Potentiation of large conductance, Ca²⁺‐activated K⁺ (BK) channels by α5β1 integrin activation in arteriolar smooth muscle

Abstract

Injury/degradation of the extracellular matrix (ECM) is associated with vascular wall remodelling and impaired reactivity, a process in which altered ECM-integrin interactions play key roles. Previously, we found that peptides containing the RGD integrin-binding sequence produce sustained vasodilatation of rat skeletal muscle arterioles. Here, we tested the hypothesis that RGD ligands work through α5β1 integrin to modulate the activity of large conductance, Ca 2+ -activated K + (BK) channels in arteriolar smooth muscle. K + currents were recorded in single arteriolar myocytes using whole-cell and single-channel patch clamp methods. ]. PP2 (but not PP3) nearly abolished the effect of FN on channel activity, suggesting that signalling between the integrin and channel involved an increase in Ca 2+ sensitivity of the channel via a membrane-delimited pathway. The effects of α5β1 integrin activation on both whole-cell and single-channel BK currents could be reproduced in HEK 293 cells expressing the BK channel α-subunit. This is the first demonstration at the single-channel level that integrin signalling can regulate an ion channel. Our results show that α5β1 integrin activation potentiates BK channel activity in vascular smooth muscle through both Ca 2+ -and c-Src-dependent mechanisms. This mechanism is likely to play a role in the arteriolar dilatation and impaired vascular reactivity associated with ECM degradation.

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

“…The presence of the ␤1 BK channel subunit may also alter the functional effect of c-src-mediated BK channel phosphorylation (36). Although we expressed only the BK channel ␣-subunit in this study, we found similar degrees of mSlo current potentiation and integrin-induced increased Ca 2ϩ sensitivity with or without co-expression of the ␤1 BK channel subunit (15); however, we did not test the role of c-src under those conditions.…”

“…␣5␤1 Integrin Activation Enhances BK Current-The activation of ␣5␤1 integrin by multivalent ligands such as FN or insoluble ␣5␤1 integrin antibody is associated with 30 -50% enhancement in whole-cell BK current in VSM cells (15). BK current enhancement could be reproduced with heterologously expressed mSlo channels ( Fig.…”

“…Potentiation of native BK channels in VSM by multivalent ␣5␤1 integrin ligands is significant at 1 min, peaks at 3-4 min, and is sustained for Ͼ8 min. The effect is blocked by the c-src inhibitor PP2 (15). Collectively, these observations suggest that the BK channel in VSM may be regulated by tyrosine kinases present in integrin-associated focal adhesion complexes.…”

“…At the same time, ␣5␤1 integrin activation leads to potentiation of L-type Ca 2ϩ current in VSM cells (in part through c-src (14)), Ca 2ϩ entry, and vasoconstriction (13). Although the effects on the two channels might appear to cancel each other, the time courses are different, with the potentiation of BK channels being more sustained (15) and developing somewhat slower than the potentiation of L-type Ca 2ϩ channels (13). Therefore, the net effect of ␣5␤1 integrin activation in VSM is probably to produce a transient increase in Ca 2ϩ influx that may subsequently trigger Ca 2ϩ release and other Ca 2ϩ -dependent intracellular signaling mechanisms.…”

“…Other studies have reported that BK channel activity is enhanced following direct channel phosphorylation by other src family kinases and by Pyk2 (proline-rich tyrosine kinase 2) (7,8). Recently, we found that the endogenous ␣5␤1 integrin ligand fibronectin (FN) potentiates BK channel current in VSM cells (15). Potentiation of native BK channels in VSM by multivalent ␣5␤1 integrin ligands is significant at 1 min, peaks at 3-4 min, and is sustained for Ͼ8 min.…”