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Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

Plog S, Klymiuk N, Binder S, Van Hook MJ, Thoreson WB, Gruber AD, Mundhenk L - PLoS ONE (2015)

Bottom Line: Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals.Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins.Moreover, the naturally occurring variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathology, Faculty of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.

ABSTRACT
The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

No MeSH data available.


Related in: MedlinePlus

CLCA4b does not evoke a calcium-dependent chloride current characteristic of other CLCA family members.(A) Currents were evoked in HEK293 cells expressing CLCA4b (wt) by a series of voltage steps (150 ms, -110 to -10 mV) before (left) and after (right) application of the calcium ionophore ionomycin (10 μM). Ionomycin stimulated an inward current at the holding potential of -50 mV and increased the amplitude of step-evoked currents. (B) Similar experiment for HEK293 cells expressing CLCA4b (mut). In A and B, zero current is indicated by the dotted lines. (C) Current-voltage relationship for the difference currents were obtained by subtracting the step-evoked currents before from those after ionomycin application. Linear extrapolation was used to determine the reversal potential (Erev) for the ionomycin-stimulated currents which were normalized to the current at the holding potential (-50 mV). (D) Mean Erev for ionomycin-stimulated currents in cells expressing CLCA4b (wt) (n = 10), CLCA4b (mut) (n = 13), CLCA1 (n = 8), serving as a positive control, or EGFP alone (n = 8), which served as a negative control. *p < 0.005, n.s. p > 0.05, independent t-test.
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pone.0140050.g005: CLCA4b does not evoke a calcium-dependent chloride current characteristic of other CLCA family members.(A) Currents were evoked in HEK293 cells expressing CLCA4b (wt) by a series of voltage steps (150 ms, -110 to -10 mV) before (left) and after (right) application of the calcium ionophore ionomycin (10 μM). Ionomycin stimulated an inward current at the holding potential of -50 mV and increased the amplitude of step-evoked currents. (B) Similar experiment for HEK293 cells expressing CLCA4b (mut). In A and B, zero current is indicated by the dotted lines. (C) Current-voltage relationship for the difference currents were obtained by subtracting the step-evoked currents before from those after ionomycin application. Linear extrapolation was used to determine the reversal potential (Erev) for the ionomycin-stimulated currents which were normalized to the current at the holding potential (-50 mV). (D) Mean Erev for ionomycin-stimulated currents in cells expressing CLCA4b (wt) (n = 10), CLCA4b (mut) (n = 13), CLCA1 (n = 8), serving as a positive control, or EGFP alone (n = 8), which served as a negative control. *p < 0.005, n.s. p > 0.05, independent t-test.

Mentions: Whole-cell recordings were obtained from CLCA4b-expressing HEK293 cells as confirmed by EGFP or RFP fluorescence (Fig 5). Shortly after application of the calcium ionophore ionomycin (10 μM), we observed a slowly-developing inward current. In cells transfected with cDNA coding for CLCA4b (wt) or CLCA4b (mut), this current reversed near the estimated equilibrium potential for calcium (Ca2+) (CLCA4b wt: +47 ± 6 mV, n = 10; CLCA4b mut: +52 ± 12 mV, n = 13; Fig 5C and 5D). In HEK cells transfected with EGFP alone, the ionomycin-evoked current reversed at +35 ± 6 mV (n = 8) which did not differ significantly from CLCA4b (wt)- or CLCA4b (mut)-transfected cells (p > 0.05). The inward current under these conditions is likely the result of the Ca2+ conductance created by insertion of the ionomycin ionophore into the membrane, suggesting that elevated intracellular [Ca2+] did not trigger a chloride (Cl-) conductance in cells expressing CLCA4b (wt), CLCA4b (mut) or EGFP alone. In contrast, we successfully recorded ionomycin-evoked anion currents in cells transfected with cDNA for CLCA1, a member of the CLCA family known to induce a calcium-activated chloride conductance [32]. As with cells transfected with CLCA4b (wt) or CLCA4b (mut), application of ionomycin was followed by an inward current, however, this current reversed at 11 ± 6 mV (n = 8; Fig 5D) and developed more quickly. This reversal potential was significantly less positive than that for CLCA4b (wt) (p<0.005, independent t-test) or for CLCA4b (mut) (p<0.01, independent t-test), and fell between the equilibrium potentials for Ca2+ and Cl-, suggesting that elevated intracellular [Ca2+] triggered a Cl- conductance in cells expressing the equine CLCA1, consistent with its previous characterization [34].


Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

Plog S, Klymiuk N, Binder S, Van Hook MJ, Thoreson WB, Gruber AD, Mundhenk L - PLoS ONE (2015)

CLCA4b does not evoke a calcium-dependent chloride current characteristic of other CLCA family members.(A) Currents were evoked in HEK293 cells expressing CLCA4b (wt) by a series of voltage steps (150 ms, -110 to -10 mV) before (left) and after (right) application of the calcium ionophore ionomycin (10 μM). Ionomycin stimulated an inward current at the holding potential of -50 mV and increased the amplitude of step-evoked currents. (B) Similar experiment for HEK293 cells expressing CLCA4b (mut). In A and B, zero current is indicated by the dotted lines. (C) Current-voltage relationship for the difference currents were obtained by subtracting the step-evoked currents before from those after ionomycin application. Linear extrapolation was used to determine the reversal potential (Erev) for the ionomycin-stimulated currents which were normalized to the current at the holding potential (-50 mV). (D) Mean Erev for ionomycin-stimulated currents in cells expressing CLCA4b (wt) (n = 10), CLCA4b (mut) (n = 13), CLCA1 (n = 8), serving as a positive control, or EGFP alone (n = 8), which served as a negative control. *p < 0.005, n.s. p > 0.05, independent t-test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608703&req=5

pone.0140050.g005: CLCA4b does not evoke a calcium-dependent chloride current characteristic of other CLCA family members.(A) Currents were evoked in HEK293 cells expressing CLCA4b (wt) by a series of voltage steps (150 ms, -110 to -10 mV) before (left) and after (right) application of the calcium ionophore ionomycin (10 μM). Ionomycin stimulated an inward current at the holding potential of -50 mV and increased the amplitude of step-evoked currents. (B) Similar experiment for HEK293 cells expressing CLCA4b (mut). In A and B, zero current is indicated by the dotted lines. (C) Current-voltage relationship for the difference currents were obtained by subtracting the step-evoked currents before from those after ionomycin application. Linear extrapolation was used to determine the reversal potential (Erev) for the ionomycin-stimulated currents which were normalized to the current at the holding potential (-50 mV). (D) Mean Erev for ionomycin-stimulated currents in cells expressing CLCA4b (wt) (n = 10), CLCA4b (mut) (n = 13), CLCA1 (n = 8), serving as a positive control, or EGFP alone (n = 8), which served as a negative control. *p < 0.005, n.s. p > 0.05, independent t-test.
Mentions: Whole-cell recordings were obtained from CLCA4b-expressing HEK293 cells as confirmed by EGFP or RFP fluorescence (Fig 5). Shortly after application of the calcium ionophore ionomycin (10 μM), we observed a slowly-developing inward current. In cells transfected with cDNA coding for CLCA4b (wt) or CLCA4b (mut), this current reversed near the estimated equilibrium potential for calcium (Ca2+) (CLCA4b wt: +47 ± 6 mV, n = 10; CLCA4b mut: +52 ± 12 mV, n = 13; Fig 5C and 5D). In HEK cells transfected with EGFP alone, the ionomycin-evoked current reversed at +35 ± 6 mV (n = 8) which did not differ significantly from CLCA4b (wt)- or CLCA4b (mut)-transfected cells (p > 0.05). The inward current under these conditions is likely the result of the Ca2+ conductance created by insertion of the ionomycin ionophore into the membrane, suggesting that elevated intracellular [Ca2+] did not trigger a chloride (Cl-) conductance in cells expressing CLCA4b (wt), CLCA4b (mut) or EGFP alone. In contrast, we successfully recorded ionomycin-evoked anion currents in cells transfected with cDNA for CLCA1, a member of the CLCA family known to induce a calcium-activated chloride conductance [32]. As with cells transfected with CLCA4b (wt) or CLCA4b (mut), application of ionomycin was followed by an inward current, however, this current reversed at 11 ± 6 mV (n = 8; Fig 5D) and developed more quickly. This reversal potential was significantly less positive than that for CLCA4b (wt) (p<0.005, independent t-test) or for CLCA4b (mut) (p<0.01, independent t-test), and fell between the equilibrium potentials for Ca2+ and Cl-, suggesting that elevated intracellular [Ca2+] triggered a Cl- conductance in cells expressing the equine CLCA1, consistent with its previous characterization [34].

Bottom Line: Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals.Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins.Moreover, the naturally occurring variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathology, Faculty of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany.

ABSTRACT
The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

No MeSH data available.


Related in: MedlinePlus