Ion permeation through the L-type Ca2+ channel in rat phaeochromocytoma cells: two sets of ion binding sites in the pore.

AUTOR(ES)

Kuo, C C

RESUMO

1. Both inward and outward unitary Li+ currents through the L-type Ca2+ channel and discrete block of such currents by either internal or external Ca2+ are recorded. Detailed kinetic analyses are obtained for all of the four experimental configurations (internal or external Ca2+ block of either inward or outward Li+ currents). 2. No matter from which side the blocking Ca2+ ion comes, the exit (unblocking) rates are always the same at the same potential in the same direction of Li+ current flow. This indicates that the high-affinity Ca2+ binding site (the blocking site) is in the pore, and internal and external Ca2+ both go to the same site to produce the block. In other words, there can only be one high-affinity Ca2+ binding site or one set of such sites (sites separated by insignificant barriers) in the pore. Furthermore, the direction of exit of the blocking Ca2+ ion is always with, not against, the Li+ current flow. This suggests ion-ion interaction (the 'long pore effect') in the high-affinity sites. Therefore there must be more than one high-affinity site in the pore. Overall it is concluded that the pore must contain a set of high-affinity Ca2+ binding sites separated by insignificant energy barriers. 3. The voltage dependence of the off- (exit) rates is very similar in amplitude for all the four experimental configurations (e-fold change per approximately 25 mV depolarization or hyperpolarization). This strong voltage dependence in every configuration cannot be explained by any Ca2+ energy profile alone and must include a certain contribution from Li+. The mechanism underlying such a contribution seems to reside in the enhancement effect of Li+ on the exist of Ca2+. 4. The on-rates (blocking rates) for external Ca2+ are always fast no matter whether the Li+ currents are outward or inward. In certain cases the rates even approach the diffusion-controlled limit (approximately 10(9) M-1 S-1). This suggests that the high-affinity sites are very easily accessible from the outside, and probably there is no other ionic site located between the external pore mouth and the high-affinity sites. 5. The on-rates for internal Ca2+ are fast and voltage independent in outward Li+ currents, but are very slow and strongly voltage dependent in inward Li+ currents.(ABSTRACT TRUNCATED AT 400 WORDS)

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