α-Calponin is a thin-filament-associated protein which has been implicated in the regulation of smooth muscle contraction. Quantification of the tissue content of rat tail arterial smooth muscle revealed approximately half the amount of α-calponin relative to actin compared with chicken gizzard and other smooth muscles, suggesting that this tissue would be particularly suitable for investigation of the effects of exogenous α-calponin on the contractile properties of permeabilized muscle strips. Rat tail arterial strips demembranated with Triton X-100 retained ≈ 90% of their complement of α-calponin, and exogenous chicken gizzard α-calponin (which conveniently has a slightly lower molecular mass than the rat arterial protein) bound to the permeabilized muscle, presumably through its high affinity for actin. Exogenous α-calponin inhibited force in demembranated muscle strips in a concentration-dependent manner when added at the peak of a submaximal Ca2+-induced contraction, with a half-maximal effect at ≈ 3 µM α-calponin. Pretreatment of demembranated muscle strips with α-calponin inhibited subsequent force development at all concentrations of Ca2+ examined over the activation range. The inhibitory effect of α-calponin was shown to be Ca2+-independent, since exogenous α-calponin also inhibited force in the absence of Ca2+ in demembranated muscle strips containing thiophosphorylated myosin. Phosphorylation of α-calponin on Ser-175 by protein kinase C has been suggested to alleviate the inhibitory effect of α-calponin on smooth muscle contraction. To test this hypothesis, the effects on Ca2+-induced and Ca2+-independent contractions of demembranated muscle strips of phosphorylated α-calponin and three site-specific mutants of α-calponin (in which Ser-175 was replaced by Ala, Asp or Thr) were compared with the effects of unphosphorylated tissue-purified and recombinant wild-type α-calponins. The recombinant wild-type protein behaved identically to the unphosphorylated tissue-purified protein, as did the S175T mutant, which is known to bind actin with high affinity and to inhibit the actin-activated myosin MgATPase in vitro. On the other hand, phosphorylated α-calponin and the S175A and S175D mutants, which bind weakly to actin and have little effect on the actin-activated myosin MgATPase in vitro, failed to cause significant inhibition of force induced by Ca2+ or myosin thiophosphorylation. These results support a role for α-calponin in the regulation of smooth muscle contraction and indicate the functional importance of Ser-175 of α-calponin as a regulatory site of phosphorylation.

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Author notes

Present address: Department of Pharmacology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan.