Vol. 54, Issue 2, 407-418, August 1998

Inhibition of Neuronal Calcium Channels by a Novel Peptide Spider Toxin, DW13.3

Kathy G. Sutton, Chet Siok, Anthony Stea, Gerald W. Zamponi, Steven D. Heck, Robert A. Volkmann, Michael K. Ahlijanian, and Terry P. Snutch

Biotechnology Laboratory, University of British Columbia, Vancouver, B.C., Canada V6T 1Z3 (K.G.S., A.S., G.W.Z., T.P.S.), and Departments of Neuroscience (C.S., M.K.A.) and Medicinal Chemistry (R.A.V.), Pfizer, Inc., Groton, Connecticut

Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently expressed calcium channels in oocytes showed that DW13.3 had the highest affinity for 1A, followed by 1B > 1C > 1E. The affinity of DW13.3 for 1B N-type currents varied by 10-fold between expressed channels and native currents. Although block occurred in a similar 1:1 manner for all subtypes, DW13.3 produced a partial block of both 1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand -conotoxin MVIIC (but not -agatoxin IVA) from its binding site in Purkinje neurons suggests that DW13.3 binds to a site close to the pore of the channel. The inhibition of different subtypes of calcium channels by DW13.3 reflects a common "macro" binding site present on all calcium channels except T-type.