ION PERMEATION THROUGH THE L-TYPE CA2- 2 SETS OF ION-BINDING SITES INTHE PORE( CHANNEL IN RAT PHEOCHROMOCYTOMA CELLS )

1. Both inward and outward unitary Li+ currents through the L-type Ca2 + channel and discrete block of such currents by either internal or ex ternal Ca2+ are recorded. Detailed kinetic analyses are obtained for a ll of the four experimental configurations (internal or external Ca2block of either inward or outward Li+ currents). 2. No matter from whi ch side the blocking Ca2+ ion comes, the exit (unblocking) rates are a lways the same at the same potential in the same direction of Li+ curr ent 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 (site s separated by insignificant barriers) in the pore. Furthermore, the d irection of exit of the blocking Ca2+ ion is always with, not against, the Li+ current flow. This suggests ion-ion interaction (the 'long po re effect') in the high-affinity sites. Therefore there must be more t han one high-affinity site in the pore. Overall it is concluded that t he pore must contain a set of high-affinity Ca2+ binding sites separat ed by insignificant energy barriers. 3. The voltage dependence of the off- (exit) rates is very similar in amplitude for all the four experi mental configurations (e-fold change per approximately 25 mV depolariz ation or hyperpolarization). This strong voltage dependence in every c onfiguration 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 Lion the exit of Ca2+. 4. The on-rates (blocking rates) for external Ca2 + are always fast no matter whether the Li+ currents are outward or in ward. In certain cases the rates even approach the diffusion-controlle d limit (approximately 10(9) m-1 s-1). This suggests that the high-aff inity sites are very easily accessible from the outside, and probably there is no other ionic site located between the external pore mouth a nd the high-affinity sites. 5. The on-rates for internal Ca2+ are fast and voltage independent in outward Li+ currents, but are very slow an d strongly voltage dependent in inward Li+ currents. The profound infl uence of Li+ current flow on the movements of Ca2+ again indicates ano ther 'long pore effect' region between the internal pore mouth and the high-affinity (blocking) sites, and the voltage dependence would sugg est the co-existence of one Ca2+ and one Li+ in this region. In other words, there must be at least two low-affinity (non-blocking) sites lo cated internal to the high-affinity sites in the pore. Therefore the L -type Ca2+ channel is a multi-ion pore with at least two different typ es (in terms of the affinity to Ca2+) of ion binding sites in the cond uction pathway.