Indirect activation of neuronal noncapacitative Ca2+ entry is the final step involved in the neurotoxic effect of Tityus serrulatus scorpion beta-toxin.
Interweaving strategies of electrophysiology, calcium imaging and immunocytochemistry bring new insights into the mode of action of the Brazilian scorpion Tityus serrulatusbeta-toxin VII. Pacemaker dorsal unpaired median neurons isolated from the cockroach central nervous system were used to study the effects of toxin VII. In current-clamp, 50 nm toxin VII produced a membrane depolarization and reduced spiking. At 200 nM, depolarization associated with multiphasic effects was seen. After artificial hyperpolarization, plateau potentials on which spontaneous electrical activity appeared were observed. In voltage clamp, toxin VII induced a negative shift of the voltage dependence of sodium current activation without significant effect on steady-state inactivation. In addition, toxin VII produced a permanent TTX-sensitive holding inward current, indicating that background sodium channels were targeted by beta-toxin. Cell-attached patch recordings indicated that these channels were switched from unclustered single openings to current fluctuating between distinct subconductance levels exhibiting increased open probability and open-time distribution. Toxin VII also produced a TTX-sensitive [Ca2+]i rise. Immunostaining with Cav2.2(alpha1b) antibodies and calcium imaging data obtained with omega-CgTx GVIA indicated that N-type high-voltage-activated calcium channels initiated calcium influx and were an essential intermediate in the pathway linking toxin VII-modified sodium channels to the activation of an additional route for calcium entry. By using inhibitors of (i) noncapacitative calcium entry (inhibitor LOE-908), (ii) NO-sensitive guanylyl cyclase (ODQ) and (iii) phosphodiesterase 2 (EHNA), together with cGMP antibodies, we demonstrated that noncapacitative calcium entry was the final step in a complex combination of events that was initiated by toxin VII-alteration of sodium channels and then involved successive activation of other membrane ion channels.
Laboratoire RCIM UPRES EA 2647, Université d'Angers, Faculté des Sciences, 2 bd Lavoisier, F-49045 Angers cedex, France. francoise.grolleau@univ-angers.fr
Françoise
F
Grolleau
Maria
M
Stankiewicz
Ewa
E
Kielbasiewicz
Marie-France
MF
Martin-Eauclaire
Céline
C
Lavialle
Jan
J
De Vente
Bruno
B
Lapied
D000465:Algorithms; D000818:Animals; D002120:Calcium Channel Agonists; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002469:Cell Separation; D003058:Cockroaches; D004594:Electrophysiology; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009474:Neurons / Q000187:drug effects*; D009498:Neurotoxins / Q000633:toxicity*; D018408:Patch-Clamp Techniques; D012604:Scorpion Venoms / Q000633:toxicity*; D015398:Signal Transduction / Q000187:drug effects
D002120:Calcium Channel Agonists; D015220:Calcium Channels; D009498:Neurotoxins; D012604:Scorpion Venoms