Abstract
The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) 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 alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(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 alpha(1G) Ca(2+) channel subunit.
Highlights
¶ Both authors contributed to this work. ** To whom correspondence should be addressed: Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium
The pore region of domain III was shown to contain a putative EF hand motif, which is conserved in all high voltage-activated (HVA) Ca2ϩ channels, and it was suggested to underlie the differential permeabilities for Ba2ϩ and Ca2ϩ of the N-type ␣1B channel subunit (23)
We explored the properties of the currents through the ␣1G subunit at different Ca2ϩ-Ba2ϩ mole fractions and tested if replacement of the T-type Ca2ϩ channel residues, which are divergent from those of the EEEE locus of HVA Ca2ϩ channels, confers anomalous mole fraction behavior on the ␣1G channels
Summary
¶ Both authors contributed to this work. ** To whom correspondence should be addressed: Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Ca2ϩ channels is a locus formed by four glutamate residues, one in each P-region of domains I–IV of the ␣1 subunit (15, 16) This so-called EEEE locus is conserved in all known ␣1 subunits that form the ion-conducting pore of the HVA Ca2ϩ channel subfamily (17–19). This EEEE locus seems to be the sole high affinity binding structure in the pore, and each glutamate residue has a differential contribution to Ca2ϩ selectivity and Cd2ϩ and proton block (20 –22). P-loop of domains III and IV, respectively These modifications on and around the EEEE locus of the ␣1C channel subunit decreased the single channel conductance for Ba2ϩ, mimicking the properties of LVA Ca2ϩ channels
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