Expression of receptor for alpha-latrotoxin in Xenopus oocytes after injection of mRNA from rat brain.
alpha-Latrotoxin, the major toxin of black widow spider venom, was suggested to bind to the specific receptor on the membrane of presynaptic cells and to activate a nonselective cation channel. The aim of this investigation was to express the receptor to alpha-latrotoxin in the membrane of Xenopus laevis oocytes. Responses to alpha-latrotoxin were studied using a double microelectrode voltage-clamp technique on X. laevis oocytes previously injected with poly(A+)-RNA from rat brain. alpha-Latrotoxin (10 nM) was shown to induce a slow activating reversible inward membrane current at a clamp potential of -60 mV. A second application of alpha-latrotoxin immediately after washing out induced a smaller response. Reversal potential of this current was near to 0 mV; it hardly changed in low Cl- external solution. Response to alpha-latrotoxin did not depend significantly on the variation of Ca2+ concentration in external solution. Ethyleneglycolbis(aminoethylether)tetra-acetate (EGTA) injection into oocytes did not decrease alpha-latrotoxin-induced current, but seemed to slow the kinetics of the response. Inorganic Ca-channel blocker Co2+ had no effect on alpha-latrotoxin response. These results indicate that alpha-latrotoxin-induced inward current flows mainly through cation nonspecific channel. A lectin concanavalin A irreversibly inhibited alpha-latrotoxin-evoked inward current. Many of these observations are similar to those reported for nerve cells after alpha-latrotoxin application. The data obtained suggest that functional receptor to alpha-latrotoxin can be successively expressed in the membrane of Xenopus oocytes providing future search of DNA encoding receptor subunits and study of receptor structure-function relationship.
Filippov
A K
AK
Institute of Biological Physics, U.S.S.R. Academy of Sciences, Pushchino, Moscow Region.
Kobrinsky
E M
EM
Tsurupa
G P
GP
Pashkov
V N
VN
Grishin
E V
EV
D000818:Animals; D001921:Brain / Q000502:physiology*; D002118:Calcium / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D003208:Concanavalin A / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D008564:Membrane Potentials / Q000187:drug effects; D008845:Microinjections; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics*; D051381:Rats; D011950:Receptors, Cholinergic / Q000235:genetics* / Q000502:physiology; D018000:Receptors, Peptide; D013111:Spider Venoms / Q000378:metabolism / Q000494:pharmacology*; D014982:Xenopus laevis
D012333:RNA, Messenger; D011950:Receptors, Cholinergic; D018000:Receptors, Peptide; D013111:Spider Venoms; C034800:alpha-latrotoxin receptor; D003208:Concanavalin A; D003035:Cobalt; D004533:Egtazic Acid; C020763:alpha-latrotoxin; D002118:Calcium