Aspartate Residues of the Glu-Glu-Asp-Asp (EEDD) Pore Locus Control Selectivity and Permeation of the T-type Ca Channel

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I–IV of the 1 subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the 1G T-type, the 1C L-type Ca 2 channel subunits, and 1G pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca channel permeation, i.e. (a) Ba over Ca permeability, (b) Ca / Ba anomalous mole fraction effect (AMFE), and (c) high Cd sensitivity, were conferred on the domain III mutant (EEED) of 1G. In contrast, the relative Ca 2 / Ba permeability and the lack of AMFE of the 1G wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd sensitivity similar to that of 1C, but currents were larger in Ca than in Ba -containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H blocked Ca currents in all mutants more efficiently than in 1G. In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in 1G wild type. We conclude that the aspartate residues of the EEDD locus of the 1G Ca 2 channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the 1G Ca 2 channel subunit.