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The characterization of a large conductance potassium channel in cultured rat melanotrophs Wong, Kathy

Abstract

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 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.

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