Intracellular Ca(2+) Inhibits Smooth Muscle L-Type Ca(2+) Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca(2+) channels by tonic elevation of cytoplasmic Ca(2+) was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba(2+) was used as charge carrier, and run down of Ca(2+) channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca(2+) in intact cells by elevation of extracellular Ca(2+) in the presence of the ionophore A23187 inhibited the activity of L-type Ca(2+) channels in cell-attached patches. Measurement of the actual level of intracellular free Ca(2+) with fura-2 revealed a 50% inhibitory concentration (IC(50)) of 260 nM and a Hill coefficient close to 4 for Ca(2+)- dependent inhibition. Ca(2+)-induced inhibition of Ca(2+) channel activity in intact cells was due to a reduction of channel open probability and availability. Ca(2+)-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca(2+) at the cytoplasmic side of inside-out patches inhibited Ca(2+) channels with an IC(50) of 2 μM and a Hill coefficient close to unity. Direct Ca(2+)-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca(2+) concentration of 1 μM inhibited Ca(2+) channel open probability and availability. Elevation of cytoplasmic Ca(2+) in the presence of PP2B, suppressed channel activity in inside-out patches with an IC(50) of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca(2+) sensitivity of Ca(2+) channels in intact cells. Our results suggest that L-type Ca(2+) channels of smooth muscle are controlled by two Ca(2+)-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca(2+) with a single membrane-associated site that may reside on the channel protein itself.