| Summary: | The characteristics of a large conductance potassium (BK) channel found in cultured
melanotrophs of the rat were studied. Single channel currents were recorded in cell-attached,
inside-out and outside-out configurations using conventional patch clamp techniques.
The current/voltage relationship was linear and revealed a single channel conductance of 261
±4 pS (mean±S.E.M.) with a reversal potential of -3.2±0.9 mV (n=7) in symmetrical potassium
solutions (150 mM). The BK channel was highly selective for potassium. In a physiological
potassium solution, (1^=3.5, Nao=140, K;=150) the permeability ratio for sodium to potassium was
<0.03. The permeability of the BK channel to ammonium ions and to the alkali metal ions, lithium,
rubidium and cesium was determined using bi-ionic solutions with complete substitution of
intracellular potassium by the test ions. The permeability sequence of these monovalent ions was
K+(l)>Rb+(0.87)>NH4
+(0.17)>>Na+(0.03)~Li+«Cs+ . Internal cesium also caused an intermediate
open channel block.
BK channel gating was sensitive both to the intracellular calcium concentration and
membrane potential. When open probability and membrane potential were fitted to the Boltzmann
equation, the half-activation potential, representing the membrane potential where the open
probability is half-maximal, was -72±7 mV, +39±19 mV and +104±14 mV in the 1 jiM (n=7),
0.5 yxM (n=5) and 0.1 uM (n=6) intracellular calcium solutions, respectively. This reveals high
sensitivity to intracellular calcium between the concentration range of 0.1 to 1 JUM. The
concentration of calcium required for a half-maximal open probability of the BK channel, at a
membrane potential of 0 mV, was estimated to be 0.6 uM.
Also from the Boltzmann fit, the voltage dependence was assessed from the values of the
slope factor. The slope factor, which represents the change in membrane potential required for an
e-fold change in open probability, was 12.3±1.4 mV, 13.6±2.6 mV, and 10.0±0.7 mV in the 1
juM, 0.5 uM and 0.1 JJM intracellular calcium solutions, respectively. Slope factors were not significantly affected by changes in intracellular calcium concentration.
Investigation of the effect of the potassium channel blocker tetraethylammonium (TEA+ ) on
outside-out patches revealed that exposure to external TEA+ caused an intermediate open channel
block. When fitted to the Hill equation the dissociation constant (KD), which represents the
concentration of T E A + required for half-maximal block, was determined to be 0.25 mM, with 95%
confidence limits (CL.) of 0.22-0.29 mM, at a membrane potential of 0 mV. The Hill coefficient,
h, which represents the number of molecules required to block the channel, was determined to be
0.81 (95% C L . 0.68-0.93), suggesting that a single molecule of T E A + was able to block the channel.
When h was constrained to be one, the K D was 0.24 mM (95% C L . 0.21-0.27 mM). Internal TEA+
caused a fast open channel block. The K D was 50.3 mM (95% C L . 44.8-56.0 mM) at a membrane
potential of 0 mV. The Hill coefficient was 0.92 (95% C L . 0.8-1.0), again suggesting that a single
molecule of T E A + is able to block the channel. When h was constrained to be one, the K D was 47.7
mM (95% C L . 44.6-50.6 mM). The effect of external 40 nM charybdotoxin was also examined on
the BK channel in the outside-out configuration. The toxin caused a slow open channel block.
In the rat melanotroph, the BK channel is likely to play a role in the repolarization of membrane
potential following an action potential. By assisting in the repolarization of the membrane potential,
the BK channel might decrease the calcium influx (required for secretion) through voltage-dependent
calcium channels and thereby indirectly assist in the cessation of hormone secretion. === Medicine, Faculty of === Cellular and Physiological Sciences, Department of === Graduate
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