The role of Kv7 channels in G-protein βγ subunit dependent vasorelaxations

J. B. Stott, I. A. Greenwood. Vascular Research Group, St George's University of London, London, UNITED KINGDOM

Introduction: Kv7 channels are important mediators of vascular responsiveness, including relaxations induced by G-protein coupled receptor (GPCR) stimulation. Activation of GPCRs promotes dissociation of heterotrimeric G-proteins (GÉ‘βγ) associated with the GPCR complex, releasing the GÉ‘ subunit from the Gβγ subunits. Both GÉ‘ and Gβγ subunits promote numerous intracellular signalling events. Whilst the pathways downstream of GÉ‘s which promote vasorelaxation are well characterised, the role of Gβγ subunit signalling in these events is not clear. Here, we investigate the role of Gβγ signalling in vasorelaxations via Kv7 channels in the vasculature.

Method: Renal and mesenteric arteries (RA and MA, respectively) were dissected from male Wistar rats (175-225g) culled by cervical dislocation, in accordance with the Animals (Scientific Procedures) Act 1986. RA and MA were mounted for isometric tension recording on pin/wire myographs (Danish Myograph Technologies). RA were preconstricted with 3µM methoxamine, and MA with 300nM U46619. Relaxations to mSIRK (to release Gβγ subunits) and isoprenaline were conducted in the presence and absence of 10µM linopirdine (Kv7 blocker) and 100µM gallein (Gβγ inhibitor). Proximity ligation assays (PLA) were performed on isolated RA and MA myocytes which were stimulated with 1µM isoprenaline (90s) in the presence or absence of 100µM gallein (30min), as per the manufacturers’ instructions.

Results: mSIRK produced relaxations of MA and RA (36.1±2.8% maximal relaxation, n=7) that were inhibited in the presence of linopirdine (13.1±2.2% maximal relaxation, n=7). Dose dependent relaxations to isoprenaline were sensitive to linopirdine in both vessels (from 79.8±6.3% to 24.3±11.4% max relaxation in RA, n=6, and from 89.9±5% to 57.5±8.7% in MA, n=7), but were sensitive to gallein only in the RA (from 76.5±10% to 33.2±12.4% max relaxation in RA, n=6, and from 71.2±11.2% to 83.1±4.9% in MA, n=8). PLA assessed the localisation of Gβ subunits with Kv7.4 in isolated RA and MA myocytes before and after treatment with isoproterenol and gallein. RA displayed a high level of basal Kv7.4-Gβ PLA puncta (7.1±0.6puncta/cell, n=21) which did not increase with isoprenaline stimulation (8.5±0.4puncta/cell, n=12), but was reduced after gallein treatment (3±0.7puncta/cell, n=15). MA displayed low basal Kv7.4-Gβ PLA puncta (3.8±0.5puncta/cell, n=28) which increased with isoprenaline stimulation (9.9±1puncta/cell, n=27), but was not reduced with gallein treatment (4.1±0.5puncta/cell, n=14).

Conclusion: Gβγ subunits are involved in mediating some vascular relaxations and this involves Kv7 channel activation. The effect of Gβγ blockade may be due to reduced modulation of vascular Kv7 channels or prevention of Gβγ mediated signalling events, or both.