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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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Relative anionic conductance and permeability in HEK293 and NCIH522 cellsA, representative currents recorded in HEK293 wild type cells during a voltage ramp protocol from a holding potential of −80 mV to +100 mV in control condition (black) and after substituting chloride with thiocyanate (grey). B, representative currents recorded in the same conditions in HEK293 cells stably transfected with PCIN5-hClCa1. Dashed lines in both A and B represent the  value of the current. C–E, summary histograms of the anionic permeability ratios (left) and the conductance ratios (right) obtained in the different cell types (see below). The relative anionic permeability is calculated from the shift of the reversal potential measured after substituting chloride with thiocyanate (SCN), iodide (I), bromide (Br), isethionate (Iseth) and gluconate (Gluc). The conductance was determined by dividing the measured current at +80 mV by the difference between the membrane potential and the membrane at which there is zero current. The relative conductance is the ratio of the conductance values obtained in different anionic conditions. C is a summary of the anionic selectivity measured in HEK293 wild type, stably transfected with PCIN5 or stably transfected with PCIN5-hClCa1 (clones 4 and 8). D is a summary of the anionic selectivity measured in the same condition as described above in HEK293 cells transiently transfected with pcDNA3 or in HEK293 transiently transfected PCDNA3-hClCa1 (hClCa1). E is a summary of the anionic selectivity measured in the same condition as described above in NCIH522 cells transiently transfected with pcDNA3 or in NCIH522 transiently transfected PCDNA3-hClCa1 (hClCa1).
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fig07: Relative anionic conductance and permeability in HEK293 and NCIH522 cellsA, representative currents recorded in HEK293 wild type cells during a voltage ramp protocol from a holding potential of −80 mV to +100 mV in control condition (black) and after substituting chloride with thiocyanate (grey). B, representative currents recorded in the same conditions in HEK293 cells stably transfected with PCIN5-hClCa1. Dashed lines in both A and B represent the value of the current. C–E, summary histograms of the anionic permeability ratios (left) and the conductance ratios (right) obtained in the different cell types (see below). The relative anionic permeability is calculated from the shift of the reversal potential measured after substituting chloride with thiocyanate (SCN), iodide (I), bromide (Br), isethionate (Iseth) and gluconate (Gluc). The conductance was determined by dividing the measured current at +80 mV by the difference between the membrane potential and the membrane at which there is zero current. The relative conductance is the ratio of the conductance values obtained in different anionic conditions. C is a summary of the anionic selectivity measured in HEK293 wild type, stably transfected with PCIN5 or stably transfected with PCIN5-hClCa1 (clones 4 and 8). D is a summary of the anionic selectivity measured in the same condition as described above in HEK293 cells transiently transfected with pcDNA3 or in HEK293 transiently transfected PCDNA3-hClCa1 (hClCa1). E is a summary of the anionic selectivity measured in the same condition as described above in NCIH522 cells transiently transfected with pcDNA3 or in NCIH522 transiently transfected PCDNA3-hClCa1 (hClCa1).

Mentions: The standard extracellular solution contained (in mm): NaCl (126), Hepes (10), sucrose (30), CaCl2 (2) and MgCl2 (2). The pH was set to 7.4 with NaOH. To quantify the chloride current, a Cl−-free external solution was used where gluconate replaced the Cl− in the standard external solution (using sodium gluconate, calcium gluconate and magnesium gluconate at the appropriate concentrations). In chloride substitution experiments described in Fig. 7, 126 mm external Cl− (Cl−o) was replaced by equimolar bromide (Br−), iodide (I−), thiocyanate (SCN−), isethionate (Ise−) or gluconate (Glc−). Patch pipettes (borosilicate glass) were pulled using a Sutter P-97 pipette puller (Instrument Company, Novato, CA, USA) and filled with a solution composed of (in mm): N-methyl-d-glucamine chloride (120), CaCl2 (4.36), MgCl2 (2), HEDTA (8) and Hepes (10), pH 7.1. We quantified the response at steady state by buffering and estimating the actual free intracellular concentration (as described in Kuruma & Hartzell, 2000). The concentration of free Ca2+ in this solution was calculated to be 10 μm using the software WinMAXC (v.2.05; Chris Patton, Stanford, CA, USA). In the experiments where different [Ca2+]i were used, the pipette solution contained: (a) 1.69 mm CaCl2 and 5 mm EGTA; (b) 3.95 mm CaCl2 and 5 mm EGTA; (c) 1.87 mm CaCl2 and 5 mm HEDTA; (d) 1.47 mm CaCl2 and 5 mm nitrilotriacetic acid (NTA); (e) 3.35 mm CaCl2 and 5 mm NTA to give a final free [Ca2+]i of: (a) 130 nm, (b) 1 μm, (c) 5 μm, (d) 100 μm and (e) 486 μm, respectively. Osmolarity was adjusted to 295 mosmol l–1 with sucrose. Currents elicited by 1 or 2 mm[Ca2+]i were recorded with a pipette solution described by Gruber et al. (1998) and Britton et al. (2002) that contained (in mm): NMDG-Cl (112), sucrose (30), Hepes (5), MgCl2 (2) and CaCl2 (1) or (2).


