Evidence that TRPC4 supports the calcium selective ICRAC-like current in human gingival keratinocytes

Abstract

We previously demonstrated that high external [Ca(2+)] activated two Ca(2+) 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 Ca(2+) currents and influx. Immunohistochemistry showed TRPC4 was present in gingival epithelium as well as in HGKs cultured in different [Ca(2+)]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 [Ca(2+)] increase obtained upon exposure to high external [Ca(2+)]. 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.

Specific TRPC6 Channel Activation, a Novel Approach to Stimulate Keratinocyte Differentiation

“…However, silencing TRPC1 did not completely block keratinocyte differentiation, suggesting that other TRPC channels may also be involved, particularly because they are known to form multimers in vivo. TRPC4 and TRPV6 have also been reported to take part in keratinocyte differentiation (15,25). Our results about the involvement of TRPC1, TRPC3, TRPC4, and TRPC6 in the high [Ca 2ϩ ] o -induced effects confirm these data.…”

“…Role of TRPC Channels in Keratinocyte Differentiation-Because their expression levels change in a differentiation-dependent manner, functional properties of TRPC channels in keratinocytes have been suggested to be involved in differentiation, which is regulated by Ca 2ϩ influx (12,14,15). Recently, TRPC1 has been implicated in the Ca 2ϩ -induced terminal differentiation of human keratinocytes in vitro (14,24).…”

“…For example Cai et al (12) detected TRPC1, TRPC5, TRPC6, and TRPC7 in gingival keratinocytes, whereas Beck et al (13) showed the expression of TRPC1, TRPC4, TRPC5, and TRPC7 in HaCaT keratinocytes. Similarly, TRPC1 as well as TRCP4 have been implicated in the Ca 2ϩ -sensing receptor triggered elevation of [Ca 2ϩ ] i (14,15). Moreover, following Ca 2ϩ -stimulated differentiation of gingival keratinocytes, elevated expression of TRPC1, TRPC5, TRPC6, and TRPC7 has been reported (12).…”

ORAI1 calcium channel orchestrates skin homeostasis

“…From this TRPC family, TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 were shown to be expressed and functional in hPK, and all of them were shown to be involved in SOCE (27,(29)(30)(31). Interestingly, although the short-term transient increase in [Ca 2+ ] i is mostly attributed to be proproliferative, for two of them, TRPC1 and TRPC4, which where shown to be SOCs, a role in SOCE and keratinocyte differentiation was shown (27,29,32,33). Among all of them, a member of the vanilloid family of TRP channels, TRPV6, has been shown to be expressed and to positively correlate with the calcium gradient (a stepwise increase in both intra-and extracellular calcium from stratum basale to stratum corneum) (34-36) and differentiation in the skin, providing a massive calcium entry into keratinocytes that is necessary to induce differentiation (37,38).…”

TRP Channels and Pruritus

“…TRPC channels are important regulators of intracellular calcium homeostasis in several cells types [47,154,362,363]. Epidermal or mucosal keratinocytes also express various TRPC channels (TRPC1, TRPC4, TRPC5, TRPC6, TRPC7), and their expression levels fluctuate in a differentiationdependent manner [364][365][366].…”