We recently reported that amino acid residues contained within a putative EF 
hand motif in the domain III S5-H5 region of the alpha(1B) subunit affected the 
relative barium:calcium permeability of N-type calcium channels (Feng, Z. P., 
Hamid, J., Doering, C., Jarvis, S. E., Bosey, G. M., Bourinet, E., Snutch, T. 
P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 5726-5730). Since this region 
partially overlaps with residues previously implicated in block of the channel 
by omega-conotoxin GVIA, we assessed the effects of mutations in the putative EF 
hand domain on channel block by omega-conotoxin GVIA and the structurally 
related omega-conotoxin MVIIA. Both of the toxins irreversibly block the 
activity of wild type alpha(1B) N-type channels. We find that in addition to 
previously identified amino acid residues, residues in positions 1326 and 1332 
are important determinants of omega-conotoxin GVIA blockade. Substitution of 
residue Glu(1332) to arginine slows the time course of development of block. 
Point mutations in position Gly(1326) to either arginine, glutamic acid, or 
proline dramatically decrease the time constant for development of the block. 
Additionally, in the G1326P mutant channel activity was almost completely 
recovered following washout. A qualitatively similar result was obtained with 
omega-conotoxin MVIIA, suggesting that common molecular determinants underlie 
block by these two toxins. Taken together the data suggest that residue 
Gly(1326) may form a barrier, which controls the access of peptide toxins to 
their blocking site within the outer vestibule of the channel pore and also 
stabilizes the toxin-channel interaction.