TY - JOUR
T1 - Regulatory roles of anoctamin-6 in human trabecular meshwork cells
AU - Banerjee, Juni
AU - Leung, Chi Ting
AU - Li, Ang
AU - Peterson-Yantorno, Kim
AU - Ouyang, Huan
AU - Daniel Stamer, W.
AU - Civan, Mortimer M.
N1 - Funding Information:
Supported by National Institutes of Health (NIH; Bethesda, MD, USA) Research Grant EY13624 and Core Grant EY01583 (M.M.C.).
Publisher Copyright:
© 2017, Association for Research in Vision and Ophthalmology Inc. All rights reserved.
PY - 2017/1/1
Y1 - 2017/1/1
N2 - PURPOSE. Trabecular meshwork (TM) cell volume is a determinant of aqueous humor outflow resistance, and thereby IOP. Regulation of TM cell volume depends on chloride ion (Cl−) release through swelling-activated channels (ICl,Swell), whose pore is formed by LRRC8 proteins. Chloride ion release through swelling-activated channels has been reported to be regulated by calcium-activated anoctamins, but this finding is controversial. Particularly uncertain has been the effect of anoctamin Ano6, reported as a Ca2+-activated Cl− (CaCC) or cation channel in other cells. The current study tested whether anoctamin activity modifies volume regulation of primary TM cell cultures and cell lines. METHODS. Gene expression was studied with quantitative PCR, supplemented by reversetranscriptase PCR andWestern immunoblots. Currents were measured by ruptured whole-cell patch clamping and volume by electronic cell sizing. RESULTS. Primary TM cell cultures and the TM5 and GTM3 cell lines expressed Ano6 3 to 4 orders of magnitude higher than the other anoctamin CaCCs (Ano1 and Ano2). Ionomycin increased cell Ca2+ and activated macroscopic currents conforming to CaCCs in other cells, but displayed significantly more positive mean reversal potentials (+5 to +12 mV) than those displayed by ICl,Swell (−14 to −21 mV) in the same cells. Nonselective CaCC inhibitors (tannic acid>CaCCinh−A01) and transient Ano6 knockdown strongly inhibited ionomycin-activated currents, ICl,Swell and the regulatory volume response to hyposmotic swelling. CONCLUSIONS. Ionomycin activates CaCCs associated with net cation movement in TM cells. These currents, ICl,Swell, and cell volume are regulated by Ano6. The findings suggest a novel clinically-relevant approach for altering cell volume, and thereby outflow resistance, by targeting Ano6.
AB - PURPOSE. Trabecular meshwork (TM) cell volume is a determinant of aqueous humor outflow resistance, and thereby IOP. Regulation of TM cell volume depends on chloride ion (Cl−) release through swelling-activated channels (ICl,Swell), whose pore is formed by LRRC8 proteins. Chloride ion release through swelling-activated channels has been reported to be regulated by calcium-activated anoctamins, but this finding is controversial. Particularly uncertain has been the effect of anoctamin Ano6, reported as a Ca2+-activated Cl− (CaCC) or cation channel in other cells. The current study tested whether anoctamin activity modifies volume regulation of primary TM cell cultures and cell lines. METHODS. Gene expression was studied with quantitative PCR, supplemented by reversetranscriptase PCR andWestern immunoblots. Currents were measured by ruptured whole-cell patch clamping and volume by electronic cell sizing. RESULTS. Primary TM cell cultures and the TM5 and GTM3 cell lines expressed Ano6 3 to 4 orders of magnitude higher than the other anoctamin CaCCs (Ano1 and Ano2). Ionomycin increased cell Ca2+ and activated macroscopic currents conforming to CaCCs in other cells, but displayed significantly more positive mean reversal potentials (+5 to +12 mV) than those displayed by ICl,Swell (−14 to −21 mV) in the same cells. Nonselective CaCC inhibitors (tannic acid>CaCCinh−A01) and transient Ano6 knockdown strongly inhibited ionomycin-activated currents, ICl,Swell and the regulatory volume response to hyposmotic swelling. CONCLUSIONS. Ionomycin activates CaCCs associated with net cation movement in TM cells. These currents, ICl,Swell, and cell volume are regulated by Ano6. The findings suggest a novel clinically-relevant approach for altering cell volume, and thereby outflow resistance, by targeting Ano6.
KW - Calcium-activated Cl current
KW - IC
KW - Intraocular pressure
KW - Outflow facility
KW - Regulatory volume decrease
KW - TMEM16F
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U2 - 10.1167/iovs.16-20188
DO - 10.1167/iovs.16-20188
M3 - Article
C2 - 28125837
AN - SCOPUS:85011085572
VL - 58
SP - 492
EP - 501
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
SN - 0146-0404
IS - 1
ER -