Proceedings of The Physiological Society
University of Birmingham (2010) Proc Physiol Soc 20, C02 and PC02
Modulation of Kv2.1 and Kv1.5 channels by AMP-activated protein kinase (AMPK)
M. L. Dallas1, J. Rafferty2, N. Ikematsu3, F. A. Ross3, D. Fedida4, D. Hardie3, C. Peers1, A. Evans2
1. Universiy of Leeds, Leeds, United Kingdom. 2. University of Edinburgh, Edinburgh, United Kingdom. 3. University of Dundee, Dundee, United Kingdom. 4. University of British Columbia, Vancouver, British Columbia, Canada.
It has been suggested that the K+ channels Kv2.1 and Kv1.5 are O2 sensitive and play an important role in hypoxic pulmonary vasoconstriction (HPV; [1-3]). Relative expression levels of Kv2.1 and Kv1.5 in smooth muscle cells may determine, in part, differences in hypoxic sensitivity of conduit versus resistance arteries. Given that our previous studies suggested that HPV requires AMPK activation , we investigated whether AMPK regulates Kv2.1 and Kv1.5 stably expressed in HEK 293 cells. From whole-cell patch clamp recordings we constructed conductance - voltage relationships for Kv2.1 as previously described . Under control conditions, the half maximal conductance (G0.5) was 10.9 ± 1.0mV (mean ± s.e.m., n=21 cells). This was shifted in the hyperpolarizing direction by pre-exposure (20 min, 37°C) of cells to the AMPK activator A-769662 (100μM; G0.5 -8.1 ± 2.1mV, n=10, P<0.01, unpaired t-test). When cells were pretreated with A-769662 in the presence of the AMPK inhibitor compound C (20μM), G0.5 (12.4 ± 1.8mV) was similar to control, i.e. compound C prevented the hyperpolarizing shift in activation caused by AMPK. We identified S440 and S537 as potential phosphorylation sites in the Kv2.1 sequence. G0.5 in S537A and S440A mutants were 6.0 ± 1.1mV (n=10) and 9.4 ± 0.9mV (n=10), respectively. Exposure to A-769662 caused a significant shift (P<0.05) in G0.5 for the S537A mutant of -11.7 ± 1.3mV (n=10). By contrast, A-769662 caused only a modest shift of the G0.5 for the S440A mutant of 3.8 ± 1.1mV (n=10). Parallel studies on phosphorylation (employing 32P incorporation) indicated that the immunoprecipitated channel protein was a direct substrate for AMPK-dependent phosphorylation at these two sites. In marked contrast to these findings for Kv2.1, AMPK markedly attenuated macroscopic Kv1.5 currents throughout their current-voltage relationship. Residual currents evoked at +30 mV measured 61.2 ± 0.2 % (n = 4, P<0.05) and 61 ± 0.1 % (n = 4, P<0.05) of control following intracellular dialysis of an active (thiophosphorylated) recombinant AMPK heterotrimer (α2β2γ1) or extracellular application of 100μM A-769662, respectively. Current inhibition was not observed upon intracellular dialysis of an inactive (α2D157A) AMPK heterotrimer, or following application of A-769662 in the presence of compound C (40μM). Parallel studies on 32P phosphorylation indicated that the immunoprecipitated channel protein was a direct substrate for AMPK-dependent phosphorylation. Our data suggest that Kv2.1 and Kv1.5 are directly modulated by AMPK and thereby regulated in a manner consistent with the differential effects of both hypoxia and mitochondrial inhibitors on Kv currents recorded in arterial smooth muscle cells of conduit and resistance sized pulmonary arteries, respectively.
Where applicable, experiments conform with Society ethical requirements