributable to release of vesicles having similar volumes, but different glutamate amounts, suggesting that [Glu]v is a main source of quantal size variation.

Perforated patch recording allows long-term monitoring of metabolite-induced electrical activity and voltage-dependent Ca2+ currents in pancreatic islet B cells

We describe the application of ‘perforated patch recording’ using the pore‐forming antibiotic nystatin, to monitor the electrical activity and underlying ionic currents of rat and human pancreatic islet B cells. We demonstrate that glucose‐induced electrical activity is seen even in single B cells during current‐clamp recordings lasting hours ‘L‐type’ Ca2+‐channel currents can also be monitored over this period of time. This technique may prove useful in examining hormone and neurotransmitter modulation of electrical activity in B cells, while minimizing the effects of cytoplasmic ‘wash‐out’.

Metabolite-regulated ATP-sensitive K+ channel in human pancreatic islet cells

In patch-clamped surface cells of human islets, we identified an inwardly rectifying, voltage-independent K+ channel that may be a crucial link between substrate metabolism and depolarization-induced insulin secretion. It is the major channel open at rest. It closes on exposure of the cell to secretagogue concentrations of glucose or other metabolic fuels and oral hypoglycemic sulfonylureas but reopens on addition of either a metabolic inhibitor that prevents substrate utilization or the hyperglycemic sulfonamide diazoxide. Onset of electrical activity coincides with channel closure by the secretagogues. In excised patches, the activity of this channel is inhibited at its cytoplasmic surface by ATP. These results suggest that in humans, as in rodents, 1) rises in cytoplasmic ATP levels during substrate metabolism trigger K+-channel closure and cell depolarization and 2) clinically useful sulfonamides modulate glucose-induced insulin secretion, in part by affecting a readily identifiable resting conductance pathway for K+.

Enhancers of cytosolic cAMP augment depolarization-induced exocytosis from pancreatic B-cells: evidence for effects distal to Ca2+ entry

SummaryWe have investigated the effects of cAMP enhancing agents on depolarization-induced membrane capacitance increases (ΔCm in single rat pancreatic B-cells. Concentrations of IBMX, 8-CPT cAMP and forskolin, which enhance cAMP and insulin release, all enhance depolarization-induced ΔCms seen in response to single voltage-clamp pulses and reduce the depression of ΔCm responses often seen with trains of pulses. These effects often occur in the absence of changes in peak Ca2+ current or the total Ca2+ charge entry during the depolarizing pulse. These data suggest that cAMP modulating maneuvers may directly affect the mechanism of insulin granule mobilization into a readily releasible store or fusion at a discharge site.