Abstract
The Shaker family voltage-dependent potassium channels (Kv1) assemble with cytosolic beta-subunits (Kvbeta) to form a stable complex. All Kvbeta subunits have a conserved core domain, which in one of them (Kvbeta2) is an aldoketoreductase that utilizes NADPH as a cofactor. In addition to this core, Kvbeta1 has an N terminus that closes the channel by the N-type inactivation mechanism. Point mutations in the putative catalytic site of Kvbeta1 alter the on-rate of inactivation. Whether the core of Kvbeta1 functions as an enzyme and whether its enzymatic activity affects N-type inactivation had not been explored. Here, we show that Kvbeta1 is a functional aldoketoreductase and that oxidation of the Kvbeta1-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP(+), induces a large increase in open channel current. The modulation is not affected by deletion of the distal C terminus of the channel, which has been suggested in structural studies to interact with Kvbeta. The rate of increase in current, which reflects NADPH oxidation, is approximately 2-fold faster at 0-mV membrane potential than at -100 mV. Thus, cofactor oxidation by Kvbeta1 is regulated by membrane potential, presumably via voltage-dependent structural changes in Kv1.1 channels.
Highlights
Kv subunits assemble with Kv1 channels in the endoplasmic reticulum early in biogenesis (9), and the two form a stable complex with a stoichiometry of (Kv1)4:(Kv)4 that is preserved during purification (10 –14)
A previous study showed that mutations in the putative catalytic site of Kv1 change the rate of channel inactivation (20), but did not show that Kv1 functions as a redox enzyme or that its activity affects channel activity
Studying in parallel the effect of these reagents on channel current, we observed a correlation between NADPH oxidation and potentiation of channel current
Summary
Protein Expression—The cDNA of the conserved core of rat Kv1 (NCBI accession number NM_017303; residues 71– 401) was cloned into a modified pQE70 vector (Qiagen Inc.) between the SphI and BamHI sites with a C-terminal 6-histidine tag cleavable by thrombin. Wild-type Kv1 has a cysteine residue at position 7 that can be oxidized on inside-out patches to affect channel inactivation, and this can be prevented by the addition of reducing reagents such as dithiothreitol (5). The holding potential was stepped to Ϫ100 mV for 30 s to allow complete channel recovery from inactivation, and current was elicited by stepping the membrane potential to ϩ60 mV. This process was repeated until the modulation reached steady state. Similar to the measurement at Ϫ100 mV, the normalized current amplitude versus the accumulative exposure time was well fit with a single-component exponential function. Isopropyl -D-thiogalactopyranoside, dithiothreitol, and kanamycin were purchased from LabScientific, Inc
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