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

Abstract

(2), or as a negative feedback mechanism to limit Ca 2ϩ entry by hyperpolarizing the plasma membrane and closing voltagedependent Ca 2ϩ channels (3) previously opened by pressure (4) or vasoconstrictors like 5-hydroxytriptamine (5-HT) and Angiotensin II (AngII) (5, 6). In vitro evidence suggests that MaxiK also may play a role in vasoconstriction as it is inhibited by the potent constrictors AngII (7) and thromboxane A2 (8) in bilayers. However, the functional role of MaxiK channels in agonist-induced contraction has not been demonstrated.Pharmacomechanical and biochemical evidence indicate that one mechanism of agonist-induced contraction may involve tyrosine phosphorylation͞dephosphorylation with phosphorylation associated with vasoconstriction (9). However, most studies have been performed by using inhibitors with broad actions (e.g., tyrphostin and genistein) (10, 11). Using more selective inhibitors for Src-family tyrosine kinases, PP1 and PP2 (12) recent studies in rat aorta (13) and mesenteric arteries (14) show that 5-HT and AngII contractions involve a Src tyrosine kinase, likely c-Src. However, the downstream effector(s) of c-Src promoting vasoconstriction are unknown.We hypothesized that MaxiK may be a potential downstream effector of c-Src favoring vasoconstriction. This is based on the facts that both c-Src tyrosine kinase and MaxiK are particularly abundant in smooth muscles including the vasculature (15-18), and that Lavendustin A (LavA), a c-Src and Lck inhibitor (10,19), increases the activity of rat tail artery MaxiK (20). Here, we provide evidence showing that agonist-induced vasoconstriction by 5-HT, AngII, and phenylephrine involves inhibition of MaxiK channels by c-Src via direct phosphorylation of the channel protein. This new signaling pathway has a significant role in human and rat vasoconstriction, providing a link between electromechanical and pharmacomechanical coupling (21). Furthermore, the results indicate that MaxiK channels can function as a rheostat controlling both vasoconstriction and vasorelaxation. Experimental ProceduresTissue. Human coronary arteries were obtained from explanted hearts (University of California, Los Angeles, Medical Center). Male 3-mo-old F344 rats were used. Protocols received institutional approval.Isometric Contraction. Arterial rings (2.0-to 3.0-mm internal diameter, 3 mm long) without endothelium were stretched to an optimal resting tension (2 g, human coronaries; 1.2 g, rat aorta) and equilibrated for 60 min in Krebs solution. Percentage relaxation after drug application was calculated for tonic contractions from: percentage relaxation ϭ (max -min)͞(maxbasal) ϫ 100; where max ϭ maximal steady-state tension after constricting agonist stimulation, min ϭ minimal tension attained after drug application, basal ϭ basal tension prior constricting agonist stimulation. For phasic contractions, percentage relaxation ϭ (cycles͞h before drug application ͞cycles͞h after drug application ) ϫ 100. Because IbTx treatment abolished phasic contractions, in this case p...

α5β1 Integrin Engagement Increases Large Conductance, Ca2+-activated K+ Channel Current and Ca2+ Sensitivity through c-src-mediated Channel Phosphorylation

“…In apparent contrast to our finding that c-src overexpression enhances the Ca 2ϩ sensitivity of mSlo, the data of Alioua et al (29) suggest that BK channel phosphorylation by endogenous c-src leads to inhibition of BK channel activity in VSM. Although different isoforms of the BK channel (mSlo and hSlo) were used in our respective studies, the two channels have ϳ96% predicted amino acid identity (GenBank TM accession numbers U09383 and U11058) with differences occurring only at two splice insert sites that do not appear to be critical for c-src phosphorylation.…”

“…Alioua et al (29) found that c-src directly phosphorylated heterologously expressed hSlo and produced a rightward shift in the G-V relationship (29). A more likely difference between our results and those of Alioua et al (29) could be related to the degree of BK channel phosphorylation induced by c-src co-expression under both experimental conditions. As shown in Fig.…”

“…However, hSlo and mSlo channels exhibit several notable functional differences, including differences in their gating currents (30,31), differences in their voltage sensitivities in the absence of Ca 2ϩ (30,32,33), and an ϳ4-fold lower sensitivity of hSlo to charybdotoxin (34,35). Alioua et al (29) found that c-src directly phosphorylated heterologously expressed hSlo and produced a rightward shift in the G-V relationship (29). A more likely difference between our results and those of Alioua et al (29) could be related to the degree of BK channel phosphorylation induced by c-src co-expression under both experimental conditions.…”

“…This sequence of events serves as a negative feedback mechanism to limit Ca 2ϩ entry by closure of voltage-dependent Ca 2ϩ channels (5). The sensitivity of BK channels to intracellular Ca 2ϩ can be modulated, directly or indirectly, through channel phosphorylation by serine-threonine kinases and tyrosine kinases (8,28,29,56).…”

Association of β-catenin with the α-subunit of neuronal large-conductance Ca 2+ -activated K+ channels

“…In Drosophila, two cytosolic regulatory proteins, Slob and dSLIP, and two protein kinases, Src and PKAc, are able to assemble with the slo ␣-subunit (27)(28)(29). In vertebrates, however, only kinases, such as Src and Syk, have been reported to interact with this subunit (30)(31)(32). To seek other proteins that associate with K Ca channels and mediate their presynaptic targeting, we used yeast two-hybrid screening to search a cDNA library made from sensory epithelia of the chicken's cochlea.…”