EXPRESSION AND FUNCTION OF PANCREATIC BETA-CELL DELAYED RECTIFIER K- ROLE IN STIMULUS-SECRETION COUPLING( CHANNELS )

Voltage-dependent delayed rectifier K+ channels regulate aspects of bo th stimulus-secretion and excitation-contraction coupling, but assigni ng specific roles to these channels has proved problematic, Using tran sgenically derived insulinoma cells (beta TC3-neo) and beta-cells puri fied from rodent pancreatic islets of Langerhans, we studied the expre ssion and role of delayed rectifiers in glucose-stimulated insulin sec retion, Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expr ession of the K+ channel gene Kv2.1 in beta TC3-neo insulinoma cells a nd purified rodent pancreatic beta-cells was detected and confirmed by immunoblotting in the insulinoma cells, beta TC3-neo cells were also found to express a related K+ channel, Kv3.2, Whole-cell patch clamp d emonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar K-d values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents, The effect of these blockers on intracellular Ca2+ co ncentration ([Ca2+](i)) was studied with fura-2 microspectrofluorimetr y and imaging techniques, In the absence of glucose, exposure to TEA ( 1-20 mM) had minimal effects on beta TC3-neo or rodent islet [Ca2+](i) , but in the presence of glucose, TEA activated large amplitude [Ca2+] (i) oscillations. In the insulinoma cells the TEA-induced [Ca2+](i) os cillations were driven by synchronous oscillations in membrane potenti al, resulting in a 4-fold potentiation of insulin secretion, Activatio n of specific delayed rectifier K+ channels can therefore suppress sti mulus secretion coupling by damping oscillations in membrane potential and [Ca2+](i) and thereby regulate secretion, These studies implicate previously uncharacterized beta-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+](i), and insulin sec retion.