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Human ClCa1 modulates anionic conduction of calcium-dependent chloride currents.

Hamann M, Gibson A, Davies N, Jowett A, Walhin JP, Partington L, Affleck K, Trezise D, Main M - J. Physiol. (Lond.) (2009)

Bottom Line: We further show that hClCa1 does not modify the permeability sequence but increases the Cl- conductance while decreasing the G(SCN-)/G(Cl-) conductance ratio from approximately 2-3 to approximately 1.We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers.Rather, hClCa1 elevates the single channel conductance of endogenous Ca(2+)-dependent Cl- channels by lowering the energy barriers for ion translocation through the pore.

View Article: PubMed Central - PubMed

Affiliation: Leicester University, Department of Cell Physiology and Pharmacology, Medical Sciences Building, PO Box 138, University Road, Leicester LE1 9HN, UK. mh86@le.ac.uk

ABSTRACT
Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca(2+)-dependent Cl- currents. The present study examines the relationship between the hClCa1 protein and Ca(2+)-dependent Cl- currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl- currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca(2+)-dependent Cl- currents and show that hClCa1 increases the amplitude of Ca(2+)-dependent Cl- currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl- conductance while decreasing the G(SCN-)/G(Cl-) conductance ratio from approximately 2-3 to approximately 1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca(2+)-dependent Cl- channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca(2+)-dependent Cl- channels by lowering the energy barriers for ion translocation through the pore.

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Taqman quantitative PCR analysis of ClCa mRNA protein subtypes in HEK293 cellsThe mRNA is extracted from HEK293 wild type (WT) and HEK293 cells stably expressing PCIN5 (PCIN5) or pCIN5-hCCla1 (clonal cultures C2 to C8). Quantitative PCR analysis was performed for A, hClCa1; B, hClCa2; C, β actin; D, GAPDH.
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fig01: Taqman quantitative PCR analysis of ClCa mRNA protein subtypes in HEK293 cellsThe mRNA is extracted from HEK293 wild type (WT) and HEK293 cells stably expressing PCIN5 (PCIN5) or pCIN5-hCCla1 (clonal cultures C2 to C8). Quantitative PCR analysis was performed for A, hClCa1; B, hClCa2; C, β actin; D, GAPDH.

Mentions: We screened for the presence of the hClCa1 gene in different HEK293 clones stably transfected with pCIN5_pl-hClCa1 DNA using a highly sensitive and quantitative PCR approach and proceeded to a quantitative analysis of the hClCa1 gene in those cell lines. Figure 1A shows that hClCa1 gene expression is detected at different levels within different HEK293 clones stably transfected with pCIN5_pl-hClCa1 DNA, ranging from 1.4 × 106 to over 10.5 × 106 copies of mRNA detected per 50 ng−1 mRNA pool for clone 6 and 4, respectively. By comparison, the hClCa1 gene is undetected in HEK293 wild type and in HEK293 stably transfected with the pCIN5 vector with less than 1 × 103 copies of mRNA detected per 50 ng mRNA pool. The quantitative analysis of hClCa2 and hClCa4 mRNAs was also performed in the same cell types and showed that by contrast to hClCa1 specifically expressed at high levels in HEK293 clones stably transfected with pCIN5_pl-hCLCA1 DNA, hClCa4 and hClCa2 mRNA was undetected in those cells, with less than 1 × 103 and 4 × 103 copies of mRNA per 50 ng−1 mRNA pool for hClCa4 and hClCa2, respectively. Those low expression levels are significantly different from control β actin and GAPDH genes ranging from 0.5 × 106 to over 2 × 106 copies of mRNA detected per 50 ng−1 mRNA pool (Fig. 1B–D). Similar experiments have also been performed in NCIH522 or HEK293 transiently transfected with GFP and pcDNA3.1(+)-hClCa1 in which we confirmed the presence of the hClCa1 (see Fig. 6A for NCIH522).


