Evaluation of a genetically encoded Cl− and pH sensor (ClopHensor) for monitoring intragranular Cl− in renal endocrine juxtaglomerular (JG) cells.
Authors: Wu andrew , Caceres Paulo , Mendez Mariela , Ortiz Pablo A. 
Published: April 2020

In most cells, intracellular cytoplasmic Cl− is maintained between 10 and 30 mM by a combination of entry and exit transport pathways. However, in endocrine cells intra‐granule Clin secretory granules is higher than cytoplasmic Cl−. This, is in part due to an intragranular positive H+ gradient driven by V‐ATPases, which generates low intra‐granular pH (4–5), and provides an electrochemical gradient for Cl− entry via CLC‐6/7. The role of V‐ATPases in exocytosis is well known, but less is known about how changes in Cl− contribute. There are very few methods available to measure intra‐granule Cl− concentration and monitor how it changes. ClopHensor is a genetically encoded protein sensor based on a Cl ‐sensitive GFP mutant (E2‐GFP) and DsRed (
Nature Methods, v7, pages 516–518, 2010). When excited by a 457 nm laser, E2‐GFP emission is sensitive to Cl− whereas at 488 nm it is sensitive to pH, whereas DsRed (insensitive to Cl− or pH) serves to control for volume changes. Renal endocrine juxtaglomerular (JG) cells, store renin, the rate‐limiting step enzyme in the production of angiotensin II, which play an essential role in blood pressure control. Renin is stored in large secretory granules and its exocytosis is stimulated primarily by increases in cAMP. We hypothesized that targeting ClopHensor to secretory granules would allow measurement of intra‐granular Cl− in JG cells and changes in its concentration during stimulation. We first tested the ability of ClopHensor to measure intracellular Cl− in M1 cells, clamped at pH 5.5 (reported for secretory granules). M1 cells transduced with adenovirus coding for ClopHensor were permeabilized to H+ with nygericin and bathed at pH 5.5, and permeabilized to Cl−. Cells were imaged by confocal microscopy under 457, 491 and 568 nm laser excitation while simultaneously acquiring emissions at 525 (E2‐GFP) and 590nm (Dsred). Under these conditions increasing Cl− from 0, to 20, 80 and 120 mM caused a logarithmic reduction in the 457/568 emission ratio (Cl‐sensitive) with a half‐max inhibition at 10.7±1.4 mM (n =6). To monitor intra‐granule Cl− we generated adenoviruses expressing NeuroPeptide Y targeting motif (NPY)‐ClopHensor. When primary mouse JG cells were transduced with NPY‐ClopHensor fluorescence was restricted to large granules. We could not permeabilize granules to Cl‐ in live JG cells Thus we measured baseline intra‐granule Cl− immediately after a daily calibration of ClopHensor in M1 cells. These results yielded an intra‐granule Cl− concentration of 77±15 mM (n=8). After measuring baseline Cl− in granules JG cells were treated with forskolin/IBMX to increase cAMP, and then 0 mM extracellular Cl− and bafilomycin. cAMP decreased intra‐granule Cl−, evidenced by a 22±5% increase (p<0.05, n= 11) in 457/568 Cl‐sensitive ratio, which was further increased by inhibiting V‐ATPse with bafilomycin by 40±6% (p<0.05, n=13). Our data show for the first time, measurements of intra‐granule Cl− in renal JG cells and suggest that NPY‐ClopHensor can be used to monitor the effect of agonists, or V‐ATPase regulators on intra‐granule Cl−. The role of intra‐granule Cl− in renin exocytosis and renin‐angiotensin system physiology remains to be studied.

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HFH Research fund: Pablo A Ortiz