Cibulsky S M, Sather W A
Department of Pharmacology and Neuroscience Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
J Gen Physiol. 2000 Sep;116(3):349-62. doi: 10.1085/jgp.116.3.349.
Selective permeability in voltage-gated Ca(2+) 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 Ca(2+) binding site in the pore, which represents an overturning of earlier models that had postulated two high-affinity Ca(2+) binding sites. The current view is based on site-directed mutagenesis work in which Ca(2+) 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 Ca(2+) 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 Ca(2+) produced substantial block of outward Li(+) current in wild-type channels, internal Ca(2+) 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 Ca(2+) binding locus in the pore of voltage-gated Ca(2+) channels.
电压门控钙通道的选择性通透取决于位于孔道中的一组四个谷氨酸残基（EEEE位点）。人们普遍认为，EEEE位点构成了孔道中唯一的高亲和力钙结合位点，这推翻了早期假设存在两个高亲和力钙结合位点的模型。当前的观点基于定点诱变研究，在该研究中，通过对EEEE位点进行单取代和双取代，钙结合亲和力减弱，而通过四聚丙氨酸（AAAA）、谷氨酰胺（QQQQ）或天冬氨酸（DDDD）取代则可消除钙结合亲和力。然而，对诱变研究的解释可能受到批评，因为EEEE位点突变可能额外破坏了第二个非EEEE位点高亲和力位点的完整性，而且由于仅从细胞外孔道入口探测突变后的孔道，这样的第二个位点可能一直未被发现。在此，我们描述了旨在检验对钙通道选择性通透的单一高亲和力位点模型的这些批评力度的实验结果。首先，半胱氨酸取代可及性实验表明，由于AAAA四聚体突变，EEEE位点附近的孔道结构并未受到广泛破坏，这反过来表明四聚体突变并未使孔道结构扭曲到可能破坏第二个高亲和力位点的程度。其次，从AAAA和QQQQ突变体的细胞内孔道入口进行探测，未检测到假定的第二个高亲和力位点。使用内面向外的膜片，我们发现，虽然微摩尔浓度的钙在野生型通道中可显著阻断外向锂电流，但高达1 mM的细胞内钙浓度在AAAA或QQQQ突变体中并未产生可检测到的外向锂电流阻断。这些结果表明，EEEE位点确实是电压门控钙通道孔道中唯一的高亲和力钙结合位点。
