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

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 alpha 5 beta 1 integrin to modulate the activity of large conductance, Ca2+-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. Activation of alpha 5 beta 1 integrin by an appropriate, insoluble alpha 5 beta 1 antibody resulted in a 30-50% increase in the amplitude of iberiotoxin (IBTX)-sensitive, whole-cell K+ current. Current potentiation occurred 1-8 min after bead-antibody application to the cell surface. Similarly, the endogenous alpha 5 beta 1 integrin ligand fibronectin (FN) potentiated IBTX-sensitive K+ current by 26%. Current potentiation was blocked by the c-Src inhibitor PP2 but not by PP3 (0.1-1 mu M). In cell-attached patches, number of open channels x open probability (NPo) of a 230-250 pS K+ channel was significantly increased after FN application locally to the external surface of cell-attached patches through the recording pipette. In excised, inside-out patches, the same method of FN application led to large, significant increases in NPo and caused a leftward shift in the NPo-voltage relationship at constant [Ca2+]. 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 alpha 5 beta 1 integrin activation on both whole-cell and single-channel BK currents could be reproduced in HEK 293 cells expressing the BK channel alpha-subunit. This is the first demonstration at the single-channel level that integrin signalling can regulate an ion channel. Our results show that alpha 5 beta 1 integrin activation potentiates BK channel activity in vascular smooth muscle through both Ca2+- 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.