The Eeee Locus Is the Sole High-Affinity Ca²⁺ Binding Structure in the Pore of a Voltage-Gated Ca²⁺ Channel: Block by Ca²⁺ Entering from the Intracellular Pore Entrance

Selective permeability in voltage-gated Ca²⁺ channels is dependent upon a quartet of pore-localized glutamate residues (EEEE locus). The EEEE locus is widely believed to comprise the sole high-affinity Ca²⁺ binding site in the pore, which represents an overturning of earlier models that had postulated two high-affinity Ca²⁺ binding sites. The current view is based on site-directed mutagenesis work in which Ca²⁺ binding affinity was attenuated by single and double substitutions in the EEEE locus, and eliminated by quadruple alanine (AAAA), glutamine (QQQQ), or aspartate (DDDD) substitutions. However, interpretation of the mutagenesis work can be criticized on the grounds that EEEE locus mutations may have additionally disrupted the integrity of a second, non-EEEE locus high-affinity site, and that such a second site may have remained undetected because the mutated pore was probed only from the extracellular pore entrance. Here, we describe the results of experiments designed to test the strength of these criticisms of the single high-affinity locus model of selective permeability in Ca²⁺ channels. First, substituted-cysteine accessibility experiments indicate that pore structure in the vicinity of the EEEE locus is not extensively disrupted as a consequence of the quadruple AAAA mutations, suggesting in turn that the quadruple mutations do not distort pore structure to such an extent that a second high affinity site would likely be destroyed. Second, the postulated second high-affinity site was not detected by probing from the intracellularly oriented pore entrance of AAAA and QQQQ mutants. Using inside-out patches, we found that, whereas micromolar Ca²⁺ produced substantial block of outward Li⁺ current in wild-type channels, internal Ca²⁺ concentrations up to 1 mM did not produce detectable block of outward Li⁺ current in the AAAA or QQQQ mutants. These results indicate that the EEEE locus is indeed the sole high-affinity Ca²⁺ binding locus in the pore of voltage-gated Ca²⁺ channels.