Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels.
TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC50 of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore.
Department of Physiology and.
Christian J
CJ
Peters
The Solomon H. Snyder Department of Neuroscience, High-Throughput Biology Center and Johns Hopkins Ion Channel Center, Johns Hopkins University, Baltimore, MD 21205; and State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Haibo
H
Yu
Department of Physiology and.
Jason
J
Tien
Department of Physiology and Howard Hughes Medical Institute, University of California, San Francisco, CA 94158;
Yuh Nung
YN
Jan
The Solomon H. Snyder Department of Neuroscience, High-Throughput Biology Center and Johns Hopkins Ion Channel Center, Johns Hopkins University, Baltimore, MD 21205; and.
Min
M
Li
Department of Physiology and Howard Hughes Medical Institute, University of California, San Francisco, CA 94158; Lily.Jan@ucsf.edu.
Lily Yeh
LY
Jan
D000409:Alanine / Q000235:genetics; D024361:Amino Acids, Basic / Q000378:metabolism*; D000818:Animals; D000838:Anions / Q000378:metabolism*; D000075369:Anoctamin-1; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D018408:Patch-Clamp Techniques; D054852:Small Molecule Libraries / Q000494:pharmacology; D013329:Structure-Activity Relationship
C531181:ANO1 protein, mouse; D024361:Amino Acids, Basic; D000838:Anions; D000075369:Anoctamin-1; D018118:Chloride Channels; D054852:Small Molecule Libraries; D000409:Alanine; D002118:Calcium
TMEM16A; calcium-activated channels; channel pharmacology; chloride channels; ion channel biophysics