In Situ Measurements of Oligomerization State of NBCe1-A in Rat Kidneys Via Spatial Fluorescence Intensity Fluctuation Analysis

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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. We developed an in situ measurement methodology to determine the oligomeric state of NBCe1-A without requiring tissue disruption using biochemical methods. We used fluorescent moment image analysis and spatial intensity distribution analysis (SpIDA) to study the oligomeric state of NBCe1-A in cultured cells expressing the cotransporter and in rat kidney tissue. Both methods allow for 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 because of their large surface area. As an independent control of monomeric brightness, we used cells expressing monomeric eGFP anchored to the membrane. Taking into account the recovered values of the monomeric eGFP quantal brightness, we show that NBCe1-A exists in monomeric and dimeric states on the cell membrane. We also used an Alexa488-alpha-bungarotoxin conjugate to label cells expressing an NBCe1A-bungarotoxin binding mutant. As a monomeric control, we immobilized Alexa488 dye on cover slips. The spatial fluorescence intensity fluctuation analysis revealed a similar distribution of aggregates as shown for eGFP data. Moreover, we immunolabeled NBCe1-A in rat kidney tissues as well as in cultured HEK293 cells expressing the cotransporter demonstrating the NBCe1-A is present in monomeric, dimeric and rarely in higher order oligomeric states. These experiments demonstrate for the first time the in situ oligomeric state(s) of NBCe1-A.