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G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity
Saturday, 2015/05/23 | 06:05:50

Jennifer B. Stott, Oleksandr V. Povstyan, Georgina Carr, Vincenzo Barrese, and Iain A. Greenwood

Significance

The Kv7.4 potassium channel is a critical regulator of vascular contractility both at rest and as a functional endpoint for a number of endogenous vasodilators. Despite its key role, nothing is known about the processes that determine Kv7.4 channel activity. We reveal an interaction between G-protein βγ subunits and Kv7.4 that is crucial for channel responses to membrane voltage. Blocking this interaction ablates channel activity, prevents β-adrenoceptor–mediated relaxation, and constricts renal arteries. Conversely, Gβγ subunits enhance Kv7.4 channels and produce arterial relaxation in a Kv7-dependent manner. This reveals a fundamental reliance of an ion channel on Gβγ subunits for basal activity, a previously unidentified finding, which has profound implications for vascular physiology and disease pathogenesis.

Abstract

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K+ currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2–250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein–coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein–coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.

 

See: http://www.pnas.org/content/112/20/6497.abstract.html?etoc

PNAS May 19, 2015 vol. 112 no. 20 6497-6502

 

Fig. 6.

Fig. 6.

In situ PLA detection of Kv7.4 and Gβ subunit protein interactions in rat renal artery myocytes and KCNQ4 stably transfected HEK cells. Representative fluorescence and bright field (Inset) confocal midcell xy sections of (A) Kv7.4 stably transfected HEK293 cells or (C) renal artery myocytes, using primary antibodies for Kv7.4 and Gβ subunit together with appropriate PLA probes. Red puncta indicate target proteins are in close proximity (<40 nm). Nuclei are shown in blue as defined by DAPI. Quantification of the mean number of PLA signals per midcell xy section in (B) Kv7.4 stably transfected HEK cells or (D) renal artery myocytes. No primary controls produced no detectable PLA signal. The number of animals (N) and number of cells (n) for each treatment is shown. *P < 0.05, ***P < 0.001 for control versus gallein according to one-way ANOVA, multiple comparisons test. Scale bar represents 10 µm.

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