Introduction: As the sodium/ myoinositol transporter (SMIT1) is a positive regulator of Kv7.4/7.5 channels in arterial smooth muscle we postulated that altering SMIT1 expression could have a major impact upon vascular reactivity. Consequently, this study aimed to characterise the effects of changes of SMIT1 membrane abundance on vascular tone and the molecular mechanisms involved.
Methods: Isometric tension recordings on 2nd order mesenteric arteries and left anterior coronary arteries from male and female Wistar Han rats were performed with morpholino based protein knockdown of SMIT1. All studies involved > 5 biological repeats. Whole artery membrane potential recording alongside electrophysiology patch-clamp recordings of Kv7.4 channels. Proximity ligation assay and whole artery antibody-based imaging.
Results: Morpholino-mediated knockdown of SMIT1 enhanced U46619- and methoxamine-mediated contractions, while attenuating relaxations to the Kv7 activator ML213, isoprenaline and CGRP in mesenteric arteries. Incubating HEK cells expressing Kv7.4 with raffinose for 1 h increased current amplitude and left shifted voltage-dependence by ~8mV when cells were co-expressed with SMIT-1 only. Similar treatments of arteries increased SMIT1 membrane abundance in smooth muscle cells, impaired receptor-mediated contractions of mesenteric and coronary arteries, augmented relaxations to ML213 and adenosine and induced membrane potential hyperpolarisation. The SGK1 inhibitor EMD638683, involved in regulating SMIT1 membrane expression, prevented the acute raffinose-induced increase in SMIT1 and the associated anti-contractile effects. Proximity ligation assays revealed that raffinose incubation increased the association of SMIT-Kv7.4/7.5 as well as Kv7.4 and Gβγ subunits.
Conclusions: Altering the membrane abundance of SMIT1 had a dramatic effect on the arterial response to vasoconstrictors and vasodilators mediated by greater coordination of Kv7 channels and Gβγ subunits. This work identified SMT1-Kv7 channel complexes and SGK1 regulation as key modulators of arterial responsiveness.