Modulation of the Ca2+-dependent K+ Channel, hslo, by the Substituted Diphenylurea NS 1608, Paxilline and Internal Ca2+

Abstract

The high-conductance Ca2+-activated K channel (BK channel) is not only regulated by a number of physiological stimuli, but it is also sensitive to pharmacological modulation. We have stably expressed the α-subunit of the human BK channel, hslo, in HEK 293 cells and studied by patch-clamp technique how its gating is modulated by the channel activator NS 1608, by the selective channel blocker paxilline, as well as by changes in [Ca2+]i and Vm. The cells expressed 200–800 hslo channels per patch. The channel activity was determined by tail current analysis, and the activation curves were fitted to single Boltzmann functions, from which a gating charge for the hslo channel of 1.2 elementary charges was deduced. The hslo channel was very sensitive to changes in [Ca2+]i within the physiological range, whereas Ca2+-independent openings were seen at Ca2+ concentrations of 15 nM or below. NS 1608 shifted the hslo channel activation curve towards negative membrane potentials with an EC50 of 2.1 μM and a maximal shift of −74 mV. The channels activated by NS 1608 were sensitive to block by paxilline, but the two molecules apparently did not interact within the same site, since paxilline reduced the size of the tail current at all voltages, whereas NS 1608 shifted the activation curve along the voltage axis. Further, the effect of paxilline was Ca2+-sensitive, whereas NS 1608 elicited identical effects in the presence of either < 0.5 nM or 500 nM [Ca2+]i. NS 1608 hyperpolarized the cells by −50 to −70 mV, and paxilline depolarized them towards 0 mV. In addition to the effects on the steady state current NS 1608 also significantly influenced the non-stationary channel kinetics. In the presence of NS 1608 the time constants for deactivation of tail currents were more than tripled at all potentials. We have shown, that NS 1608 modulates steady-state BK currents and channel gating kinetics through a Ca2+-independent interaction with the α-subunit of the channel. Copyright © 1996 Elsevier Science Ltd