Block of the L-type Ca2+ channel pore by external and internal Mg2+ in rat phaeochromocytoma cells.

1. Three to eight micromolar external Mg2+ produces discrete block of the unitary inward currents through the L-type Ca2+ channel carried by 300 mM external Li+. Like the Ca2+ block, increasing Li+ concentration decreases the Mg2+ on-rate and increases the Mg2+ off-rate. 2. These kinetic changes are saturating and the apparent dissociation constant (Kd) for the on-rates in 75 mM Li+ (in activity), the same as that in the case of Ca2+ block. This suggests that Mg2+ and Ca2+ produce the discrete block at the same site. The apparent Kd for the off-rates is 300 mM, much smaller than that in the case of Ca2+ block. This indicates that Mg2+ exerts much less repulsion on the Li+ ion in the neighbouring (enhancement) site than Ca2+, although Mg2+ and Ca2+ both have two charges. The theoretical fits to the off-rates also suggest that Mg2+ can exit the blocking sites at a rate of several hundred per second in the absence of any enhancement effect. 3. Seventeen to forty-eight micromolar internal Mg2+ produces discrete block of the outward unitary currents carried by 300 mM internal Li+. The off-rates are in general approximately 20 times faster as compared to the Mg2+ off-rates in the inward currents. This finding suggests that Mg2+ in the high-affinity sites can much more easily exit to the outside than to the inside, implying significantly higher energy barriers on the inner side of the high-affinity sites for Mg2+. 4. At least 5-10 mM internal Mg2+ is needed to produce discrete block of the inward unitary currents carried by 215 mM external Na+. The off-rates in such experiments are generally the same as those in the case of external Mg2+ block of inward currents. This suggests that internal and external Mg2+ both reach the same site, namely the high-affinity Ca2+ binding sites in the pore, to produce the discrete block. 5. Other than discrete block, 5-10 mM internal Mg2+ also decreases the size of the inward unitary current. This is most probably due to a fast block at the more internally located low-affinity sites in the pore. The fractional decrease of the currents is voltage dependent and can be fitted by a rectangular hyperbola to calculate the apparent Kd, which increases e-fold per 45 mV hyperpolarization, indicating an electrical distance of 0.3 between the low-affinity sites and the internal pore mouth.(ABSTRACT TRUNCATED AT 400 WORDS)