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Characterization of the high-affinity Ca2+ binding sites in the L-type Ca2+ channel pore in rat phaeochromocytoma
Abstract:
1. Two major types of ion-ion interaction are demonstrated in the pore of the L-type Ca2+ channel, the 'lock-in' and the 'enhancement' effect. The former denotes that the ion at a certain site in the pore cannot move if the neighbouring site is occupied by the other ion. The latter denotes that the ion occupying a certain site may facilitate the exist of the other ion in the neighbouring site. 2. With inward currents carried by 300 mM external Li+, the exit rates of the blocking Ca2+ ion are decreased by approximately 4 times when the internal Li+ concentration is increased from 55 to 300 mM. With outward currents carried by 300 mM internal Li+, the exist rates of the blocking Ca2+ ion are decreased by approximately 2.5 times when the external Li+ concentration is increased from 55 to 300 mM. These findings demonstrate the 'lock-in' effect. 3. When inward currents are carried in Li+ in the cell-attached configuration, the on-rates of the external Ca2+ are decreased in a rectangular hyperbolic fashion with increasing external Li+ concentration (apparent dissociation constant (Kd) approximately 75 mM in activity), suggesting competition between Ca2+ and Li+ for a certain site. On the other hand, the off-rates of the blocking Ca2+ ion are increased linearly with increasing external Li+ concentration between 75 and 850 mM, and the line extrapolates to the zero point, indicating that Ca2+ exist is negligible at zero Li+. This finding demonstrates not only the existence of the enhancement effect in the channel, but also the indispensability of such an effect for Ca2+ to exit the pore. Moreover, a linear relationship up to 850 mM Li+ suggests that the affinity of Li+ to the enhancement sites is very low (apparent Kd very high) when a Ca2+ ion is present in the neighbouring site. 4. The unitary conductances of inward Ba2+ currents in the cell-attached configuration are increased with increasing Ba2+ concentration. The apparent Kd obtained from a rectangular hyperbolic fit to the data is approximately 6 mM Ba2+ (in activity). When inward Ba2+ currents are blocked by Cd2+, the on-rates of Cd2+ are decreased with increasing Ba2+ concentration also in a rectangular hyperbolic fashion, and the apparent Kd is approximately 5.5 mM. The similar Kd from these two different experiments suggests the high-affinity set can accommodate no more than two Ba2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)