NaPi2b diffusion and activity could be regulated by membrane fluidity in the microvilli of the small intestine
Authors: Al‐Juboori Saif I. , Lei Tim , Hogg‐Cornejo Veronica , Sutherland Eileen , Levi Moshe , Dobrinskikh Evgenia 
Published: November 2023

Inorganic phosphate (Pi) plays an important role in growth, development, bone formation and cellular metabolism. A physiological phosphate balance is maintained through the multi‐organ communication among intestine, kidneys and bones. Dysregulation of phosphate balance can induce many disorders ranging from osteoporosis to cardiovascular calcification. Pi cannot easily cross the cell‐membrane barrier and needs a special cell transporter to carry it into the cell. Sodium‐dependent phosphate (NaPi) co‐transporters play a key role in the regulation of phosphate metabolism by mediating phosphate absorption and reabsorption in the small intestine and kidney. Despite important biological functions, the molecular regulation of the intestinal sodium‐dependent phosphate co‐transport is not fully understood. Even though NaPi2b expression levels are lower in the duodenal than jejunal Brush Border Membranes (BBMs), phosphate uptake is similar; indicating similar activity of the co‐transporters. Since it is very hard to study proteins dynamics in the microvilli because of their packed structure and movement, Giant Unilamellar Vesicles (GUVs) are convenient biomimetic systems of the membrane that are increasingly being used to quantitatively address biophysical and biochemical processes related to protein function. The cell membrane influences many cell functions, and membrane fluidity plays a key role in regulation of membrane dynamics under physiological conditions and in the pathogenesis of different diseases. Plasma membrane contains many lipid species, which could be organized as liquid‐disordered (ld) phase, solid‐ordered (so) phase, and liquid‐ordered (lo) which is enriched in sphingolipids and cholesterol (rafts). Membrane fluidity can be measured by using special probes such as di‐4‐ANEPPDHQ, which has a blue shift in emission for the membrane liquid‐ordered phase relative to membranes in liquid‐disordered phase. We performed Generalized Polarization (GP) measurements in BBMs isolated from the duodenum and jejunum of 8 weeks old Sprague‐Dawley rats fed with 0.6% phosphate diet. We found that duodenal and jejunal BBMs were differently organized. Duodenal membrane contained mixed regions of ordered and disordered domains with average GP −0.472±0.017, whereas jejunal membrane had two distinct layers with average GP −0.555±0.013, indicating a more fluid phase. Diffusion within the membrane is important for cellular processes and protein function. We measured the NaPi‐2b diffusion using Fluorescence Correlational Spectroscopy (FCS) on the same GUVs co‐labeled with Alexa647 conjugated NaPi‐2b antibody. NaPi‐2b diffusion dynamics were markedly different in the BBMs isolated from the two regions of the small intestine. The diffusion coefficients of NaPi‐2b in duodenum and jejunum GUVs were 10.2 μm2/s and 0.58 μm2/s respectively, despite the fact that jejunal membrane was more fluid. This could indicate that NaPi‐2b proteins reside in specific lipid order domains within the microvilli of the two regions, resulting in different diffusion in the membrane, and therefore different activity.