Abstract
The activation mechanism of the recently cloned human transient receptor potential vanilloid type 6 (TRPV6) channel, originally termed Ca(2+) transporter-like protein and Ca(2+) transporter type 1, was investigated in whole-cell patch-clamp experiments using transiently transfected human embryonic kidney and rat basophilic leukemia cells. The TRPV6-mediated currents are highly Ca(2+)-selective, show a strong inward rectification, and reverse at positive potentials, which is similar to store-operated Ca(2+) entry in electrically nonexcitable cells. The gating of TRPV6 channels is strongly dependent on the cytosolic free Ca(2+) concentration; lowering the intracellular free Ca(2+) concentration results in Ca(2+) influx, and current amplitude correlates with the intracellular EGTA or BAPTA concentration. This is also the case for TRPV6-mediated currents in the absence of extracellular divalent cations; compared with endogenous currents in nontransfected rat basophilic leukemia cells, these TRPV6-mediated monovalent currents reveal differences in reversal potential, inward rectification, and slope at very negative potentials. Release of stored Ca(2+) by inositol 1,4,5-trisphosphate and/or the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin appears not to be involved in TRPV6 channel gating in both cell lines but, in rat basophilic leukemia cells, readily activates the endogenous Ca(2+) release-activated Ca(2+) current. In conclusion, TRPV6, expressed in human embryonic kidney cells and in rat basophilic leukemia cells, functions as a Ca(2+)-sensing Ca(2+) channel independently of procedures known to deplete Ca(2+) stores.
Highlights
Calcium is involved in a multitude of intracellular signal transduction mechanisms ranging from contraction to secretion
The gating of TRPV6 channels is strongly dependent on the cytosolic free Ca2؉ concentration; lowering the intracellular free Ca2؉ concentration results in Ca2؉ influx, and current amplitude correlates with the intracellular EGTA or BAPTA concentration
Ca2ϩ currents mediated by recombinant TRPV6 and endogenous CRAC channels had similar biophysical properties and could not clearly be discriminated by their current-voltage relationship
Summary
Cell Culture, Transfected cDNA, and Transfection—HEK-293 (ATCC, 1573-CRL) and RBL-1 cells (ATTC, 1378-CRL) were from the American Type Culture Collection (Manassas, VA). The intracellular Mg-ATP concentration was increased to 6 mM for the experiments in the absence of external divalent cations This was done to minimize contamination of monovalent currents presumably mediated by endogenously expressed TRPM7 channels in HEK and RBL cells [19]. Ca2ϩ currents elicited by voltage ramps were leak-subtracted by subtracting either the first ramp in TRPV6-transfected cells or by averaging the first two to four ramps after establishing the whole-cell mode in untransfected cells (depending on how fast ICRAC developed) and subtracting the mean from all subsequent traces Drugs—All chemicals were purchased from Sigma, except BAPTA (Molecular Probes)
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