Coupling of c-Src to large conductance voltage- and Ca~(2+)-activated K~+ channels as a new mechanism of agonist-induced vasoconstriction

摘要

The voltage-dependent and Ca~(2+)-activated K~+ channel (MaxiK, BK) and the cellular proto-oncogene pp60~(c-src) (c-Src) are abundant proteins in vascular smooth muscle. The role of MaxiK channels as a vasorelaxing force is well established, but their role in vasoconstriction is unclear. Because Src participates in regulating vasoconstriction, we investigated whether c-Src inhibits MaxiK as a mechanism for agonist-induced vasoconstriction. Functional experiments in human and rat show that inhibitors of Src (Lavendustin A, PP2) but not inactive compounds (Lavendustin B, PP3) induce a pronounced relaxation of coronary or aortic smooth muscle precontracted with 5-hydroxytriptamine, phenylephrine, or Angiotensin II. Iberiotoxin, a MaxiK blocker, antagonizes the relaxation induced by Lavendustin A or PP2, indicating that c-Src inhibits the Iberiotoxin-sensitive component, likely MaxiK channels. In agreement, coronary muscle MaxiK currents were enhanced by Lavendustin A. To investigate the molecular mechanism of c-Src action on MaxiK channels, we transiently expressed its a subunit, hSIo, with or without c-Src in HEK293T cells. The voltage sensitivity of hSIo was right-shifted by ≈16 mV. hSIo inhibition by c-Src is due to channel direct phosphorylation because: (ⅰ) excised patches exposed to protein tyrosine phosphatase (CD45) resulted in a partial reversal of the inhibitory effect by ≈10 mV, and (ⅱ) immunoprecipitated hSIo channels were recognized by an anti-phosphotyrosine Ab. Furthermore, coexpression of hSIo and c-Src demonstrate a striking colocalization in HEK293T cells. We propose that MaxiK channels via direct c-Src-dependent phosphorylation play a significant role supporting vasoconstriction after activation of G protein-coupled receptors by vasoactive substances and neurotransmitters.