Abstract 425: Role of Nucleotide Binding and Catalysis in Regulating β-Subunit Mediated Inactivation of Voltage-Gated K+ Channels
Kv channels interacts withβ subunits that belong to the aldoketo reductase superfamily. Kvβ regulates inactivation, expression, and localization of Kv channels, and β-mediated inactivation of Kv channels is sensitive to intracellular redox state, but underlying mechanisms remain unclear. To examine redox regulation, Kvβ 3 was co-expressed with Kv1.5 channels in COS-7 cells. Kvβ 3 increased the inactivation of whole-cell Kv currents from 4.3±1% to 71±4% (n=10; P<0.01), inclusion of 0.1 mmol/L NAD(P)H increased Kv inactivation to 87±.5% (n=0, P<.01) whereas inactivation was 11±3% in the presence of 1 mM NAD(P)+. Cells expressing Kv1.5+β 3 patched with a hypoxic mixture of nucleotides in the pipette (NADPH80, NADP+50, NADH1000, NAD+200 μM) displayed only 10±1% inactivation, whereas currents of cells patched with normoxic complement of nucleotides (NADPH100, NADP+30, NADH50, NAD+1000 μM) were inactivated by 78±3%, indicating that the Kvα-β couple responds to physiologic changes in nucleotide concentrations. These changes appear to be “sensed” by the cytosolic C-terminus domain of Kvαbecause deletion of the last 58 residues of the C-terminus abolished NAD(P)(H)-mediated changes in inactivation. In addition to changes in bulk nucleotide concentration, Kv channel could also respond to local changes in nucleotides bound to Kvβ. We found that isolated recombinant Kvβ 2 displayed low, but significant, catalytic activity with several aldehydes and ketones including 9,10-phenanthroquinone and methylglyoxal. Incubation of Kvβ with substrates led to a time-dependent decrease in the absorbance of Kvβ-NADPH binary complex. In multiple turnover experiments, the catalytic rate was 0.17min−1, which could shift the equilibrium between NADPH-NADP+bound β-subunit, thus affecting inactivation within several minutes of substrate appearance. These findings suggest inactivation is regulated in part by differential nucleotide binding to Kvβ that could be affected by endogenous aldehydes or intracellular metabolism and that the redox forms of Kvβ modulate inactivation via the C-terminal domain of Kvα. Redox modulation of Kv current due to nucleotide binding and catalysis provides a new paradigm of metabolism-excitation coupling and oxygen-sensing.