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

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 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 alpha1A, followed by alpha1B > alpha1C > alpha1E. The affinity of DW13.3 for alpha1B 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 alpha1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand omega-conotoxin MVIIC (but not omega-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.

Effects of Methylmercury on Human Neuronal L-Type Calcium Channels Transiently Expressed in Human Embryonic Kidney Cells (HEK-293)

“…In contrast, the recombinant L-type Ca 2ϩ channel current expressed in HEK-293 cells could be blocked only incompletely by MeHg. This difference in pharmacological sensitivity between native and cloned, heterologously expressed channels has also been reported for calcicludine reduction of L-type (Stotz et al, 2000), -agatoxin IVA reduction of P/Q-type (Bourinet et al, 1999), and peptide spider toxin DW13.3 reduction of N-type (Sutton et al, 1998) currents. One possible reason for this apparent difference in our experiments could be the use of a ␤ subunit with properties different from the native ␤ subunits that associate with the corresponding native L-type Ca 2ϩ channels; ␤ subunits are known to modulate channel kinetics (Hofmann et al, 1999).…”

Pharmacologically active spider peptide toxins

“…Larger peptides (>8 kDa) with VSCC specificity have also been found in spider venoms. The SpPTx DW13.3 isolated from the venom of the spider Filistata hibernalis causes a potent but transient block of native calcium channels [55]. DW13.3 had the highest affinity for P/Qtype, followed by N-> L-> R-type with no effect on Ttype currents recorded from GH3 cells.…”

Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca 2+ channels by using knockout mice

“…The fraction of channels blocked by -CTX GVIA is less in mouse than in rat, and the fraction of channels subsequently blocked by -CTX MVIIC is more, suggesting that in mouse there is not only a large N-type Ca 2ϩ channel population but also a significant population of P͞Q-type channels that is not seen in rat. As judged from our nifedipine results, the contribution of L-type Ca 2ϩ channels (18%) may be a little greater in mouse than in rat neurons [often Ͻ10%, (21,32)]. …”