Hyperpolarization-activated inward potassium and calcium-sensitive chloride currents in beating pacemaker insect neurosecretory cells (dorsal unpaired median neurons)

Abstract

Hyperpolarization-activated inward currents were studied in single adult cockroach Periplaneta americana pacemaker neurosecretory cells, identified as dorsal unpaired median neurons using the whole-cell patch-clamp technique. Under current clamp, injection of negative current produced a hyperpolarization of the cell membrane with a sag in the membrane potential toward the resting value. Under voltage clamp, the whole-cell current–voltage relationship exhibited an unexpected biphasic aspect. The global hyperpolarization-activated inward current could be dissociated by means of 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid and tetraethylammonium chloride sensitivity, ionic selectivity, voltage dependence and activation threshold as inward potassium and calcium-sensitive chloride currents. The inward potassium current was activated around −80 mV. The reversal potential followed the potassium equilibrium potential when the extracellular potassium concentration was raised. This current was not dependent on the external sodium concentration and was sensitive to 10 mM tetraethylammonium chloride or 5 mM barium chloride. The hyperpolarization-activated inward calcium-sensitive chloride current was activated in a range of potential 20 mV more positive than the potassium current. The estimated reversal potential (−71 mV) was very close to the equilibrium potential for chloride ions (−73 mV). Intracellularly applied 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and external application of 1 mM zinc chloride, calcium-free saline or high concentrations of intracellular 1,2-bis-(2-aminophenoxy)ethane- N, N, N′, N′-tetra-acetate blocked the inward chloride current. Current-clamp experiments indicated that the inward potassium current accounted for inward rectification of dorsal unpaired median neurons. Our findings report, for the first time in pacemaker neurosecretory cells, the co-existence of two distinct hyperpolarization-activated inward currents which have specialized function in pacemaker activity.