INTRACELLULAR CA2-MUSCLE L-TYPE CA2+ CHANNELS BY ACTIVATION OF PROTEIN PHOSPHATASE TYPE 2B AND BY DIRECT INTERACTION WITH THE CHANNEL( INHIBITS SMOOTH)

Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic C a2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and ru n down of Ca2+ channel activity in inside-out patches was prevented wi th calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells b y elevation of extracellular Ca2+ in the presence of the ionophore A23 187 inhibited the activity of L-type Ca2+ channels in cell-attached pa tches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-indu ced inhibition of Ca2+ channel activity in intact cells was due to a r eduction of channel open probability and availability. Ca2+-induced in hibition was not affected by the protein kinase inhibitor H-7 (10 mu M ) or the cytoskeleton disruptive agent cytochalasin B (20 mu M), but p revented by cyclosporin A (1 mu g/ml), an inhibitor of protein phospha tase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of in side-out patches inhibited Ca2+ channels with an IC50 of 2 mu M and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in c ell-free patches was due to a reduction of open probability, whereas a vailability was barely affected. Application of purified protein phosp hatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 mu M inhibited Ca2+ channel open probabi lity and availability. Elevation of cytoplasmic Ca2+ in the presence o f PP2B, suppressed channel activity in inside-out patches with an IC50 of similar to 380 nM and a Hill coefficient of similar to 3; i.e., ch aracteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanis ms. These mechanisms are based on (a) a protein phosphatase 2B-mediate d dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the cha nnel protein itself.