Intracellular Ca2+ inhibits smooth muscle L-type Ca2+ channels by activation of protein phosphatase type 2B and by direct interaction with the channel.
Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. 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+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 microM) or the cytoskeleton disruptive agent cytochalasin B (20 microM), but prevented by cyclosporin A (1 microg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 microM and a Hill coefficient close to unity. Direct Ca2+-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 Ca2+ concentration of 1 microM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of approximately 380 nM and a Hill coefficient of approximately 3; i.e., characteristics 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 mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.
Schuhmann
K
K
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, A-8010, Graz, Austria.
Romanin
C
C
Baumgartner
W
W
Groschner
K
K
D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D015220:Calcium Channels / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques
D015220:Calcium Channels; D016572:Cyclosporine; D019703:Calcineurin; D002118:Calcium