Title

Purification and Characterization of Two Sodium Channels from Rat Brain With Different Alpha Subunits and Distinct Functional Properties

Document Type

Presentation

Publication Date

2-1988

Abstract

Voltage-dependent Na channels have been purified from electric eel electroplax, rat brain, and rat and rabbit skeletal muscle transverse tubules using binding of radiolabeled saxitoxin (STX) or other neurotoxins to assay for the channel protein. Each Na channel consists of a single large alpha subunit (M.W. about 260K) and, except for the electric eel, one or two beta subunits (M.W. about 35 - 39K). Using a modification of the procedure described by Hartshorne and Catterall (J.B.C. 259: 1667, 1984) and HPLC in the final steps to improve resolution, we separated two 3H-STX binding proteins with different alpha subunits (M.W. 235K and 265K) as revealed by SDS-PAGE. Both channel types had similar beta subunits. These channels were reconstituted in planar lipid bilayers for characterization of channel function. In the presence of veratridine (VER), the Na channel with the 235K alpha subunit had a conductance of 10 pS and resembled that described by Garber and Miller (J.G.P. 89: 459, 1987). Extracellular scorpion venom (Leiurus quinquestriatus) caused a small (15%) increase in single channel conductance and an increase in the probability of being open. In contrast, the Na channel with the 265K alpha subunit had a conductance of 5 pS in the presence of VER and underwent about an 80% increase in single channel conductance in the presence of scorpion venom (to 9 pS) together with an increase in the probability of being open. Both 10 pS (M.W. 235K) and 5 pS (M.W. 265K) Na channels displayed voltage-dependent gating and were blocked by nanomolar concentrations of STX. Supported by NIH.

Comments

Presented at the 32nd Annual Meeting of the Biophysical Society, Phoenix, AZ.

Presentation Number M-AM-C5.

Copyright © 1988 The Biophysical Society. Published by Elsevier Inc. All rights reserved.

DOI

10.1016/S0006-3495(88)83093-5