Severe defects in absorptive ion transport in distal colons of mice that lack ClC-2 channels

Abstract

Background & aims: The fluid secretion model predicts that intestinal obstruction disorders can be alleviated by promoting epithelial Cl(-) secretion. The adenosine 3',5'-cyclic monophosphate (cAMP)-activated anion channel CFTR mediates Cl(-)-dependent fluid secretion in the intestine. Although the role of the ClC-2 channel has not been determined in the intestine, this voltage-gated Cl(-) channel might compensate for the secretory defects observed in patients with cystic fibrosis and other chronic constipation disorders. We investigated whether mice that lack ClC-2 channels (Clcn2(-/-)) have defects in intestinal ion transport.

Methods: Immunolocalization and immunoblot analyses were used to determine the cellular localization and the amount of ClC-2 expressed in mouse early distal colon (EDC) and late distal colon (LDC). Colon sheets from wild-type and Clcn2(-/-) littermates were mounted in Ussing chambers to determine transepithelial bioelectrical parameters and Na(+), K(+), and Cl(-) fluxes.

Results: Expression of ClC-2 was higher in the basolateral membrane of surface cells in the EDC compared with the LDC, with little expression in crypts. Neither cAMP nor Ca(2+)-induced secretion of Cl(-) was affected in the EDC or LDC of Clcn2(-/-) mice, whereas the amiloride-sensitive short-circuit current was increased approximately 3-fold in Clcn2(-/-) EDC compared with control littermates. Conversely, electroneutral Na(+), K(+), and Cl(-) absorption was dramatically reduced in colons of Clcn2(-/-) mice.

Conclusions: Basolateral ClC-2 channels are required for colonic electroneutral absorption of NaCl and KCl. The increase in the amiloride-sensitive short-circuit current in Clcn2(-/-) mice revealed a compensatory mechanism that is activated in the colons of mice that lack the ClC-2 channel.

Figure 1. Amiloride-sensitive I_SC and R_TE in EDC and LDC

Representative current clamp experiments performed in EDC and LDC from wildtype (Panel A) and Clcn2^−/− (Panel B) epithelial sheets showing transepithelial potentials before and after addition of 10 μM amiloride to the mucosal side. C. Calculated amiloride-sensitive I_SC in EDC and LDC from wildtype and Clcn2^−/− mice. D. Transepithelial resistance values for EDC and LDC from wildtype and Clcn2^−/− mice before addition of amiloride. Indomethacin (5 μM) was present in the serosal side. Values are given as mean ± SEM from n=10 per experimental condition, except for Clcn2^−/− EDC (n=9). (*), p<0.05, t test. (**), p<0.01, t test between different and same genotypes, respectively.

Figure 2. cAMP- and Ca²⁺-activated Cl⁻ secretion in EDC from wildtype and Clcn2^−/− mice

Cl⁻ secretion was elicited by stimulating an increase in cAMP (100 μM IBMX +10 μM Forsk) and Ca²⁺ (50 μM CCh) in wildtype EDC (Panel A, n=10) and Clcn2^−/− EDC (Panel B, n=9). Before addition of CCh, 10 μM chromanol 293B was added to the serosal side to block basolateral cAMP-activated K⁺ channels, and consequently, cAMP-induced Cl⁻ secretion. Indomethacin (5 μM) was present in the serosal side. Representative experiments are shown in panels A and B.

Figure 3. Unidirectional and net Na⁺, Cl⁻ and K⁺⁻ fluxes in wildtype and Clcn2^−/− EDC

J_MS, J_SM and J_net for Na⁺ (A), Cl⁻ (B) and K⁺ (C) fluxes studies performed under short circuit conditions. Values are given as mean ± SEM; comparison of the same condition between wildtype and Clcn2^−/− mice where (*), p<0.05, and (**), p<0.01, t test. Amiloride (10 μM) was present in the apical side and 1 μM tetrodotoxin in the basolateral side.

Figure 4. ClC-2 channels are expressed in the mouse distal colon surface epithelium

Immunolocalization studies performed in EDC (upper panels) and LDC (lower panels) from wildtype (left panels) and Clcn2^−/− (right panels) mice. Bars = 50 μm.

Figure 5. Differential cellular expression of ClC-2 along the mouse distal colon

A. Higher magnification of the surface epithelium from wildtype EDC (left panel) and LDC (right panel). Bars = 20 μm. B. Western blot assays for ClC-2 protein abundance using biotinylated plasma membrane (left panel) and whole cell lysates (right panel) in the EDC and LDC from wildtype and Clcn2^−/− mice.