title: Influence of intracellular Ca2+ and alternative splicing on the pharmacological profile of ANO1 channels.
abstract: Anoctamin-1 (ANO1) is a Ca(2+)-activated Cl(-) channel expressed in many types of cells. Splice variants of ANO1 have been shown to influence the biophysical properties of conductance. It has been suggested that several new antagonists of ANO1 with relatively high affinity and selectivity might be useful for experimental and, potentially, therapeutic purposes. We investigated the effects of intracellular Ca(2+) concentration ([Ca(2+)]i) at 100-1,000 nM, a concentration range that might be achieved in cells during physiological activation of ANO1 channels, on blockade of ANO1 channels expressed in HEK-293 cells. Whole cell and excised patch configurations of the patch-clamp technique were used to perform tests on a variety of naturally occurring splice variants of ANO1. Blockade of ANO1 currents with aminophenylthiazole (T16Ainh-A01) was highly dependent on [Ca(2+)]i Increasing [Ca(2+)]i reduced the potency of this blocker. Similar Ca(2+)-dependent effects were also observed with benzbromarone. Experiments on excised, inside-out patches showed that the diminished potency of the blockers caused by intracellular Ca(2+) might involve a competitive interaction for a common binding site or repulsion of the blocking drugs by electrostatic forces at the cytoplasmic surface of the channels. The degree of interaction between the channel blockers and [Ca(2+)]i depends on the splice variant expressed. These experiments demonstrate that the efficacy of ANO1 antagonists depends on [Ca(2+)]i, suggesting a need for caution when ANO1 blockers are used to determine the role of ANO1 in physiological functions and in their use as therapeutic agents.
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada; and.
authors.forename: Tae Sik
authors.initials: TS
authors.lastname: Sung
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada; and.
authors.forename: Kate
authors.initials: K
authors.lastname: O'Driscoll
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada; and.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Zheng
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada; and.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Yapp
authors.affiliation: Department of Pharmacology, Center for Cardiovascular Research, University of Nevada School of Medicine, Reno, Nevada.
authors.forename: Normand
authors.initials: N
authors.lastname: Leblanc
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada; and.
authors.forename: Sang Don
authors.initials: SD
authors.lastname: Koh
authors.affiliation: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada; and ksanders@medicine.nevada.edu.
authors.forename: Kenton M
authors.initials: KM
authors.lastname: Sanders
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics*; D000818:Animals; D000075369:Anoctamin-1; D001553:Benzbromarone / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018408:Patch-Clamp Techniques / Q000379:methods; D011743:Pyrimidines / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
chemical_list: C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D011743:Pyrimidines; C578466:T16AInh-A01; D013844:Thiazoles; D001553:Benzbromarone; D002118:Calcium
keywords: Ca2+-activated Cl− channels; Cl− channel blocker; anion channel; ion channel