Canonical transient receptor potential 6 (TRPC6), a redox-regulated cation channel.

This study examined the effect of H(2)O(2) on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H(2)O(2) significantly stimulated Ca(2+) entry in a dose-dependent manner. Electrophysiological experiments showed that H(2)O(2) significantly increased TRPC6 channel open probability and whole-cell currents. H(2)O(2) also evoked a robust inward current in A7r5 vascular smooth muscle cells, which was nearly abolished by knockdown of TRPC6 using a small interfering RNA. Catalase substantially attenuated arginine vasopressin (AVP)-induced Ca(2+) entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H(2)O(2) response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide- and H(2)O(2)-induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-sn-glycerol effect on TRPC6 was significantly greater in the presence of H(2)O(2). Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H(2)O(2). Similarly, in cells transfected with TRPC6-EGFP, confocal microscopy showed a significant increase in fluorescence intensity in the region of the cell membrane and adjacent to the membrane. AVP also increased the fluorescence intensity on the surface of the cells co-transfected with TRPC6-EGFP and V1 receptor, and this response was inhibited by catalase. These data indicate that H(2)O(2) activates TRPC6 channels via modification of thiol groups of intracellular proteins. This cysteine oxidation-dependent pathway not only stimulates the TRPC6 channel by itself but also sensitizes the channels to diacylglycerol and promotes TRPC6 trafficking to the cell surface.

Interaction between TRPC1/TRPC4 assembly and STIM1 contributes to store-operated Ca2+ entry in mesangial cells.

Although Orai1 protein was recently identified as the component of CRAC channels in hematopoietic cells, store-operated channels (SOC) in other cell types may have a different molecular entity. Also, the activation mechanism of SOC remains unclear, in general. In the present study, we tested the hypothesis that TRPC1 and TRPC4 proteins were functional subunits of SOC in glomerular mesangial cells (MCs) and that STIM1 was required for the channel activation through interaction with the TRPC proteins. In cultured human MCs, cell-attached patch clamp and fura-2 fluorescence measurements showed that single knockdown of either TRPC1 or TRPC4 significantly attenuated thapsigargin-induced membrane currents and Ca2+ entry as well as Ang II-induced channel activity. Double knockdown of both TRPCs resulted in a comparable inhibition on store-operated Ca2+ entry with single knockdown of either TRPC. Consistent with our previous report, co-immunoprecipitation showed a physical interaction between TRPC1 and TRPC4. Furthermore, we found that knockdown of STIM1 using RNAi significantly reduced the thapsigargin-stimulated membrane currents. Co-immunoprecipitation showed that STIM1 interacted with TRPC4, but not TRPC1. In addition, simultaneous inhibition of STIM1 and TRPC1 resulted in a comparable effect on SOC with single inhibition of either one of them. Taken together, we conclude that in glomerular mesangial cells, the TRPC1/TRPC4 complexes constitute the functional subunits of SOC and that the interaction between STIM1 and TRPC4 may be the mechanism for the activation of the channels.

Ca2+-sensitive transcriptional regulation: direct DNA interaction by DREAM.

Calcium is a major regulator of cell function, acting as a second messenger to relay signals from the cell surface to other parts of the cell. It plays an integral role in contraction of muscle cells and it regulates cell growth and proliferation, as well as cell death (1). The present review will discuss how Ca2+ mediates these functions through the regulation of gene expression. This can be accomplished by Ca2+-sensitive protein kinases as well as phosphatases which activate transcription factors. It can also be mediated by direct interaction of Ca2+-sensitive proteins with regulatory elements within the DNA sequence itself. Special attention will be given to recent advances in research in the down-stream regulatory element (DRE) and its Ca2+-sensitive modulator DREAM (downstream regulatory element antagonist modulator; also named calsenilin or K+ channel interacting protein 3 (KChIP3)) (2).

Mediation of angiotensin II-induced Ca2+ signaling by polycystin 2 in glomerular mesangial cells.

