Blood calcium concentration is maintained within a narrow range despite large variations in dietary input and body demand. The Transient Receptor Potential ion channel TRPV5 has been implicated in this process. We report here that TRPV5 is stimulated by the mammalian hormone klotho. Klotho, a beta-glucuronidase, hydrolyzes extracellular sugar residues on TRPV5, entrapping the channel in the plasma membrane. This maintains durable calcium channel activity and membrane calcium permeability in kidney. Thus, klotho activates a cell surface channel by hydrolysis of its extracellular N-linked oligosaccharides.
Extracellular pH has long been known to affect the rate and magnitude of ion transport processes among others via regulation of ion channel activity. The Ca 2؉ -selective transient receptor potential vanilloid 5 (TRPV5) channel constitutes the apical entry gate in Ca 2؉ -transporting cells, contributing significantly to the overall Ca 2؉ balance. Here, we demonstrate that extracellular pH determines the cell surface expression of TRPV5 via a unique mechanism. By a comprehensive approach using total internal reflection fluorescence microscopy, cell surface protein labeling, electrophysiology, 45 Ca 2؉ uptake assays, and functional channel recovery after chemobleaching, this study shows that upon extracellular alkalinization, a pool of TRPV5-containing vesicles is rapidly recruited to the cell surface without collapsing into the plasma membrane. These vesicles contain functional TRPV5 channels since extracellular alkalinization is accompanied by increased TRPV5 activity. Conversely, upon subsequent extracellular acidification, vesicles are retrieved from the plasma membrane, simultaneously resulting in decreased TRPV5 activity. Thus, TRPV5 accesses the extracellular compartment via transient openings of vesicles, suggesting that rapid responses of constitutive active TRP channels to physiological stimuli rely on vesicular "kiss and linger" interactions with the plasma membrane.The superfamily of transient receptor potential (TRP) channels is involved in diverse physiological processes, ranging from sensory activity to fertility and epithelial ion transport (15). The highly Ca 2ϩ -selective TRP vanilloid 5 (TRPV5) channel constitutes the apical entry gate in Ca 2ϩ -transporting cells and facilitates renal Ca 2ϩ influx from the prourine (10). Several lines of evidence indicate that TRPV5 activity is sensitive to pH. First, acid-based homeostasis is known to affect renal Ca 2ϩ handling as reflected by altered Ca 2ϩ excretion in kidneys during chronic acidosis or alkalosis, which is mediated at least in part by changes in TRPV5 gene expression (16). Second, in vitro studies indicated that intra-and extracellular pH directly regulate the activity of TRPV5. Acidification inhibited, whereas alkalinization stimulated, TRPV5 activity, likely mediated by conformational changes of the channel pore helix (24-26). An intrinsic physiological effect of extracellular pH is the regulation of trafficking processes like endo-and exocytosis and lysosomal trafficking (8,12,14). Since several TRP channels display constitutive activity, controlled recruitment of these channels towards the plasma membrane is important for the translation of physiological stimuli into increased ion permeability of the plasma membrane. For instance, an essential process during insulin-like growth factor-I stimulation of cell growth is TRPV2 recruitment facilitating Ca 2ϩ entry during progression through the cell cycle (11). In Drosophila photoreceptors, the TRP-like subunit is shuttled between the plasma membrane and an intracellular compartment by a light...
Fluoride ion channels of the Fluc family combat toxicity arising from accumulation of environmental F-. Although crystal structures are known, the densely packed pore region has precluded delineation of the ion pathway. Here we chart out the Fluc pore and characterize its chemical requirements for transport. A ladder of H-bond donating residues creates a 'polar track' demarking the ion-conduction pathway. Surprisingly, while track polarity is well conserved, polarity is nonetheless functionally dispensable at several positions. A threonine at one end of the pore engages in vital interactions through its b-branched methyl group. Two critical central phenylalanines that directly coordinate F-through a quadrupolar-ion interaction cannot be functionally substituted by aromatic, non-polar, or polar sidechains. The only functional replacement is methionine, which coordinates F-through its partially positive g-methylene in mimicry of phenylalanine's quadrupolar interaction. These results demonstrate the unusual chemical requirements for selectively transporting the strongly H-bonding F-anion.
The transient receptor potential vanilloid 5 (TRPV5) channel determines urinary Ca 2 þ excretion, and is therefore critical for Ca 2 þ homeostasis. Interestingly, mice lacking the serine protease tissue kallikrein (TK) exhibit robust hypercalciuria comparable to the Ca 2 þ leak in TRPV5 knockout mice. Here, we delineated the molecular mechanism through which TK stimulates Ca 2 þ reabsorption. Using TRPV5-expressing primary cultures of renal Ca 2 þ -transporting epithelial cells, we showed that TK activates Ca 2 þ reabsorption. The stimulatory effect of TK was mimicked by bradykinin (BK) and could be reversed by application of JE049, a BK receptor type 2 antagonist. A cell permeable analog of DAG increased TRPV5 activity within 30 min via protein kinase C activation of the channel since mutation of TRPV5 at the putative PKC phosphorylation sites S299 and S654 prevented the stimulatory effect of TK. Cell surface labeling revealed that TK enhances the amount of wild-type TRPV5 channels, but not of the TRPV5 S299A and S654A mutants, at the plasma membrane by delaying its retrieval. In conclusion, TK stimulates Ca 2 þ reabsorption via the BK-activated PLC/ DAG/PKC pathway and the subsequent stabilization of the TRPV5 channel at the plasma membrane.
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