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

Yan Yang,Xin Wu,Peichun Gui,Jianbo Wu,Jian-Zhong Sheng,Shizhang Ling,Andrew P Braun,George E Davis,Michael J Davis

doi:10.1074/jbc.m109.033506

Abstract

Large conductance, calcium-activated K(+) (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous alpha5beta1 integrin ligand, enhances BK channel current through both Ca(2+)- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel alpha-subunits (mSlo) are acutely potentiated following alpha5beta1 integrin activation. The effect occurs in a Ca(2+)-dependent manner, 1-3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca(2+) concentrations >or=1 microm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca(2+) sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by alpha5beta1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel alpha-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca(2+) sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions.

Highlights

Large conductance, Ca2ϩ-activated Kϩ channels (BK,3 Maxi-K) represent a subset of the superfamily of Kϩ-selective ion channels widely expressed in neurons, smooth muscle, and other tissues
In vascular smooth muscle (VSM), BK channels are important targets of endothelium-derived vasoactive factors, and their activation by depolarization and/or increases in intracellular calcium leads to smooth muscle hyperpolarization and vasodilation [1,2,3,4]
Integrins are a widely expressed class of adhesion receptor whose interactions with extracellular matrix (ECM) proteins lead to activation of signaling cascades, including protein tyrosine phosphorylation [9]

Summary

EXPERIMENTAL PROCEDURES

Construction of cDNA Plasmids—cDNAs encoding the murine brain BK channel ␣-subunit (mSlo) and green fluorescent protein (GFP) were separately subcloned into the polylinker region of the SV40 promoter-based mammalian expression plasmid SR␣ using standard techniques [7]. The washed slides were first incubated with mouse anti-human integrin ␣5␤1 monoclonal antibody (1:100) and rabbit anti-BK channel polyclonal antibody (1:100) for 60 min at room temperature. Cells were washed with PBS and incubated for 45 min at room temperature with Alexa Fluor 488 goat anti-rabbit IgG antibody to detect BK channels and with Alexa Fluor 568 goat anti-mouse IgG to label ␣5␤1 integrins. The nitrocellulose membrane was incubated with primary anti-src mouse monoclonal antibody 327 (1:200) in TTBS containing 1% skim milk powder for 1 h at room temperature, followed by 5-min washes with TTBS. Membranes were incubated for 1 h with secondary antibody (goat anti-mouse IgG) diluted (1:4000) in TTBS containing 1% skim milk powder, followed by 5-min washes with TTBS. Except as otherwise stated, were obtained from Sigma

RESULTS

Findings

DISCUSSION