| Summary: | The objective of these experiments was to investigate the actions of a
number of novel putative class III antiarrhythmic agents on the large conductance
calcium-activated potassium channel (BK ) in cultured hippocampal neurons.
The experiments were carried out in three steps. In the first set of
experiments (BK)ca channel, isolated from cultured rat hippocampal CA1 neuronal
somatic membrane, was first identified and its physiological and pharmacological
properties were characterized at a single channel level. This (BKca ) channel had an
average single channel conductance of 80 pS with physiological transmembrane
K+ (140 mM inside and 5 mM outside). It was very selective to K+over Na+, a
ratio of 100 to 7 was determined from this experiment. Calcium, at the internal
side of the membrane, was necessary to activate the BKca channel. With low
internal calcium concentration, depolarization could promote the rate of channel
openings. An e-fold increase in p0 was found with 1 µ M internal calcium and 15
mV depolarization. With 200 µM internal calcium, p0 was virtually independent of
voltage. 0.1 mM external TEA showed fast blockade of this channel. Internal TEA
and 4-AP (internal or external) showed no effect on BKca.
In the second set of experiments, the actions of putative class III drugs on
BKca was studied. The drugs were RP-62719, UK-68798, tedisamil (KC-8857) and
risotilide (WY-48986) at concentrations 0.1 ~ 10 µ M. All these agents, applied both
to the inside or to the outside of the patch membrane, resulted in the opening of
the BKca channel to exhibit rapid flicking from open to nonconducting levels. This
effect was dose-dependent and for KC-8857 and UK-68798 was evident at
concentrations of 0.1 µ M. The blocking rate constants were determined from a
simple open channel blockade scheme and were not dependent on voltages.
Single channel conductance and ionic selectivity were not affected by the drugs.
The potencies for channel block of the drugs acting either externally or internally were in the order UK-68798>tedisamil>RP-62719> risotilide with UK-68798
reducing the mean open time of BKca by one-half at a concentration near 0.4 µ M.
In the final set of experiments, the thermodynamics associated with RP-
62719 block BKca channel was studied in order to better understand the
molecular mechanisms of channel block. The Q10 associated with the channel
mean open time was found to be 2.2 with 5 µ M RP-62719 at the inner surface of
the patch membrane. The blocking and unblocking rate constants were
determined using the simple open channel block scheme. Thermodynamic
analysis, using transition rate theory, showed that the blocking rate constant was
associated with a large increase in entropy. The relatively high temperature
dependence for channel blockade was not consistent with a rate-limiting process
established by simple diffusion of the agent to a channel blocking site. Channel
block may involve conformational changes in the channel protein as a
consequence of hydrophobic interactions between drug and channel sites.
|
|---|
