omega-agatoxin-iva and iberiotoxin

To characterize the voltage-gated ion channels in human beta-cells from nondiabetic donors and their role in glucose-stimulated insulin release.. Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated beta-cells. The ion channel complement was determined by quantitative PCR.. Human beta-cells express two types of voltage-gated K(+) currents that flow through delayed rectifying (K(V)2.1/2.2) and large-conductance Ca(2+)-activated K(+) (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of K(V)2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na(+) currents (Na(V)1.6/1.7) 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 beta-cells are equipped with L- (Ca(V)1.3), P/Q- (Ca(V)2.1), and T- (Ca(V)3.2), but not N- or R-type Ca(2+) channels. Blockade of L-type channels abolished glucose-stimulated insulin release, while inhibition of T- and P/Q-type Ca(2+) channels reduced glucose-induced (6 mmol/l) secretion by 60-70%. Membrane potential recordings suggest that L- and T-type Ca(2+) channels participate in action potential generation. Blockade of P/Q-type Ca(2+) channels suppressed exocytosis (measured as an increase in cell capacitance) by >80%, whereas inhibition of L-type Ca(2+) channels only had a minor effect.. Voltage-gated T-type and L-type Ca(2+) channels as well as Na(+) channels participate in glucose-stimulated electrical activity and insulin secretion. Ca(2+)-activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca(2+) influx through P/Q-type Ca(2+) channels.

Topics: Cells, Cultured; Cobalt; Electrophysiology; Gene Expression Profiling; Humans; Insulin; Insulin Secretion; Insulin-Secreting Cells; Islets of Langerhans; Large-Conductance Calcium-Activated Potassium Channels; omega-Agatoxin IVA; omega-Conotoxin GVIA; Peptides; Reverse Transcriptase Polymerase Chain Reaction; Scorpion Venoms

The types of Ca(2+)-dependent K(+) channel involved in the prolonged afterhyperpolarization (AHP) in a subgroup of sympathetic neurons have been investigated in guinea pig celiac ganglia in vitro. The conductance underlying the prolonged AHP (gKCa2) was reduced to a variable extent in 100 nM apamin, an antagonist of SK-type Ca(2+)-dependent K(+) channels, and by about 55% in 20 nM iberiotoxin, an antagonist of BK-type Ca(2+)-dependent K(+) channels. The reductions in gKCa2 amplitude by apamin and iberiotoxin were not additive, and a resistant component with an amplitude of nearly 50% of control remained. These data imply that, as well as apamin- and iberiotoxin-sensitive channels, other unknown Ca(2+)-dependent K(+) channels participate in gKCa2. The resistant component of gKCa2 was not abolished by 0.5-10 mM tetraethylammonium, 1 mM 4-aminopyridine, or 5 mM glibenclamide. We also investigated which voltage-gated channels admitted Ca(2+) for the generation of gKCa2. Blockade of Ca(2+) entry through L-type Ca(2+) channels has previously been shown to reduce gKCa2 by about 40%. Blockade of N-type Ca(2+) channels (with 100 nM omega-conotoxin GVIA) and P-type Ca(2+) channels (with 40 nM omega-agatoxin IVA) each reduced the amplitude of gKCa2 by about 35%. Thus Ca(2+) influx through multiple types of voltage-gated Ca(2+) channel can activate the intracellular mechanisms that generate gKCa2. The slow time course of gKCa2 may be explained if activation of multiple K(+) channels results from Ca(2+) influx triggering a kinetically invariant release of Ca(2+) from intracellular stores located close to the membrane.

Topics: 4-Aminopyridine; Animals; Apamin; Calcium; Calcium Channel Blockers; Calcium Channels; Female; Ganglia, Sympathetic; Glyburide; Guinea Pigs; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Male; Membrane Potentials; Neurons; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Calcium-Activated; Small-Conductance Calcium-Activated Potassium Channels; Tetraethylammonium