SINGLE-MICROELECTRODE VOLTAGE-CLAMP MEASUREMENTS OF PANCREATIC BETA-CELL MEMBRANE IONIC CURRENTS IN-SITU

A conventional patch clamp amplifier was used to test the feasibility of measuring whole-cell ionic currents under voltage clamp conditions from beta-cells in intact mouse islets of Langerhans perifused with bi carbonate Krebs buffer at 37 degrees C. Cells impaled with a high resi stance microelectrode (ca. 0.150 G Ohm) were identified as beta-cells by the characteristic burst pattern of electrical activity induced by 11 mM glucose. Voltage-dependent outward K+ currents were enhanced by glucose both in the presence and absence of physiological bicarbonate buffer and also by bicarbonate regardless of the presence or absence o f glucose. For comparison with the usual patch clamp protocol, similar measurements were made from single rat beta-cells at room temperature ; glucose did not enhance the outward currents in these cells. Voltage -dependent inward currents were recorded in the presence of tetraethyl ammonium (TEA), an effective blocker of the K+ channels known to be pr esent in the beta-cell membrane. Inward currents exhibited a fast comp onent with activation-inactivation kinetics and a delayed component wi th a rather slow inactivation; inward currents were dependent on Ca2in the extracellular solution. These results suggest the presence of e ither two types of voltage-gated Ca2+ channels or a single type with f ast and slow inactivation. We conclude that it is feasible to use a si ngle intracellular microelectrode to measure voltage-gated membrane cu rrents in the beta-cell within the intact islet at 37 degrees C, under conditions that support normal glucose-induced insulin secretion and that glucose enhances an as yet unidentified voltage-dependent outward K+ current.