Human ClCa1 modulates anionic conduction of calcium-dependent chloride currents.

Hamann M, Gibson A, Davies N, Jowett A, Walhin JP, Partington L, Affleck K, Trezise D, Main M - J. Physiol. (Lond.) (2009)

Taqman quantitative PCR analysis of ClCa mRNA protein subtypes in HEK293 cellsThe mRNA is extracted from HEK293 wild type (WT) and HEK293 cells stably expressing PCIN5 (PCIN5) or pCIN5-hCCla1 (clonal cultures C2 to C8). Quantitative PCR analysis was performed for A, hClCa1; B, hClCa2; C, β actin; D, GAPDH.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691903&req=5

fig01: Taqman quantitative PCR analysis of ClCa mRNA protein subtypes in HEK293 cellsThe mRNA is extracted from HEK293 wild type (WT) and HEK293 cells stably expressing PCIN5 (PCIN5) or pCIN5-hCCla1 (clonal cultures C2 to C8). Quantitative PCR analysis was performed for A, hClCa1; B, hClCa2; C, β actin; D, GAPDH.
Mentions: We screened for the presence of the hClCa1 gene in different HEK293 clones stably transfected with pCIN5_pl-hClCa1 DNA using a highly sensitive and quantitative PCR approach and proceeded to a quantitative analysis of the hClCa1 gene in those cell lines. Figure 1A shows that hClCa1 gene expression is detected at different levels within different HEK293 clones stably transfected with pCIN5_pl-hClCa1 DNA, ranging from 1.4 × 106 to over 10.5 × 106 copies of mRNA detected per 50 ng−1 mRNA pool for clone 6 and 4, respectively. By comparison, the hClCa1 gene is undetected in HEK293 wild type and in HEK293 stably transfected with the pCIN5 vector with less than 1 × 103 copies of mRNA detected per 50 ng mRNA pool. The quantitative analysis of hClCa2 and hClCa4 mRNAs was also performed in the same cell types and showed that by contrast to hClCa1 specifically expressed at high levels in HEK293 clones stably transfected with pCIN5_pl-hCLCA1 DNA, hClCa4 and hClCa2 mRNA was undetected in those cells, with less than 1 × 103 and 4 × 103 copies of mRNA per 50 ng−1 mRNA pool for hClCa4 and hClCa2, respectively. Those low expression levels are significantly different from control β actin and GAPDH genes ranging from 0.5 × 106 to over 2 × 106 copies of mRNA detected per 50 ng−1 mRNA pool (Fig. 1B–D). Similar experiments have also been performed in NCIH522 or HEK293 transiently transfected with GFP and pcDNA3.1(+)-hClCa1 in which we confirmed the presence of the hClCa1 (see Fig. 6A for NCIH522).

Bottom Line: We further show that hClCa1 does not modify the permeability sequence but increases the Cl- conductance while decreasing the G(SCN-)/G(Cl-) conductance ratio from approximately 2-3 to approximately 1.We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers.Rather, hClCa1 elevates the single channel conductance of endogenous Ca(2+)-dependent Cl- channels by lowering the energy barriers for ion translocation through the pore.

View Article: PubMed Central - PubMed

Affiliation: Leicester University, Department of Cell Physiology and Pharmacology, Medical Sciences Building, PO Box 138, University Road, Leicester LE1 9HN, UK. mh86@le.ac.uk

ABSTRACT
Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca(2+)-dependent Cl- currents. The present study examines the relationship between the hClCa1 protein and Ca(2+)-dependent Cl- currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl- currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca(2+)-dependent Cl- currents and show that hClCa1 increases the amplitude of Ca(2+)-dependent Cl- currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl- conductance while decreasing the G(SCN-)/G(Cl-) conductance ratio from approximately 2-3 to approximately 1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca(2+)-dependent Cl- channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca(2+)-dependent Cl- channels by lowering the energy barriers for ion translocation through the pore.

Show MeSH
Related in: MedlinePlus