Secreted CLCA1 modulates TMEM16A to activate Ca2+-dependent chloride currents in human cells

Calcium-activated chloride channel regulator 1 (CLCA1) activates calcium-dependent chloride currents; neither the target, nor mechanism, is known. We demonstrate that secreted CLCA1 activates calcium-dependent chloride currents in HEK293T cells in a paracrine fashion, and endogenous TMEM16A/Anoctamin1 conducts the currents. Exposure to exogenous CLCA1 increases cell surface levels of TMEM16A and cellular binding experiments indicate CLCA1 engages TMEM16A on the surface of these cells. Altogether, our data suggest that CLCA1 stabilizes TMEM16A on the cell surface, thus increasing surface expression, which results in increased calcium-dependent chloride currents. Our results identify the first Cl(−) channel target of the CLCA family of proteins and establish CLCA1 as the first secreted direct modifier of TMEM16A activity, delineating a unique mechanism to increase currents. These results suggest cooperative roles for CLCA and TMEM16 proteins in influencing the physiology of multiple tissues, and the pathology of multiple diseases, including asthma, COPD, cystic fibrosis, and certain cancers. DOI: http://dx.doi.org/10.7554/eLife.05875.00

Novel roles for chloride channels, exchangers, and regulators in chronic inflammatory airway diseases

Chloride transport proteins play critical roles in inflammatory airway diseases, contributing to the detrimental aspects of mucus overproduction, mucus secretion, and airway constriction. However, they also play crucial roles in contributing to the innate immune properties of mucus and mucociliary clearance. In this review, we focus on the emerging novel roles for a chloride channel regulator (CLCA1), a calcium-activated chloride channel (TMEM16A), and two chloride exchangers (SLC26A4/pendrin and SLC26A9) in chronic inflammatory airway diseases

Cantu syndrome–associated SUR2 (ABCC9) mutations in distinct structural domains result in KATP channel gain-of-function by differential mechanisms

The complex disorder Cantu syndrome (CS) arises from gainof-function mutations in either KCNJ8 or ABCC9, the genes encoding the Kir6.1 and SUR2 subunits of ATP-sensitive potassium (KATP) channels, respectively. Recent reports indicate that such mutations can increase channel activity by multiple molecular mechanisms. In this study, we determined the mechanism by which KATP function is altered by several substitutions in distinct structural domains of SUR2: D207E in the intracellular L0-linker and Y985S, G989E, M1060I, and R1154Q/R1154W in TMD2. We engineered substitutions at their equivalent positions in rat SUR2A (D207E, Y981S, G985E, M1056I, and R1150Q/R1150W) and investigated functional consequences using macroscopic rubidium (86Rb-) efflux assays and patchclamp electrophysiology. Our results indicate that D207E increases KATP channel activity by increasing intrinsic stability of the open state, whereas the cluster of Y981S/G985E/M1056I substitutions, as well as R1150Q/R1150W, augmented Mg-nucleotide activation. We also tested the responses of these channel variants to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS. None of the D207E, Y981S, G985E, or M1056I substitutions had a significant effect on glibenclamide sensitivity. However, Gln and Trp substitution at Arg-1150 significantly decreased glibenclamide potency. In summary, these results provide additional confirmation that mutations in CS-Associated SUR2 mutations result in KATP gain-of-function. They help link CS genotypes to phenotypes and shed light on the underlying molecular mechanisms, including consequences for inhibitory drug sensitivity, insights that may inform the development of therapeutic approaches to manage CS

Effect of diaminopropionic acid (Dap) on the biophysical properties of a modified synthetic channel-forming peptide

Citation: Bukovnik, U., Sala-Rabanal, M., Francis, S., Frazier, S. J., Schultz, B. D., Nichols, C. G., & Tomich, J. M. (2013). Effect of diaminopropionic acid (Dap) on the biophysical properties of a modified synthetic achannel-forming peptide. Molecular Pharmaceutics, 10(10), 3959-3966.Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK₄-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, incorporated into cell membranes and was nonimmunogenic, but lacked the structural properties for high conductance and anion selectivity when assembled into a pore. Further studies suggested that the threonine residues at positions 13, 17, and 20 line the pore of assembled p22-T19R, S22W, and here we used 2,3-diaminopropionic acid (Dap) substitutions to introduce positive charges to the pore-lining interface of the predicted p22-T19R, S22W channel. Dap-substituted p22-T19R, S22W peptides retained the α-helical secondary structure characteristic of their parent peptide, and induced short-circuit transepithelial currents when exposed to the apical membrane of Madin-Darby canine kidney (MDCK) cells; the sequences containing multiple Dap-substituted residues induced larger currents than the peptides with single or no Dap substitutions. To gain further insights into the effects of Dap residues on the properties of the putative pore, we performed two-electrode voltage clamp electrophysiology on Xenopus oocytes exposed to p22-T19R, S22W or its Dap-modified analogues. We observed that Dap-substituted peptides also induced significantly larger voltage-dependent currents than the parent compound, but there was no apparent change in reversal potential upon replacement of external Na⁺, Cl⁻ or K⁺, indicating that these currents remained nonselective. These results suggest that the introduction of positively charged side chains in predicted pore-lining residues does not improve anion-to-cation selectivity, but results in higher conductance, perhaps due to higher oligomerization numbers