Activation of an apical Ca²⁺-dependent Cl⁻ channel (CaCC) is the rate-limiting step for fluid secretion in many exocrine tissues. Here, we compared the properties of native CaCC in mouse submandibular salivary gland acinar cells to the Ca²⁺-gated Cl⁻ currents generated by Tmem16A and Best2, members from two distinct families of Ca²⁺-activated Cl⁻ channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca²⁺-activated Cl⁻ currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca²⁺-activated Cl⁻ current found in native salivary acinar cells. Best2^−/− and Tmem16A^−/− mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca²⁺-activated Cl⁻ current in submandibular gland acinar cells from Best2-deficient mice were the same as in wild type cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable with that in wild type mice. In contrast, submandibular gland acinar cells from Tmem16A^−/− mice lacked a Ca²⁺-activated Cl⁻ current and a Ca²⁺-mobilizing agonist failed to stimulate Cl⁻ efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wild type and Best2^−/− mice. Our results demonstrate that both Tmem16A and Best2 generate Ca²⁺-activated Cl⁻ current in vitro with similar properties to those expressed in native cells, yet only Tmem16A appears to be a critical component of the acinar Ca²⁺-activated Cl⁻ channel complex that is essential for saliva production by the submandibular gland.

The Cl– channel ClC-2 is expressed in transporting epithelia and has been proposed as an alternative route for Cl– efflux that might compensate for the malfunction of CFTR in cystic fibrosis. There is controversy concerning the cellular and membrane location of ClC-2, particularly in intestinal tissue. The aim of this paper is to resolve this controversy by immunolocalization studies using tissues from ClC-2 knockout animals as control, ascertaining the sorting of ClC-2 in model epithelial cells and exploring the possible molecular signals involved in ClC-2 targeting. ClC-2 was exclusively localized at the basolateral membranes of surface colonic cells or villus duodenal enterocytes. ClC-2 was sorted to the basolateral membranes in MDCK, Caco-2 and LLC-PK1-μ1B, but not in LLC-PK1-μ1A cells. Mutating a di-leucine motif (L812L813) to a di-alanine changed the basolateral targeting of ClC-2 to an apical location. The basolateral membrane localization of ClC-2 in absorptive cells of the duodenum and the colon is compatible with an absorptive function for this Cl– channel. Basolateral targeting information is contained in a di-leucine motif (L812L813) within CBS-2 domain at the C-terminus of ClC-2. It is speculated that ClC-2 also contains an apical sorting signal masked by L812L813. The proposal that CBS domains in ClC channels might behave as regulatory sites sensing intracellular signals opens an opportunity for pharmacological modulation of ClC-2 targeting.

Functional and structural studies demonstrate that Cl− channels of the ClC family have a dimeric double-barrelled structure, with each monomer contributing an identical pore. Single protopore gating ...

Fluid secretion by exocrine glands requires the coordinated activity of multiple water and ion transporter and channel proteins. The molecular cloning of many of the transporter molecules involved in fluid secretion has yielded a better understanding of the fluid secretion process. Mouse salivary glands are easily accessible model systems for the study of exocrine gland secretion at the cellular and organ level. Indeed, the characterization of mice with null mutations in many of the water and ion transporter and channel genes has demonstrated the physiological roles of individual proteins. This overview will focus on recent developments in determining the molecular identification of the proteins that are involved in the fluid secretion process. J. Med. Invest. 56 Suppl.: 192-196, December, 2009

Significance: There are no effective treatments for dry mouth, a common problem that affects millions of people in the United States. Salivary gland hyposecretion is caused by multiple diseases, including Sjögren's syndrome, as well as by radiation therapy and medications. The resulting dry mouth is often associated with dysphagia, reduced taste sensation, and opportunistic infections. The mammalian fluid secretion model predicts that Ca ²⁺ -activated Tmem16A and/or cAMP-dependent Cftr Cl ^- channels are critical in this process. We demonstrate that activation of the Tmem16A channel is required for muscarinic, Ca ²⁺ -dependent salivation, but that β-adrenergic receptor-mediated salivation is independent of Tmem16A, Cftr, and ClC-2 Cl ^- channels. A better understanding of the β-adrenergic stimulated secretion mechanism may prove important in the development of therapeutic targets for dry mouth.