Ca(+) influx across the plasma membrane is a major component of mesangial cell (MC) response to vasoconstrictors. Polycystin 2 (PC2), the protein product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, has been shown to function as a nonselective cation channel in a variety of cell types. The present study was performed to test the hypothesis that PC2 and its binding partners constitute a Ca(2+)-permeable channel and contribute to ANG II-induced Ca(2+) signaling in MCs. Western blot and immunocytochemistry showed PC2 expression in cultured human MCs. The existence of PC2 in MCs was further confirmed by immunohistochemsitry in rat kidney sections. Coimmunoprecipitation displayed a selective interaction of PC2 with canonical transient receptor potential (TRPC) proteins TRPC1 and TRPC4. Cell-attached patch-clamp experiments revealed that ANG II-induced membrane currents were enhanced by overexpression of pkd2 but significantly inhibited by knock down of pkd2, 30 microM Gd(3+) (a PC2 channel blocker), and dominant-negative pkd2 mutant (pkd2-D511V). Corresponding to the increase in channel currents, ANG II stimulation increased expression of PC2 on the cell surface of MCs and interaction with TRPC1 and TRPC4. Furthermore, ANG II-induced MC contraction was significantly reduced in pkd2-knocked down MCs. These data suggest that PC2 selectively assembles with TRPC1 and TRPC4 to form channel complexes mediating ANG II-induced Ca(2+) responses in MCs.

Downregulation of TRPC6 protein expression by high glucose, a possible mechanism for the impaired Ca2+ signaling in glomerular mesangial cells in diabetes.

The present study was performed to investigate whether transient receptor potential (TRPC)6 participated in Ca(2+) signaling of glomerular mesangial cells (MCs) and expression of this protein was altered in diabetes. Western blots and real-time PCR were used to evaluate the expression level of TRPC6 protein and mRNA, respectively. Cell-attached patch-clamp and fura-2 fluorescence measurements were utilized to assess angiotensin II (ANG II)-stimulated membrane currents and Ca(2+) responses in MCs. In cultured human MCs, high glucose significantly reduced expression of TRPC6 protein, but there was no effect on either TRPC1 or TRPC3. The high glucose-induced effect on TRPC6 was time and dose dependent with the maximum effect observed on day 7 and at 30 mM glucose, respectively. In glomeruli isolated from streptozotocin-induced diabetic rats, TRPC6, but not TRPC1, was markedly reduced compared with the glomeruli of control rats. Furthermore, TRPC6 mRNA in MCs was also significantly decreased by high glucose as early as 1 day after treatment with maximal reduction on day 4. Patch-clamp experiments showed that ANG II-stimulated membrane currents in MCs were significantly attenuated or enhanced by knockdown or overexpression of TRPC6, respectively. Fura-2 fluorescence measurements revealed that the ANG II-induced Ca(2+) influxes were markedly inhibited in MCs with TRPC6 knockdown, reminiscent of the impaired Ca(2+) entry in response to ANG II in high glucose-treated MCs. These results suggest that the TRPC6 protein expression in MCs was downregulated by high glucose and the deficiency of TRPC6 protein might contribute to the impaired Ca(2+) signaling of MCs seen in diabetes.

Canonical transient receptor potential 1 channel is involved in contractile function of glomerular mesangial cells.

Contractility of mesangial cells (MC) is tightly controlled by [Ca(2+)](i). Ca(2+) influx across the plasma membrane constitutes a major component of mesangial responses to vasoconstrictors. Canonical transient receptor potential 1 (TRPC1) is a Ca(2+)-permeable cation channel in a variety of cell types. This study was performed to investigate whether TRPC1 takes part in vasoconstrictor-induced mesangial contraction by mediating Ca(2+) entry. It was found that angiotensin II (AngII) evoked remarkable contraction of the cultured MC. Downregulation of TRPC1 using RNA interference significantly attenuated the contractile response. Infusion of AngII or endothelin-1 in rats caused a decrease in GFR. The GFR decline was significantly reduced by infusion of TRPC1 antibody that targets an extracellular domain in the pore region of TRPC1 channel. However, the treatment of TRPC1 antibody did not affect the AngII-induced vasopressing effect. Electrophysiologic experiments revealed that functional or biologic inhibition of TRPC1 significantly depressed AngII-induced channel activation. Fura-2 fluorescence-indicated that Ca(2+) entry in response to AngII stimulation was also dramatically inhibited by TRPC1 antibody and TRPC1-specific RNA interference. These results suggest that TRPC1 plays an important role in controlling contractile function of MC. Mediation of Ca(2+) entry might be the underlying mechanism for the TRPC1-associated MC contraction.