Abstract
The contribution of endogenous and recombinant transient receptor potential vanilloid type 6 (TRPV6) channels to Ca2+ entry across the plasma membrane was studied in the human lymph node prostate cancer cell line (LNCaP). LNCaP cells do express the TRPV6 gene, and Ca2+ entry currents in these cells were detected after active and passive Ca2+ store depletion by intracellular application of inositol 1,4,5-trisphosphate, Ca2+ chelators, and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. This store-operated Ca2+ current (ISOC) had biophysical properties similar to those of the Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukemia cells such as the activation mechanism, inward rectification, and Ca2+ selectivity. These properties are also shared by the Ca2+-sensing Ca2+ current (ITRPV6) recorded after heterologous expression of TRPV6 cDNA in human embryonic kidney and rat basophilic leukemia cells (Bödding, M., Wissenbach, U., Flockerzi, V. (2002) J. Biol. Chem. 277, 36656-36664). TRPV6 cDNA transfection of LNCaP cells restored recombinant ITRPV6, which can be distinguished from ISOC by the mechanism of activation, the voltage dependence of monovalent currents in the absence of external divalent cations, and the changes in Ca2+ current densities due to different membrane potentials. In addition, ISOC was not affected by antiandrogen or 1,25-dihydroxyvitamin D3 treatment of LNCaP cells, which up-regulates TRPV6 gene expression, or by androgen treatment, which has the opposite effect. Therefore, native channels responsible for ISOC are different from those for recombinant ITRPV6 and do not appear to be affected if one of their assumed subunits, TRPV6, is up- or down-regulated, suggesting a rather rigid subunit composition in vivo.
Highlights
Calcium is necessary for several intracellular signal transduction mechanisms such as muscle contraction and secretion of neurotransmitters
There were differences between both channels, including the protocols necessary to activate ICRAC in rat basophilic leukemia (RBL) cells and to activate recombinant ITRPV6 recorded after overexpressing its cDNA in RBL cells [1] or human embryonic kidney (HEK) cells [1, 9]
LNCaP cells endogenously express the TRPV6 gene; but unlike recombinant ITRPV6 in HEK or RBL cells, ISOC is not increased under elevated Ca2ϩ buffering conditions, and the activation kinetics correlate with the expected time course of active or passive store depletion, which is not the case for recombinant ITRPV6
Summary
Calcium is necessary for several intracellular signal transduction mechanisms such as muscle contraction and secretion of neurotransmitters. TRPV6 cDNA transfection of LNCaP cells restored recombinant ITRPV6, which can be distinguished from ISOC by the mechanism of activation, the voltage dependence of monovalent currents in the absence of external divalent cations, and the changes in Ca2؉ current densities due to different membrane potentials.
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