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In Situ Measurement of Oligomerization State of NBCe1‐A in Kidney Tissues using Spatial Fluorescence Intensity Fluctuation Analysis
Author(s) -
Sergeev Mikhail,
Godin Antoine G,
Kao Liyo,
Abuladze Natalia,
Wiseman Paul W,
Kurtz Ira
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.815.3
Subject(s) - chemistry , cotransporter , biophysics , monomer , in situ , fluorescence , membrane , green fluorescent protein , microbiology and biotechnology , biochemistry , biology , sodium , gene , physics , organic chemistry , quantum mechanics , polymer
NBCe1‐A plays an important role in absorbing sodium bicarbonate across the basolateral membrane of the proximal tubule. We have previously showed that minimal functional unit of NBCe1‐A is a monomer, and based on in‐vitro biochemical studies in HEK293 cells, the oligomeric state of the cotransporter was shown to be predominantly dimeric with monomeric and higher oligomeric forms also present. In the present study, we developed an in situ measurement methodology to determine the oligomeric state of NBCe1‐A without requiring tissue disruption. We used fluorescent moment image analysis and spatial intensity distribution analysis (SpIDA) to study the oligomeric state of NBCe1‐A. Both methods allow for unbiased quantitative measurement of fluorescent particle densities and oligomerization states within individual images acquired with laser‐scanning microscopy. Initially we examined basal membranes of highly adherent CHO K1 cells expressing eGFP‐tagged NBCe1‐A, and showed that NBCe1‐A existed primarily in the monomeric state and negligibly in the dimeric state on the cell membrane. We immunolabeled NBCe1‐A in kidney tissues demonstrating the NBCe1‐A is present in dimeric and rarely in higher order oligomeric states. As a control, we compared the results obtained from LSM images of tissues of NBCe1‐A knockout mouse. These experiments demonstrate for the first time the in situ oligomeric state(s) of NBCe1‐A.