SOCE is modulated by AQP2 and TRPV4 affecting both Ca2+ influx and clearance in renal cells

LÓPEZ GONZÁLEZ, MACARENA; DI GIUSTO, GISELA; CAPURRO, CLAUDIA; FORD, PAULA; PIZZONI, ALEJANDRO; RIVAROLA, VALERIA

Tucumán

Congreso; III Latin American Federation of Biophysical Societies; 2016

Sociedad Argentina de Biofísica

Store-operated Ca2+ entry (SOCE) is a homeostatic process regulated by the filling state of the intracellular Ca2+stores. This mechanism is strongly influenced by changes of the driving force for Ca2+ influx. In addition, SOCE is modulated through the plasma membrane Ca2+ ATPase (PMCA)-mediated cytosolic Ca2+[Ca2+]i clearance with important consequences in downstream signaling pathways1.In a previous communication we described that AQP2 can modulate SOCE by interacting with TRPV4, affecting K+ channels and in consequence membrane potential (Vm). The aim of the present work was to further characterize if SOCE modulation by AQP2 depends on changes of TRPV4 localization, the driving force for Ca2+ entry and Ca2+ clearance rates. We used two renal cell lines; one not expressing AQPs (WT-RCCD1) and another one transfected with AQP2 (AQP2-RCCD1). We performed immunofluorescence studies to determine if Ca2+store depletion with thapsigargin (1 μM) can change subcellular TRPV4 localization. Also, FURA-2 and DIBAC4(3) dyes were used to monitor [Ca2+]i and Vm changes respectively. We found that after Ca2+store depletion, TRPV4 was enriched only in the membrane of cells expressing AQP2. In addition, SOCE sensitivity to changes in the driving force was evaluated by varying sequentially either the electrical (high extracellular K+ medium, HK) or the chemical (low extracellular Ca2+ medium, LC) driving force for short periods in the same cells. WT cells responded strongly reducing [Ca2+]I but surprisingly, AQP2 cells showed a minor reduction on [Ca2+]i upon depolarization following introduction of HK medium. Contrarily, both cell lines respond similarly to LC medium. Experiments designed to evaluate Ca2+ clearance revealed that AQP2 expressing cells remove Ca2+ from citosol slower than WT cells. These results suggest that AQP2 can modulate SOCE by influencing either Ca2+ influx and Ca2+ clearance from cytosol.