Abstract
We previously demonstrated that high external [Ca2+] activated two Ca2+ currents in human gingival keratinocytes (HGKs): an initial small I-CRAC-like current and a second large nonspecific cation current (Fatherazi S, Belton CM, Cai S, Zarif S, Goodwin PC, Lamont RJ, Izutsu KT; Pflugers Arch 448:93-104, 2004). It was recently shown that TRPC1, a member of the transient receptor potential protein family, is a component of the store-operated calcium entry mechanism in keratinocytes. To further elucidate the molecular identity of these channels, we investigated the expression of TRPC4 in gingival tissue and in cultured keratinocytes, and the effect of knockdown of TRPC4 expression on the Ca2+ currents and influx. Immunohistochemistry showed TRPC4 was present in gingival epithelium as well as in HGKs cultured in different [Ca2+]s. Results from tissue and cultured HGKs demonstrated TRPC4 expression decreased with differentiation. Knockdown of TRPC4 in proliferating HGKs with antisense oligonucleotides significantly reduced the intracellular [Ca2+] increase obtained upon exposure to high external [Ca2+]. Antisense knockdown of TRPC4 expression was confirmed by reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence microscopy of transfected HGKs. Immunofluorescence microscopy and patch clamp measurements in Lucifer-yellow-tagged, antisense-treated HGKs showed attenuation of TRPC4 expression levels as well as attenuation of the I-CRAC-like current in the same cell, whereas the large nonspecific cation current was unchanged but significantly delayed. Cells transfected with a scrambled TRPC4 oligonucleotide showed no change in either the I-CRAC-like or nonspecific currents. The results indicate that TRPC4 is an important component of the I-CRAC-like channel in HGKs.