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)

Relative anionic conductance and permeability in HEK293 and NCIH522 cellsA, representative currents recorded in HEK293 wild type cells during a voltage ramp protocol from a holding potential of −80 mV to +100 mV in control condition (black) and after substituting chloride with thiocyanate (grey). B, representative currents recorded in the same conditions in HEK293 cells stably transfected with PCIN5-hClCa1. Dashed lines in both A and B represent the  value of the current. C–E, summary histograms of the anionic permeability ratios (left) and the conductance ratios (right) obtained in the different cell types (see below). The relative anionic permeability is calculated from the shift of the reversal potential measured after substituting chloride with thiocyanate (SCN), iodide (I), bromide (Br), isethionate (Iseth) and gluconate (Gluc). The conductance was determined by dividing the measured current at +80 mV by the difference between the membrane potential and the membrane at which there is zero current. The relative conductance is the ratio of the conductance values obtained in different anionic conditions. C is a summary of the anionic selectivity measured in HEK293 wild type, stably transfected with PCIN5 or stably transfected with PCIN5-hClCa1 (clones 4 and 8). D is a summary of the anionic selectivity measured in the same condition as described above in HEK293 cells transiently transfected with pcDNA3 or in HEK293 transiently transfected PCDNA3-hClCa1 (hClCa1). E is a summary of the anionic selectivity measured in the same condition as described above in NCIH522 cells transiently transfected with pcDNA3 or in NCIH522 transiently transfected PCDNA3-hClCa1 (hClCa1).
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fig07: Relative anionic conductance and permeability in HEK293 and NCIH522 cellsA, representative currents recorded in HEK293 wild type cells during a voltage ramp protocol from a holding potential of −80 mV to +100 mV in control condition (black) and after substituting chloride with thiocyanate (grey). B, representative currents recorded in the same conditions in HEK293 cells stably transfected with PCIN5-hClCa1. Dashed lines in both A and B represent the value of the current. C–E, summary histograms of the anionic permeability ratios (left) and the conductance ratios (right) obtained in the different cell types (see below). The relative anionic permeability is calculated from the shift of the reversal potential measured after substituting chloride with thiocyanate (SCN), iodide (I), bromide (Br), isethionate (Iseth) and gluconate (Gluc). The conductance was determined by dividing the measured current at +80 mV by the difference between the membrane potential and the membrane at which there is zero current. The relative conductance is the ratio of the conductance values obtained in different anionic conditions. C is a summary of the anionic selectivity measured in HEK293 wild type, stably transfected with PCIN5 or stably transfected with PCIN5-hClCa1 (clones 4 and 8). D is a summary of the anionic selectivity measured in the same condition as described above in HEK293 cells transiently transfected with pcDNA3 or in HEK293 transiently transfected PCDNA3-hClCa1 (hClCa1). E is a summary of the anionic selectivity measured in the same condition as described above in NCIH522 cells transiently transfected with pcDNA3 or in NCIH522 transiently transfected PCDNA3-hClCa1 (hClCa1).
Mentions: The standard extracellular solution contained (in mm): NaCl (126), Hepes (10), sucrose (30), CaCl2 (2) and MgCl2 (2). The pH was set to 7.4 with NaOH. To quantify the chloride current, a Cl−-free external solution was used where gluconate replaced the Cl− in the standard external solution (using sodium gluconate, calcium gluconate and magnesium gluconate at the appropriate concentrations). In chloride substitution experiments described in Fig. 7, 126 mm external Cl− (Cl−o) was replaced by equimolar bromide (Br−), iodide (I−), thiocyanate (SCN−), isethionate (Ise−) or gluconate (Glc−). Patch pipettes (borosilicate glass) were pulled using a Sutter P-97 pipette puller (Instrument Company, Novato, CA, USA) and filled with a solution composed of (in mm): N-methyl-d-glucamine chloride (120), CaCl2 (4.36), MgCl2 (2), HEDTA (8) and Hepes (10), pH 7.1. We quantified the response at steady state by buffering and estimating the actual free intracellular concentration (as described in Kuruma & Hartzell, 2000). The concentration of free Ca2+ in this solution was calculated to be 10 μm using the software WinMAXC (v.2.05; Chris Patton, Stanford, CA, USA). In the experiments where different [Ca2+]i were used, the pipette solution contained: (a) 1.69 mm CaCl2 and 5 mm EGTA; (b) 3.95 mm CaCl2 and 5 mm EGTA; (c) 1.87 mm CaCl2 and 5 mm HEDTA; (d) 1.47 mm CaCl2 and 5 mm nitrilotriacetic acid (NTA); (e) 3.35 mm CaCl2 and 5 mm NTA to give a final free [Ca2+]i of: (a) 130 nm, (b) 1 μm, (c) 5 μm, (d) 100 μm and (e) 486 μm, respectively. Osmolarity was adjusted to 295 mosmol l–1 with sucrose. Currents elicited by 1 or 2 mm[Ca2+]i were recorded with a pipette solution described by Gruber et al. (1998) and Britton et al. (2002) that contained (in mm): NMDG-Cl (112), sucrose (30), Hepes (5), MgCl2 (2) and CaCl2 (1) or (2).

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