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A3736 - Knockout of TRPC4 Reduces Cytosolic Ca2+ Transients and Delays Store-Operated Ca2+ Entry
Author Block: N. Xu1, C. M. Francis1, M. T. Lin2, T. Stevens1; 1Physiology and Cell Biology, Center for Lung Biology, University of South Alabama College of Medicine, Mobile, AL, United States, 2Physiology and Cell Biology, University of South Alabama College of Medicine, Mobile, AL, United States.
Rationale: Pulmonary endothelial cells express the canonical transient receptor potential 4 (TRPC4) protein on the plasma membrane. Activation of the TRPC4-containing store-operated Ca2+ entry (ISOC) channel promotes Ca2+ influx, leading to increased permeability in extra-alveolar vessels. However, Ca2+ influx through TRPC4 channels appears to be highly dynamic, with rapid oscillations that do not necessarily increase global cytosolic Ca2+. The relationship between TRPC4 activation and dynamic cytosolic Ca2+ oscillations that increase permeability are poorly understood. Here, we tested the hypothesis that Ca2+influx through TRPC4 channels is necessary to sustain dynamic Ca2+oscillations. Methods: Spinning Disk confocal imaging was performed to measure cytosolic Ca2+ transients. Fischer rat wild-type and TRPC4-knockout pulmonary microvascular endothelial cells were seeded on cover glasses and grown to confluence in 3~5 days. Monolayers were loaded with Fluo-4 AM, an intracellular Ca2+ indicator. Fluorescence intensity was measured to assess intracellular Ca2+ dynamics. Thapsigargin (100 nM and 500 nM) was added to activate ISOC channels. In separate experiments, whole-cell patch-clamp recordings were used to monitor transmembrane currents every 30 s. Once a stable recording was achieved (~15 cycles), thapsigargin (100 nM or 500 nM) was added to the external bath solution to activate ISOC channels. Whole-cell membrane currents were recorded for at least another 30 cycles. Results: Imaging data revealed the heterogeneous nature of Ca2+ signals both in wild-type and knockout cells. TRPC4 knockout significantly decreased the duration, amplitude, and signal spread of cytosolic Ca2+ transients, both under basal conditions and after thapsigargin (100 nM and 500 nM) challenge. TRPC4 expression significantly increased the duration of the Ca2+ transients responding to thapsigargin challenge when compared to the baseline. Consistent with these data, patch clamp recordings showed that thapsigargin induces faster Ca2+ entry responses in wild-type cells than in the knockout counterparts. Wild-type cells were more likely to respond to thapsigargin than were the knockout cells. Conclusion: TRPC4 knockout reduces cytosolic Ca2+ transients and delays the onset of store-operated Ca2+ entry.