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 q uartet of pore-localized glutamate residues (EEEE locus). The EEEE locus is widely believed to comprise the sole high-affinity Ca2+ binding site in th e pore, which represents an overturning of earlier models that had postulat ed 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 quadru ple alanine (AAAA), glutamine (QQQQ), or aspartate (DDDD) substitutions. Ho wever, interpretation of the mutagenesis work can be criticized on the grou nds that EEEE locus mutations may have additionally disrupted the integrity of a second, non-EEEE locus high-affinity site, and that such a second sit e may have remained undetected because the mutated pore was probed only fro m the extracellular pore entrance. Here, we describe the results of experim ents designed to test the strength of these criticisms of the single high-a ffinity locus model of selective permeability in Ca2+ channels. First, subs tituted-cysteine accessibility experiments indicate that pore structure in the vicinity of the EEEE locus is not extensively disrupted as a consequenc e of the quadruple AAAA mutations, suggesting in turn that the quadruple mu tations 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 oriente d pore entrance of AAAA and QQQQ mutants. Using inside-out patches, we foun d that, whereas micromolar Ca2+ produced substantial block of outward Li+ c urrent in wild-type channels, internal Ca2+ concentrations up to 1 mM did n ot produce detectable block of outward Li+ current in the AAAA or QQQQ muta nts. These results indicate that the EEEE locus is indeed the sole high-aff inity Ca2+ binding locus in the pore of voltage-gated Ca2+ channels.