Role of aspartate residues in Ca²⁺ affinity and permeation of the distal ECaC1

Abstract

The Ca(2+) affinity and permeation of the epithelial Ca(2+) channel (ECaC1) were investigated after expression in Xenopus oocytes. ECaC1 displayed anomalous mole-fraction effects. Extracellular Ca(2+) and Mg(2+) reversibly inhibited ECaC1 whole cell Li(+) currents: IC(50) = 2.2 +/- 0.4 microM (n = 9) and 235 +/- 35 microM (n = 10), respectively. These values compare well with the Ca(2+) affinity of the L-type voltage-gated Ca(2+) (Ca(V)1.2) channel measured under the same conditions, suggesting that high-affinity Ca(2+) binding is a well-conserved feature of epithelial and voltage-gated Ca(2+) channels. Neutralization of D550 and E535 in the pore region had no significant effect on Ca(2+) and Mg(2+) affinities. In contrast, neutralization of D542 significantly decreased Ca(2+) affinity (IC(50) = 1.1 +/- 0.2 mM, n = 6) and Mg(2+) affinity (IC(50) > 25 +/- 3 mM, n = 4). Despite a 1,000-fold decrease in Ca(2+) affinity in D542N, Ca(2+) permeation properties and the Ca(2+)-to-Ba(2+) conductance ratio remained comparable to values for wild-type ECaC1. Together, our observations suggest that D542 plays a critical role in Ca(2+) affinity but not in Ca(2+) permeation in ECaC1.

Outer Pore Topology of the ECaC-TRPV5 Channel by Cysteine Scan Mutagenesis

“…Site-directed Mutagenesis of the Rabbit ECaC-TRPV5-The cDNAs coding for the wild-type ECaC-TRPV5 (GenBank TM AJ133128) (17) and the wild-type CaT2-TRPV5 (GenBank TM AF209196) (18) were obtained after reverse transcription of rabbit distal tubule mRNA as reported before (7). ECaC-TRPV5 and CaT2-TRPV5 were subcloned into the pT7TS vector (generously provided by Dr. Paul A. Krieg, University of Texas) using exonuclease III (19) for optimal expression in Xenopus laevis oocytes.…”

“…Run-off transcripts were prepared using methylated cap analog m 7 G(5Ј)ppp(5Ј)G and T7 RNA polymerase with the mMessage mMachine ® transcription kit (Ambion, Austin, TX). Expression of CaT2-TRPV5 Wild Type, ECaC-TRPV5 Wild Type, and Mutants in Xenopus Oocytes-Female Xenopus laevis clawed frog (Nasco, Fort Atkinson, WI) were anesthetized by immersion in 0.1% tricaine or MS-222 (3-aminobenzoic acid ethyl ester, Sigma) for 15 min before surgery as detailed before (7,20). cRNA was injected at a concentration of 0.46 -4.6 ng per oocyte depending upon the channel (wild type or mutant) being expressed.…”

“…Whole Cell Recordings-Whole cell currents were measured at room temperature with a two-electrode voltage-clamp amplifier (OC-725, Warner Instruments) as described before (7). Voltage and current electrodes (0.1-0.2 M⍀ tip resistance) were filled with 3 M KCl; 1 mM EGTA; 10 mM HEPES (pH 7.4).…”

“…In particular, ECaC-TRPV5 displays a high Ca 2ϩ affinity with a K d of Ϸ2 M (7) that is comparable to the K d of Ϸ1 M for voltage-dependent Ca V channels (9). A single residue in the S5-S6 linker (Asp 542 ) was found to account for the high Ca 2ϩ affinity of ECaC-TRPV5 (7). The absence of the aspartate residue at the equivalent position in the pore region of TRPV1-4 channels might explain, together with the presence of a lysine residue, the Ϸ20-fold lower Ca 2ϩ selectivity of TRPV1-4 channels (10).…”

Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

“…All of the nonpolar amino acids were replaced by a polar amino acid, Ser, and the polar amino acids were replaced by a small, nonpolar 1 The abbreviations used are: GFP, green fluorescence protein; GST, glutathione S-transferase. amino acid, Ala. Gly is classified as a polar amino acid and was thus replaced by Ala. Because it has been reported that the negatively charged amino acids Glu and Asp in the transmembrane domain contribute to ion selectivity in voltage-gated Ca 2ϩ channels (30,31) and ECaC (32,33) and to interaction with channel blockers (34,35), the remarkable Asp 341 was replaced by each of 7 amino acids, Glu, Lys, Arg, Thr, Asn, Ala, and Leu, and the positively charged amino acid His 353 was also replaced by each of 3 amino acids, Asp, Arg, and Ala. The mutant genes bearing these single amino acid substitutions were then introduced into cells of the mid1-⌬5 mutant, and the resulting transformants were examined for viability and Ca 2ϩ accumulation after exposure to ␣-factor.…”