Expression of Polycystins in LLC‐PK1 Cells Does Not Increase Flow‐Activated Calcium Fluxes
Authors: Stavola Lindsey Katharine , Prætorius Helle , Caplan Michael J 
Published: November 2023

The primary cilium is thought to play a critical role as a mechanosensor that detects fluid flow in the kidney. The molecular basis of this mechanosensitivity has yet to be fully elucidated. Portions of the cellular populations of the polycystin 1 (PC1) and polycystin 2 (PC2) proteins, which are encoded by the Pkd1 and Pkd2 genes that are mutated in Autosomal Dominant Polycystic Kidney Disease, localize to the primary cilium. PC1 is a ~450 kDa membrane protein with a large N terminal extracellular domain and 11 predicted transmembrane spans. PC2 spans the membrane six times and is a member of the TRP family of nonselective calcium‐permeable cation channels. PC1 and PC2 form a complex with one another, and this association plays an important role in their trafficking to the primary cilium. It has been suggested that the ciliary population of polycystin proteins functions as a mechanosensitive ion channel in renal epithelia, and that the ciliary polycystins play an obligate role in producing cilium‐dependent flow‐activated cytosolic calcium transients.We generated LLC‐PK1 porcine renal epithelial cell lines that stably express PC1 and PC2. Immunofluorescence analysis reveals that both of these proteins are abundantly detected in the primary cilium. We conducted live cell imaging of the intracellular Ca2+ concentration ([Ca2+]i) using these LLC‐PK1 cells loaded with the fluorescent Ca2+‐sensitive dye Fluo4‐AM. We find that overexpression of ciliary PC1 and PC2 does not increase the magnitude or duration of the [Ca2+]i transients elicited in response to increase in physiological fluid flow (10 μl/s corresponding to a linear velocity above the membrane of 70 μm/s). These data suggest that, at least when expressed in this cell type, exogenous polycystins do not by themselves further add to the flow‐activated [Ca2+]i increase. Previous data have suggested that TRPV4 works together with polycystins and that a complex containing TRPV4 and the polycystins confers mechanosensitivity on the cilium. To test this possibility, we co‐expressed TRPV4 tagged with the S‐Tag at its C‐terminus with PC1 and PC2 in LLC‐PK1 cells. Immunofluorescence using an antibody directed against the S‐Tag revealed that little or no TRPV4 protein was detectable in the primary cilium, but accumulated instead at the basolateral plasma membrane. Interestingly, however, expression of TRPV4 in LLC‐PK1 cells caused much larger flow‐induced and ATP‐induced [Ca2+]i transients than was detected in cells expressing PC1 and PC2 alone. These results demonstrate that exogenously expressed polycystins are not sufficient to further enhance ciliary mechanosensation and suggest that TRPV4, may play an indirect role in this process and in supporting cellular Ca2+ signaling.Support or Funding InformationThe NSF GRFP Graduate Research Opportunities Worldwide (GROW), The Danish Council for Independent Research, The Lundbeck Foundation and the research centre MEMBRANES, The William Townsend Porter Predoctoral Fellowship from the American Physiological Society