INHIBITION BY CALPONIN OF ISOMETRIC FORCE IN DEMEMBRANATED VASCULAR SMOOTH-MUSCLE STRIPS - THE CRITICAL ROLE OF SERINE-175

alpha-Calponin is a thin-filament-associated protein which has been im plicated in the regulation of smooth muscle contraction. Quantificatio n of the tissue content of rat tail arterial smooth muscle revealed ap proximately half the amount of alpha-calponin relative to actin compar ed with chicken gizzard and other smooth muscles, suggesting that this tissue would be particularly suitable for investigation of the effect s of exogenous alpha-calponin on the contractile properties of permeab ilized muscle strips. Rat tail arterial strips demembranated with Trit on X-100 retained approximate to 90% of their complement of alpha-calp onin, and exogenous chicken gizzard alpha-calponin (which conveniently has a slightly lower molecular mass than the rat arterial protein) bo und to the permeabilized muscle, presumably through its high affinity for actin. Exogenous alpha-calponin inhibited force in demembranated m uscle strips in a concentration-dependent manner when added at the pea k of a submaximal Ca2+-induced contraction, with a half-maximal effect at approximate to 3 mu M alpha-calponin. Pretreatment of demembranate d muscle strips with alpha-calponin inhibited subsequent force develop ment at all concentrations of Ca2+ examined over the activation range. The inhibitory effect of alpha-calponin was shown to be Ca2+-independ ent, since exogenous alpha-calponin also inhibited force in the absenc e of Ca2+ in demembranated muscle strips containing thiophosphorylated myosin. Phosphorylation of alpha-calponin on Ser-175 by protein kinas e C has been suggested to alleviate the inhibitory effect of alpha-cal ponin on smooth muscle contraction. To test this hypothesis, the effec ts on Ca2+-induced and Ca2+-independent contractions of demembranated muscle strips of phosphorylated alpha-calponin and three site-specific mutants of alpha-calponin (in which Ser-175 was replaced by Ala, Asp or Thr) were compared with the effects of unphosphorylated tissue-puri fied and recombinant wild-type alpha-calponins. The recombinant wild-t ype protein behaved identically to the unphosphorylated tissue-purifie d protein, as did the S175T mutant, which is known to bind actin with high affinity and to inhibit the actin-activated myosin MgATPase in vi tro. On the other hand, phosphorylated alpha-calponin and the S175A an d S175D mutants, which bind weakly to actin and have little effect on the actin-activated myosin MgATPase in vitro, failed to cause signific ant inhibition of force induced by Ca2+ or myosin thiophosphorylation. These results support a role for alpha-calponin in the regulation of smooth muscle contraction and indicate the functional importance of Se r-175 of alpha-calponin as a regulatory site of phosphorylation.