Margatoxin binds to a homomultimer of K(V)1.3 channels in Jurkat cells. Comparison with K(V)1.3 expressed in CHO cells.
Voltage-gated potassium (K(V)) channels play key roles in setting the resting potential and in the activation cascade of human peripheral T lymphocytes. Margatoxin (MgTX), a 39-amino acid peptide from Centruroides margaritatus, is a potent inhibitor of lymphocyte K(V) channels. The binding of monoiodotyrosinyl margatoxin ([125I]MgTX) to plasma membranes prepared from either Jurkat cells, a human leukemic T cell line, or CHO cells stably transfected with the Shaker-type voltage-gated K+ channel, K(V)1.3, has been used to investigate the properties of lymphocyte K(V) channels. These data were compared with [125I]MgTX binding to heterotetrameric K(V) channels in rat brain synaptic plasma membranes [Knaus, H. G., et al. (1995) Biochemistry 34, 13627-13634]. The affinity for [125I]MgTX is 100-200 fM in either Jurkat or CHO/K(V)1.3 membranes, and the receptor density is 20-120 fmol/mg in Jurkat membranes or 1000 fmol/mg in CHO/K(V)1.3 membranes. In contrast to rat brain, [125I]MgTX binding to Jurkat and CHO/K(V)1.3 membranes exhibits an absolute requirement for K+, with no potentiation of binding by Na+. K(V)1.3 was the only K(V)1 series channel present in either CHO/K(V)1.3 or Jurkat plasma membranes as determined by immunoprecipitation of [125I]MgTX binding or by Western blot analyses using sequence-specific antibodies prepared against members of the K(V)1 family. The relative potencies of a series of peptidyl K(V) channel inhibitors was essentially the same for inhibition of [125I]MgTX binding to Jurkat, CHO, or rat brain membranes and for blocking 86Rb+ efflux from the CHO/K(V)1.3 cells, except that alpha-dendrotoxin was more potent at blocking binding to rat brain membranes than in the other assays. The characteristics of [125I]MgTX binding, the antibody profiles, and the effects of the peptidyl K(V) inhibitors all indicate that the [125I]MgTX receptor in Jurkat lymphocytes is comprised of a homomultimer of K(V)1.3, unlike the heteromultimeric arrangement of the receptor in rat brain.
Helms
L M
LM
Department of Membrane Biochemistry and Biophysics, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Felix
J P
JP
Bugianesi
R M
RM
Garcia
M L
ML
Stevens
S
S
Leonard
R J
RJ
Knaus
H G
HG
Koch
R
R
Wanner
S G
SG
Kaczorowski
G J
GJ
Slaughter
R S
RS
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D006801:Humans; D007455:Iodine / Q000378:metabolism; D019169:Jurkat Cells; D007700:Kinetics; D051664:Kv1.3 Potassium Channel; D009498:Neurotoxins / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D051381:Rats; D012413:Rubidium / Q000378:metabolism; D012604:Scorpion Venoms / Q000378:metabolism*
C496973:KCNA3 protein, human; C496975:Kcna3 protein, rat; D051664:Kv1.3 Potassium Channel; D009498:Neurotoxins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D012604:Scorpion Venoms; C105268:margatoxin receptor; C077910:margatoxin; D007455:Iodine; D012413:Rubidium