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 bicarbonate Krebs buffer at 37 degrees C. Cells impaled with a high resistance microelectrode (ca. 0.150 G omega) 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 of 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 tetraethylammonium (TEA), an effective blocker of the K+ channels known to be present in the beta-cell membrane. Inward currents exhibited a fast component with activation-inactivation kinetics and a delayed component with a rather slow inactivation; inward currents were dependent on Ca2+ in the extracellular solution. These results suggest the presence of either two types of voltage-gated Ca2+ channels or a single type with fast and slow inactivation. We conclude that it is feasible to use a single intracellular microelectrode to measure voltage-gated membrane currents 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.
Rojas
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Laboratory of Cell Biology and Genetics, National Institutes of Health, NIDDK, Bethesda, Maryland 20893.
Stokes
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CL
Mears
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Atwater
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D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D015220:Calcium Channels / Q000032:analysis / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D005947:Glucose / Q000494:pharmacology; D007515:Islets of Langerhans / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008839:Microelectrodes; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000032:analysis / Q000187:drug effects / Q000502:physiology*; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D001639:Bicarbonates; D015220:Calcium Channels; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D013757:Tetraethylammonium Compounds; D019789:Tetraethylammonium; D005947:Glucose