Pyridine Nucleotide Dependence of Kv Beta - Induced Kv Inactivation: Role of Kv Alpha C-Terminus

Abstract

Binding of ancillary β-subunits (Kvβ) to the N-terminal T1 domain of Kv1 and Kv4 regulates channel function and localization. The β subunits of Kv channels belong to the aldo-keto reductase superfamily (AKR6). These proteins bind NAD(P)(H) with high affinity, but the mechanisms by which nucleotides regulate channel gating are unclear. Herein we report that when coexpressed with Kv1.5 in COS-7 cells, Kvβ3 shifts the half-activation potential and imparts inactivation to slowly inactivating Kv1.5 current. Addition of NAD(P)H to the patch pipette increased rate and extent of inactivation, whereas NAD(P)+ reduced inactivation. These results conform to a model assuming that NAD(P)(H) binding regulates rate and extent of inactivation synergistically by altering the number of Kvβ monomers involved in inactivation. Deletion of 56 C-terminal amino acids of Kv1.5 (KvΔC56) did not significantly affect Kv association with Kvβ or Kvβ-mediated inactivation. KvΔC56 did not, however, respond to changes in intracellular pyridine nucleotide concentration when co-expressed with Kvβ3 and neither NAPDH nor NADP+ altered rate or extent of inactivation. Glutathione-S-transferase (GST) fusion protein containing peptides from the last 38 (Ile565-Leu602) and 60 (Arg543-Leu602), but not 19 (Asp584-Leu602), amino acids of Kv1.5 C-terminus precipitated Kvβ2 and Kvβ3 in pull-down assays from lysates of transformed bacteria. The C-terminal peptide (GST-C60) also precipitated Kvβ1 and Kvβ2 from mouse brain extracts. The GST-C60 construct did not bind to apoKvβ2, and it displayed higher affinity for Kvβ2:NADPH than for the Kvβ2:NADP+ binary complex. These results suggest that nucleotide binding provides an efficient mechanism to adjust potassium flux in response to metabolic changes. The C-terminal domain of Kv1.5 from Arg543-Asp584 interacts with Kvβ and this interaction may be involved in sensing different conformational states of Kvβ bound to either reduced or oxidized pyridine nucleotides.