From loss of Ca2+-influx to altered organelle temperature and energy failure: A neonatal-lethal TRPV4 mutant (L618P).

TRPV4 is a thermosensitive and non-selective cation channel which is expressed widely. So far, several point mutations in TRPV4 have been identified and all these mutants induce pathophysiological defects that range from mild-phenotype to neonatal lethality, suggesting that TRPV4 mutation-induced channelopathies have differential levels of phenotypic penetrations. The reasons for such variability are still poorly understood. In this work, we aim to characterize TRPV4-L618P, a mutation that has been linked with neonatal lethality. This mutation also induces multiple skeletal disorders and metatropic dysplasias, even in the somatic mosaic form. We could successfully generate a F11 (a peripheral Dorsal Root Ganglion neuron-derived) cell line that expresses this mutant after a long-term selection, suggesting that TRPV4-L618P is not lethal for the cells in culture condition. Using Ca2+-imaging experiments, we show that TRPV4-L618P mutant cannot be activated by TRPV4-specific agonists in different conditions, and thus behave as a 'loss-of-function'-like mutant, at least for Ca2+-ions. However, basal level expression of TRPV4-L618P alters the thermal spectrum of sub-cellular organelles. TRPV4-L618P increases the relative temperature of mitochondria, and ER, and decreases for plasma membrane and lysosome. Cells expressing TRPV4-L618P mutant do not show an alteration in the number of lysosomes but show increased lysosomal pH. Low-level expression of TRPV4-L618P significantly reduces mitochondrial Ca2+, membrane potential (ψ), mitochondrial ATP and cellular GTP, but increases ROS generation. We propose that at the cellular level, TRPV4-L618P-induced defects are mostly caused by lower energetics, reduced sub-cellular organelle functions, altered thermal homeostasis, lower Ca2+-buffering by mitochondria and greater mitochondrial problems rather than higher cytosolic Ca2+.