ISA Channel Complexes Include Four Subunits Each of DPP6 and Kv4.2

Heun Soh,Steve A.N Goldstein

doi:10.1074/jbc.m706964200

Abstract

Kv4 potassium channels produce rapidly inactivating currents that regulate excitability of muscles and nerves. To reconstitute the neuronal A-type current I(SA), Kv4 subunits assemble with DPP6, a single transmembrane domain accessory subunit. DPP6 alters function-accelerating activation, inactivation, and recovery from inactivation-and increases surface expression. We sought here to determine the stoichiometry of Kv4 and DPP6 in complexes using functional and biochemical methods. First, wild type channels formed from subunit monomers were compared with channels carrying subunits linked in tandem to enforce 4:4 and 4:2 assemblies (Kv4.2-DPP6 and Kv4.2-Kv4.2-DPP6). Next, channels were overexpressed and purified so that the molar ratio of subunits in complexes could be assessed by direct amino acid analysis. Both biophysical and biochemical methods indicate that I(SA) channels carry four subunits each of Kv4.2 and DPP6.

Highlights

Kv4 subunits are molecular components of the neuronal voltage-gated potassium current ISA (1–3)
The stoichiometry of channels naturally assembled with dipeptidyl aminopeptidase 6 (DPP6) and Kv4.2 subunits is first inferred through comparison of the activity of channels formed by Kv4.2 or linked Kv4.2-Kv4.2, Kv4.2-DPP6, or Kv4.2-Kv4.2DPP6 pore-forming subunits with or without DPP6 and with or without KChIP2
Auxiliary subunits are an important feature in potassium channel physiology because they influence channel location, abundance, sensitivity to stimulation, and pharmacology in vivo (22–24)

Summary

EXPERIMENTAL PROCEDURES

Molecular Biology—Human charybdotoxin-sensitive Kv4.2 (15, 16), KChIP2 (accession number AF199598), and DPP6-s (previously DPPX and DPL-1, accession number NP_001927) were expressed in pRAT, a dual purpose vector, for expression in mammalian cells and in vitro transcription. The cells were incubated in cross-linking buffer containing 150 mM KCl, 50 mM HEPES, pH 7.4, and 1 mg/ml dimethyl 3,3Јdithiobispropionimidate (DTBP; Pierce) for 1 h at room temperature to preserve protein-protein interactions between Kv4.2 and DPP6 (4). The cells from 10 –20 plates were solubilized for 1 h at 4 °C in lysis buffer containing 2.5% CHAPS, 100 mM NaCl, 40 mM KCl, 1 mM EDTA, 20 mM HEPES-KOH, pH 7.4, and 10% glycerol with complete protease inhibitors (Roche Applied Science). Protein was eluted with 0.7% CHAPS, 100 mM NaCl, 40 mM KCl, 1 mM EDTA, 20 mM HEPES-KOH, pH 7.4, complete protease inhibitor, and 0.1 mg/ml 1d4 peptide (Yale University Keck Facility, New Haven, CT).

RESULTS

Inactivation Time to peak at expressed

DISCUSSION