March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Functional Study of SLC4A11 in HEK293 cells
Author Affiliations & Notes
  • Diego G. Ogando
    School of Optometry, Indiana University, Bloomington, Indiana
  • Supriya S. Jalimarada
    School of Optometry, Indiana University, Bloomington, Indiana
  • Eranga N. Vithana
    Singapore Eye Research Institute, Singapore, Singapore
  • Joseph A. Bonanno
    School of Optometry, Indiana University, Bloomington, Indiana
  • Footnotes
    Commercial Relationships  Diego G. Ogando, None; Supriya S. Jalimarada, None; Eranga N. Vithana, None; Joseph A. Bonanno, None
  • Footnotes
    Support  NIH EY08834
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 6005. doi:
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      Diego G. Ogando, Supriya S. Jalimarada, Eranga N. Vithana, Joseph A. Bonanno; Functional Study of SLC4A11 in HEK293 cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6005.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Mutations in SLC4A11 are associated with Congenital Hereditary Dystrophy and Fuchs Corneal Dystrophy. All members of the Slc4 family except SLC4A11 have been demonstrated to transport HCO3-. In this study we tested the following hypotheses: 1) SLC4A11 is a Na+:HCO3- co-transporter, 2) SLC4A11 is a Na+: OH- co-transporter or a Na+: H+ exchanger.

Methods: : HEK293 cells were transfected with the construct phCMV2-HA-SLC4A11 (T) or empty vector (C). Expression on the membrane surface was confirmed by immunofluorescence and by membrane protein isolation. Forty eight hours post-transfection the cells were loaded with BCECF or SBFI dyes in order to measure pHi and Na+i changes in CO2/HCO3- -rich (BR) or HCO3- -free (BF) Ringer.

Results: : In C cells addition of CO2/HCO3- induce a fast acidification (-0.54 pH/min) of 0.19 pH units; followed by a slower alkalinization (0.044 pH units/min), reaching steady state at a slightly lower pH (Delta pH: -0.074). Significantly higher or lower steady-state pHi in BR would be consistent with Na:nHCO3 cotransport. However, in T cells only the following differences from C were found: less acidification (0.14 pH units, p=0.018), slightly higher alkalinization recovery rate (0.073 pH/min, p=0.098) and the same steady-state pHi as starting in BF. In C cells addition of NH4Cl (10mM) in BF induced a fast alkalinization (0.68 pH/min) of 0.23 pH units followed by a slow acidification (0.079 pH/min). Removal of NH4Cl produced a fast acidification (-0.42 pH/min) of 0.27 pH units, followed by a slower alkalinization (0.045 pH/min at pH 7.3). In T cells the following differences from C were found: faster acidification (0.172 pH/ min, p=0.04) in the presence of NH4Cl, indicative of NH4+ transport by SLC4A11, greater extent of acidification upon NH4Cl removal (0.40 pH units, p=0.003) and increased alkalinization recovery (0.116 pH/min at pH 7.3, p=0.04). The recovery is accompanied by an 82% higher rate of Na+ influx (p=0.002). The amiloride analogue EIPA at 1 µM inhibits the alkalinization recovery rate in C (0.013 pH/min, pH 7.3; 3.5 fold) and surprisingly also in T (0.022 pH/min, pH: 7.3; 5.3 fold).

Conclusions: : (1) Overexpression of SLC4A11 does not provide Na:nHCO3- cotransport. (2) SLC4A11 is a cation transporter that includes Na+ & NH4+ as substrates. (3) SLC4A11 increased recovery from acid loads, consistent with SLC4A11 acting as a Na+: OH- or a Na+: H+ transporter. (4) SLC4A11 is very sensitive to EIPA, in contrast to a previous report.

Keywords: ion transporters • pH • cornea: endothelium 

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