Purification, visualization, and biophysical characterization of Kv1.3 tetramers.
The voltage-gated K+ channel of T-lymphocytes, Kv1.3, was heterologously expressed in African Green Monkey kidney cells (CV-1) using a vaccinia virus/T7 hybrid expression system; each infected cell exhibited 10(4) to 5 x 10(5) functional channels on the cell surface. The protein, solubilized with detergent (3-[cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or cholate), was purified to near-homogeneity by a single nickel-chelate chromatography step. The Kv1.3 protein expressed in vaccinia virus-infected cells and its purified counterpart are both modified by a approximately 2-kDa core-sugar moiety, most likely at a conserved N-glycosylation site in the external S1-S2 loop; absence of the sugar does not alter the biophysical properties of the channel nor does it affect expression levels. Purified Kv1.3 has an estimated size of approximately 64 kDa in denaturing SDS-polyacrylamide electrophoresis gels, consistent with its predicted size based on the amino acid sequence. By sucrose gradient sedimentation, purified Kv1.3 is seen primarily as a single peak with an approximate mass of 270 kDa, compatible with its being a homotetrameric complex of the approximately 64-kDa subunits. When reconstituted in the presence of lipid and visualized by negative-staining electron microscopy, the purified Kv1.3 protein forms small crystalline domains consisting of tetramers with dimensions of approximately 65 x 65 A. The center of each tetramer contains a stained depression which may represent the ion conduction pathway. Functional reconstitution of the Kv1.3 protein into lipid bilayers produces voltage-dependent K+-selective currents that can be blocked by two high affinity peptide antagonists of Kv1.3, margatoxin and stichodactylatoxin.
Spencer
R H
RH
Departments of Microbiology and Molecular Genetics and of Physiology and Biophysics, University of California, Irvine, California 92697, USA.
Sokolov
Y
Y
Li
H
H
Takenaka
B
B
Milici
A J
AJ
Aiyar
J
J
Nguyen
A
A
Park
H
H
Jap
B K
BK
Hall
J E
JE
Gutman
G A
GA
Chandy
K G
KG
D000818:Animals; D002522:Chlorocebus aethiops; D019826:Cholic Acid; D002793:Cholic Acids; D002846:Chromatography, Affinity; D003064:Cnidarian Venoms / Q000494:pharmacology; D006031:Glycosylation; D051664:Kv1.3 Potassium Channel; D009498:Neurotoxins / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000302:isolation & purification*; D024642:Potassium Channels, Voltage-Gated; D011487:Protein Conformation; D012604:Scorpion Venoms / Q000494:pharmacology; D012995:Solubility
D002793:Cholic Acids; D003064:Cnidarian Venoms; D051664:Kv1.3 Potassium Channel; D009498:Neurotoxins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D012604:Scorpion Venoms; C059713:neurotoxin I, Stichodactyla helianthus; C077910:margatoxin; D019826:Cholic Acid; D011188:Potassium