Kvβ Subunit Oxidoreductase Activity and Kv1 Potassium Channel Trafficking

Belvin Gong,Claire R Campomanes,Marcia E Hershberger,Dana E Antonucci,Kenneth J Rhodes,James S Trimmer,Karen I Carroll,Louis N Manganas

doi:10.1074/jbc.m110276200

Abstract

Voltage-gated Kv1 potassium channels consist of pore-forming alpha subunits and cytoplasmic Kv beta subunits. The latter play diverse roles in modulating the gating, stability, and trafficking of Kv1 channels. The crystallographic structure of the Kv beta2 subunit revealed surprising structural homology with aldo-keto reductases, including a triosephosphate isomerase barrel structure, conservation of key catalytic residues, and a bound NADP(+) cofactor (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell 90, 943-952). Each Kv1-associated Kv beta subunit (Kv beta 1.1, Kv beta 1.2, Kv beta 2, and Kv beta 3) shares striking amino acid conservation in key catalytic and cofactor binding residues. Here, by a combination of structural modeling and biochemical and cell biological analyses of structure-based mutations, we investigate the potential role for putative Kv beta subunit enzymatic activity in the trafficking of Kv1 channels. We found that all Kv beta subunits promote cell surface expression of coexpressed Kv1.2 alpha subunits in transfected COS-1 cells. Kv beta1.1 and Kv beta 2 point mutants lacking a key catalytic tyrosine residue found in the active site of all aldo-keto reductases have wild-type trafficking characteristics. However, mutations in residues within the NADP(+) binding pocket eliminated effects on Kv1.2 trafficking. In cultured hippocampal neurons, Kv beta subunit coexpression led to axonal targeting of Kv1.2, recapitulating the Kv1.2 localization observed in many brain neurons. Similar to the trafficking results in COS-1 cells, mutations within the cofactor binding pocket reduced axonal targeting of Kv1.2, whereas those in the catalytic tyrosine did not. Together, these data suggest that NADP(+) binding and/or the integrity of the binding pocket structure, but not catalytic activity, of Kv beta subunits is required for intracellular trafficking of Kv1 channel complexes in mammalian cells and for axonal targeting in neurons.

Highlights

Voltage-dependent potassium channels of the Shaker or Kv1 family play a fundamental role in the mammalian nervous system by determining resting membrane potential, frequency of action potential firing, and neurotransmitter release [2]
Previous studies have shown that coexpression with either Kv␤1.1 or Kv␤2 increased 125I-dendrotoxin binding in cells expressing Kv1.2 [15] and that the autonomous highly conserved core domain is sufficient to mediate increases in current amplitude of coexpressed Kv1.4 ␣ subunits [21]
We addressed whether each wild-type Kv␤ subunit harboring this core domain would display the effects on Kv1.2 intracellular trafficking and surface expression observed previously for Kv␤2 [15]

Summary

Introduction

Voltage-dependent potassium channels of the Shaker or Kv1 family play a fundamental role in the mammalian nervous system by determining resting membrane potential, frequency of action potential firing, and neurotransmitter release [2]. These data suggest that NADP؉ binding and/or the integrity of the binding pocket structure, but not catalytic activity, of Kv␤ subunits is required for intracellular trafficking of Kv1 channel complexes in mammalian cells and for axonal targeting in neurons.

Results

Conclusion