Abstract
Molecular cloning studies have revealed that heterogeneity of T-type Ca2+ currents in native tissues arises from the three isoforms of Ca(v)3 channels: Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3. From pharmacological analysis of the recombinant T-type channels, low concentrations (<50 microM) of nickel were found to selectively block the Ca(v)3.2 over the other isoforms. To date, however, the structural element(s) responsible for the nickel block on the Ca(v)3.2 T-type Ca2+ channel remain unknown. Thus, we constructed chimeric channels between the nickel-sensitive Ca(v)3.2 and the nickel-insensitive Ca(v)3.1 to localize the region interacting with nickel. Systematic assaying of serial chimeras suggests that the region preceding domain I S4 of Ca(v)3.2 contributes to nickel block. Point mutations of potential nickel-interacting sites revealed that H191Q in the S3-S4 loop of domain I significantly attenuated the nickel block of Ca(v)3.2, mimicking the nickel-insensitive blocking potency of Ca(v)3.1. These findings indicate that His-191 in the S3-S4 loop is a critical residue conferring nickel block to Ca(v)3.2 and reveal a novel role for the S3-S4 loop to control ion permeation through T-type Ca2+ channels.
Highlights
In low voltage-activated channels two of these glutamates were replaced by aspartates (D), creating an EEDD locus
Replacement of these aspartates in Cav3.1 channels with glutamates confers a cadmiumsensitive block to Cav3.1, which requires much higher concentrations of cadmium to be blocked than HVA calcium channels [29]
Our previous studies indicated that nickel blocks Cav3 channels in part by binding within the permeation path [19], presumably because of binding at the EEDD locus
Summary
Chemicals—Nickel (II) chloride hexahydrate (NiCl21⁄76H2O) was obtained from Sigma HGGG/E137Q—The forward and reverse primers to amplify the upper fragments covering from the 5Ј-polylinker to 499 (nucleotide number of Cav3.2) were 5Ј-TAATACGACTCACTATAGGG-3Ј (T7 promoter sequence) and 5Ј-TCAAAGGCCTGCAGGATGTTGCAGCGCTC-3Ј, respectively. The plasmid HGGG/E137Q was constructed by inserting the PCR-generated DNA cassette digested with ClaI and SalI into the plasmid Cav3.1 pGEM-HEA, which was opened by ClaI (5Ј-polylinker) and SalI (1552, Cav3.1). HGGG/H191Q—The forward and reverse primers to amplify the upper fragments covering from the 5Ј-polylinker to 682 (nucleotide number of Cav3.2) were T7 promoter sequence and 5Ј-GGCTCACGTTCTGTCCGTCCAACGAGTACTC-3Ј, respectively. H191Q was constructed by inserting the PCR-generated DNA cassette digested with ClaI and SalI into the plasmid Cav3.1 pGEM-HEA, which was opened by ClaI (5Ј-polylinker) and SalI (1552, Cav3.1). The plasmid Cav3.1/Q172H was constructed by inserting the PCR-generated DNA cassette digested with ClaI and HindIII into the plasmid Cav3.1 pGEM-HEA, which was opened by ClaI (5Ј-polylinker) and HindIII (1755, Cav3.1). Data are presented as means Ϯ S.E. and tested for significance using Student’s unpaired t test
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
