Solid tumors tend to have a more glycolytic metabolism leading to an accumulation of acidic metabolites in their cytosol, and consequently, their intracellular pH (pHi) turns critically lower if the cells do not handle the acid excess. Recently, it was proposed that the voltage gated proton channels (HV1) can regulate the pHi in several cancers. Here we report the functional expression of voltage gated proton channels in a human glioblastoma multiforme (GBM) cell line, the most common and lethal brain tumor. T98G cells presented an outward, slow activating voltage-dependent proton current, which was also ΔpH-dependent and inhibited by ZnCl2, characterizing it as being conducted by HV1 channels. Furthermore, blocking HV1 channels with ZnCl2 significantly reduced the pHi, cell survival, and migration, indicating an important role for HV1 for tumor proliferation and progression in GBM. Overall, our results suggest that HV1 channels can be a new therapeutic target for GBM.
Myogenic contraction of renal arterioles is an important regulatory mechanism for renal blood flow autoregulation. We have previously demonstrated that integrin-mediated mechanical force increases the occurrence of Ca sparks in freshly isolated renal vascular smooth muscle cells (VSMCs). To further test whether generation of Ca sparks is a downstream signal of mechanotransduction in pressure induced myogenic constriction, the relationship between Ca spark frequency and transmural perfusion pressure was investigated in intact VSMCs of pressurized rat afferent arterioles. Spontaneous Ca sparks were found in VSMCs when afferent arterioles were perfused at 80 mmHg. The spark frequency was significantly increased when perfusion pressure was increased to 120 mmHg. Similar increase of spark frequency was also observed in arterioles stimulated with β-integrin activating antibody. Moreover, spark frequency was significantly higher in arterioles of spontaneous hypertensive rats (SHR) at 80 and 120 mmHg. Spontaneous membrane current recorded with whole-cell perforated-patch in renal VSMCs showed predominant activity of spontaneous transient inward currents (STICs) instead of spontaneous transient outward currents (STOCs) when holding potential was set close to physiological resting membrane potential. Real-time PCR and immunofluorescence confirmed the expression of Ca-activated Cl channel (Cl) TMEM16A in renal VSMCs. Inhibition of TMEM16A with T16Ainh-A01 impaired pressure-induced myogenic contraction in perfused afferent arterioles. Our study, hence, for the first time detected Ca sparks in intact VSMCs of afferent arterioles and their frequencies were positively modulated by the perfusion pressure. Our results suggest that Ca sparks may couple to Cl channels and trigger pressure-induced myogenic constriction via membrane depolarization.
determined the cryo-EM structures of zebrafish Otop1 (3.0 A ˚) and chicken OTOP3 (3.3 A ˚), both embedded in lipid nanodiscs. The structures reveal that the Otopetrin family adopt a unique dimeric fold, with each subunit divided into two structurally related halves. Notably we observe cholesterol(-like) molecules occluding the wide central tunnel formed between the two subunits. Using all-atom molecular dynamics simulations, we show that cholesterol molecules at the resolved positions within the central tunnel represent stable binding poses, thereby excluding water and other solutes. We then examined the putative proton conducting pathways in our MD simulations, using areas susceptible to water penetration as a first approximation. The two halves of both Otop1 and OTOP3 are partially hydrated as expected, due to their vestibular-shaped openings to the cytoplasmic side, and the presence of polar and charged residues. Surprisingly, a third region of water penetration is found along the intrasubunit interface between the two halves in a subunit. In functional studies, a key glutamate found in this region is intolerant to charge-neutralisation mutations. These results also correlate to the extent of hydration in this region in the simulations. Altogether, we show that there might be three potential pathways (or their combinations) contributing to proton currents in Otopetrins. We also study novel mechanically-activated (MA) ion channels found in mammals and plants, via a combination of structural and computational studies. Recently we solved the structures of OSCA channels in plants and work is underway for other MA channels.
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