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

  1. Kathy G. Sutton1,
  2. Chet Siok2,
  3. Anthony Stea1,
  4. Gerald W. Zamponi1,
  5. Steven D. Heck4,
  6. Robert A. Volkmann3,
  7. Michael K. Ahlijanian2 and
  8. Terry P. Snutch1
  1. 1Biotechnology 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 2Neuroscience (C.S., M.K.A.) and 3Medicinal Chemistry (R.A.V.), 4Pfizer, Inc., Groton, Connecticut

    Abstract

    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 spiderFilistata 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 α1A, followed by α1B > α1C > α1E. The affinity of DW13.3 for α1B 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 α1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand ω-conotoxin MVIIC (but not ω-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.

    Footnotes

    • Send reprint requests to: Dr. Terry P. Snutch, Biotechnology Laboratory Rm. 237, 6174 University Blvd., University of British Columbia, Vancouver, B.C., Canada V6T 1Z3. E-mail:snutch{at}zoology.ubc.ca

    • This work was supported by an Izaak Walton Killam Postdoctoral Fellowship (K.G.S.) and Medical Research Council (MRC) of Canada Postdoctoral Fellowships (G.W.Z. and A.S.). G.W.Z. also holds a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research. T.P.S. is the recipient of an MRC Scientist Award and is supported by a grant from the MRC.

    • Abbreviations:
      ω-CgTX
      ω-conotoxin
      ω-Aga
      agatoxin
      HEK
      human embryonic kidney
      ES-MS
      electrospray-mass spectrometry
      HPLC
      high performance liquid chromatography
      EGTA
      ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      DMEM
      Dulbecco’s modified eagle medium
      MEM
      minimum essential medium
      FCS
      fetal calf serum
      BAPTA
      1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
      • Received March 9, 1997.
      • Accepted May 8, 1998.
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    1. Molecular Pharmacology August 1, 1998 vol. 54 no. 2 407-418
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