EAVK segment "c" sequence confers Ca2+-dependent changes to the kinetics of full-length human Ano1.
Anoctamin1 (Ano1 and TMEM16A) is a calcium-activated chloride channel specifically expressed in the interstitial cells of Cajal (ICC) of the gastrointestinal tract muscularis propria. Ano1 is necessary for normal electrical slow waves and ICC proliferation. The full-length human Ano1 sequence includes an additional exon, exon "0," at the NH2terminus. Ano1 with exon 0 [Ano1(0)] had a lower EC50for intracellular calcium ([Ca2+]i) and faster chloride current (ICl) kinetics. The Ano1 alternative splice variant with segment "c" encoding exon 13 expresses on the first intracellular loop four additional amino acid residues, EAVK, which alterIClat low [Ca2+]iExon 13 is expressed in 75-100% of Ano1 transcripts in most human tissues but only 25% in the human stomach. Our aim was to determine the effect of EAVK deletion on Ano1(0)IClparameters. By voltage-clamp electrophysiology, we examinedIClin HEK293 cells transiently expressing Ano1(0)with or without the EAVK sequence [Ano1(0)ΔEAVK]. The EC50values of activating and deactivatingIClfor [Ca2+]iwere 438 ± 7 and 493 ± 9 nM for Ano1(0)but higher for Ano1(0)ΔEAVK at 746 ± 47 and 761 ± 26 nM, respectively. Meanwhile, the EC50values for the ratio of instantaneous to steady-stateIClwere not different between variants. Congruently, the time constant of activation was slower for Ano1(0)ΔEAVK than Ano1(0)currents at intermediate [Ca2+]iThese results suggest that EAVK decreases the calcium sensitivity of Ano1(0)current activation and deactivation by slowing activation kinetics. Differential expression of EAVK in the human stomach may function as a switch to increase sensitivity to [Ca2+]ivia faster gating of Ano1.NEW & NOTEWORTHYCalcium-activated chloride channel anoctamin1 (Ano1) is necessary for normal slow waves in the gastrointestinal interstitial cells of Cajal. Exon 0 encodes the NH2terminus of full-length human Ano1 [Ano1(0)], while exon 13 encodes residues EAVK on its first intracellular loop. Splice variants lack EAVK more often in the stomach than other tissues. Ano1(0)without EAVK [Ano1(0)ΔEAVK] has reduced sensitivity for intracellular calcium, attributable to slower kinetics. Differential expression of EAVK may function as a calcium-sensitive switch in the human stomach.
Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
Peter R
PR
Strege
Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
Simon J
http://orcid.org/0000-0001-5311-5993
SJ
Gibbons
Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
Amelia
A
Mazzone
Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
Cheryl E
CE
Bernard
Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota.
Arthur
A
Beyder
Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota farrugia.gianrico@mayo.edu.
Gianrico
G
Farrugia
D017398:Alternative Splicing; D000075369:Anoctamin-1; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005091:Exons; D005753:Gastric Mucosa / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D056885:Interstitial Cells of Cajal / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D020033:Protein Isoforms; D013270:Stomach / Q000166:cytology; D014162:Transfection
C476213:ANO1 protein, human; D000075369:Anoctamin-1; D018118:Chloride Channels; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D002118:Calcium
TMEM16A; electrophysiology; ion channels