Interactions between the C-terminus of Kv1.5 and Kvβ regulate pyridine nucleotide-dependent changes in channel gating

Voltage-gated potassium (Kv) channels are tetrameric assemblies of transmembrane Kv proteins with cytosolic N- and C-termini. The N-terminal domain of Kv1 proteins binds to beta-subunits, but the role of the C-terminus is less clear. Therefore, we studied the role of the C-terminus in regulating Kv1.5 channel and its interactions with Kv beta-subunits. When expressed in COS-7 cells, deletion of the C-terminal domain of Kv1.5 did not affect channel gating or kinetics. Coexpression of Kv1.5 with Kv beta 3 increased current inactivation, whereas Kv beta 2 caused a hyperpolarizing shift in the voltage dependence of current activation. Inclusion of NADPH in the patch pipette solution accelerated the inactivation of Kv1.5-Kv beta 3 currents. In contrast, NADP(+) decreased the rate and the extent of Kv beta 3-induced inactivation and reversed the hyperpolarizing shift in the voltage dependence of activation induced by Kv beta 2. Currents generated by Kv1.5 Delta C+Kv beta 3 or Kv1.5 Delta C+Kv beta 2 complexes did not respond to changes in intracellular pyridine nucleotide concentration, indicating that the C-terminus is required for pyridine nucleotide-dependent interactions between Kv beta and Kv1.5. A glutathione--transferase (GST) fusion protein containing the C-terminal peptide of Kv1.5 did not bind to apoKv beta 2, but displayed higher affinity for Kv beta 2:NADPH than Kv beta 2:NADP(+). The GST fusion protein also precipitated Kv beta proteins from mouse brain lysates. Pull-down experiments, structural analysis and electrophysiological data indicated that a specific region of the C-terminus (Arg543-Val583) is required for Kv beta binding. These results suggest that the C-terminal domain of Kv1.5 interacts with beta-subunits and that this interaction is essential for the differential regulation of Kv currents by oxidized and reduced nucleotides.