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Original Article |
Block by Ca2+ Entering from the Intracellular Pore Entrance
Neuroscience Center, B-138, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262.303-315-2503
william.sather{at}uchsc.edu
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.
Key Words: ion selectivity • selectivity filter • L-type Ca2+ channel • patch clamp • cysteine mutagenesis
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