The EEEE Locus Is the Sole High-affinity Ca 2 1 Binding Structure in the Pore of a Voltage-gated Ca 2 1 Channel Block by Ca 2 1 Entering from the Intracellular Pore Entrance

Selective permeability in voltage-gated Ca 2 1 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 1 binding site in the pore, which represents an overturning of earlier models that had postulated two high-affinity Ca 2 1 binding sites. The current view is based on site-directed mutagenesis work in which Ca 2 1 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 1 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 1 produced substantial block of outward Li 1 current in wild-type channels, internal Ca 2 1 concentrations up to 1 mM did not produce detectable block of outward Li 1 current in the AAAA or QQQQ mutants. These results indicate that the EEEE locus is indeed the sole high-affinity Ca 2 1 binding locus in the pore of voltage-gated Ca 2 1 channels. key words: ion selectivity • selectivity filter • L-type Ca 2 1 channel • patch clamp • cysteine mutagenesis I N T R O D U C T I O N For voltage-gated Ca 2 1 channels, selective permeability is believed to involve interaction between two or more permeant ions. However, the number of pore-localized ion binding sites that may mediate these interactions has been contentious, with assertions of two or more binding sites (Almers and McCleskey, 1984; Hess and Tsien, 1984; Hess et al., 1986; Lansman et al., 1986; Friel and Tsien, 1989; Yue and Marban, 1990; Rosenberg and Chen, 1991; Kuo and Hess, 1993a,b,c), one binding site (Kostyuk et al., 1983; Kostyuk and Mironov, 1986; Lux et al., 1990; Armstrong and Neyton, 1991), or no discrete binding sites (Nonner and Eisenberg, 1998) made by various researchers. Two-site models have been the most widely considered (McCleskey, 1999; Miller, 1999), and they are similar in nature to the multi-ion, multi-site models that have been used to describe block and flux in voltage-gated K 1 channels (Hodgkin and Keynes, 1955; Hille and Schwarz, 1978; Neyton and Miller, 1988a,b). The general validity of these kinds of multi-ion, multi-site models has been strongly supported by the recently obtained crystal structure of a bacterial K 1 channel (Doyle et al., 1998). This structure reveals a narrow, single-file selectivity filter containing a pair of discrete sites of dehydrated metal ion localization (binding) separated by a short distance of z 7.5 Å. This small separation very likely allows significant ion–ion interaction to occur within the K 1 channel’s pore, as has also been postulated for Ca 2 1 channels. In the case of Ca 2 1 channels, tight binding of one Ca 2 1 ion within the single-file region of the pore obstructs permeation by foreign ions (selectivity), whereas Ca 2 1 –Ca 2 1 interaction inside the pore overcomes tight Ca 2 1 binding to generate high Ca 2 1 throughput (unitary Ca 2 1 current). These two processes can be observed in an experimental setting, where the probability of pore occupancy by Ca 2 1 can be manipulated: with approximately micromolar Ca 2 1 in the bath, the probability of single occupancy by Ca 2 1 is high, so that Ca 2 1 effectively blocks monovalent metal cation (e.g., Na 1 ) flux; with approximately millimolar Ca 2 1 in the bath, the probability of double occupancy by Ca 2 1 is high, so that Ca 2 1 –Ca 2 1 interactions within the pore are frequent Address correspondence to William A. Sather, Neuroscience Center, B-138, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262. Fax: 303-315-2503; E-mail: william.sather@uchsc.edu on A ril 9, 2017 D ow nladed fom Published August 14, 2000