|
Strobaek D, Christophersen P, Holm NR, Moldt P, Ahring PK,
Johansen TE, Olesen SP. Neuropharmacology. 1996;35(7):903-14.
Modulation of the Ca(2+)-dependent K+ channel, hslo, by the
substituted diphenylurea NS 1608, paxilline and internal Ca2+.
NeuroSearch A/S, Glostrup, Denmark.
The high-conductance Ca(2+)-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 alpha-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 Ca(2+)-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 microM 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 Ca(2+)-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 Ca(2+)-independent interaction with the
alpha-subunit of the channel.
PMID: 8938721 |
