Proceedings of The Physiological Society

Physiology 2015 (Cardiff, UK) (2015) Proc Physiol Soc 34, C39

Oral Communications

Differential Kv7 subunit interaction in vascular smooth muscle cells of mesenteric and renal arteries in the normotensive and spontaneously hypertensive rat

H. Spiers1, G. Carr1, I. A. Greenwood1


Background: KCNQ encoded Kv7 voltage-gated potassium channels (mainly Kv7.1, Kv7.4, Kv7.5), expressed in a range of vasculature, regulate vascular tone1 and are compromised in hypertension. Kv7 channel regulation is still being elucidated. Kv7 channels are tetramers of individual channel subunits; therefore these channels can be regulated by changes in subunit composition. We used Proximity Ligation Assay (PLA) technology2 to investigate the molecular architecture of Kv7 channels in renal (RA) and mesenteric artery (MA) vascular smooth muscle cells (VSMCs) in normotensive (NT) rats and compare any differences. We also investigated the molecular architecture of channels in the same arterial beds in spontaneously hypertensive rats (SHRs) for comparison to the NT rat. Methods: RA and MA were dissected from rats, sacrificed by cervical dislocation whilst complying with ethical requirements, and single VSMCs enzymatically isolated. The Duolink in situ PLA detection kit 563 was used to determine protein interactions for Kv7.4, Kv7.5 and Kv7.1. Cells were incubated with primary antibody pairs (rabbit Kv7.4+goat Kv7.4; rabbit Kv7.4+goat Kv7.5; rabbit Kv7.5+goat Kv7.5; rabbit Kv7.4+mouse Kv7.1; rabbit Kv7.5+mouse Kv7.1; rabbit Kv7.1+mouse Kv7.1; 1:200). Controls omitted primary antibodies or employed only 1 antibody. Cells were labeled with specialised secondary probes attached to synthetic oligonucleotides that hybridised in close proximity (<40nm), generating a red spot, viewed by confocal microscopy. The number of puncta was assessed and compared using 1-way ANOVA and Bonferroni's multiple comparisons test. Results/Conclusion: Kv7.4/Kv7.4 (47.79±11.04, n=14) and Kv7.4/Kv7.5 (43.36±6.94, n=14) combinations produced a high puncta level in MA VSMCs from NT rats indicating a 3:1 heterotetramer of Kv7.4:Kv7.5 subunit channel is predominant. The same combinations (Kv7.4/Kv7.4 56.21±14.65, n=14; Kv7.4/Kv7.5 62.50±11.16, n=20), along with the Kv7.5/Kv7.5 (59.13±10.06, n=20) and Kv7.4/Kv7.1 (41.50±6.97, n=21) combinations, produced the highest puncta levels in RA VSMCs from NT rats inferring the predominant species is a 1:1 heterotetramer of Kv7.4:Kv7.5 subunits with Kv7.4/Kv7.1 subunit interactions highlighted. The important potential interactions of Kv7.1 with other Kv7s were further emphasised by the fact that these interactions were different in the SHR, where Kv7.4/7.5 interactions were low (7.50±1.95, n=14; 10.33±2.71, n=9; SHR MA and RA VSMCs respectively). The study is the first to highlight differences in Kv7 channel stoichiometry between MA and RA for NT rats and the potential interactions of Kv7.1 with other Kv7s and how these interactions change in hypertension. The study could form the basis of further research into therapeutic targets that are tissue specific in hypertension treatment.

Where applicable, experiments conform with Society ethical requirements