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1.  Evaluation of Genetic Association and Expression Reduction of TRPC1 in the Development of Diabetic Nephropathy 
American Journal of Nephrology  2008;29(3):244-251.
The TRPC1 gene on chromosome 3q22–24 resides within the linkage region for diabetic nephropathy (DN) in type 1 (T1D) and type 2 diabetes mellitus (T2D). A recent study has demonstrated that TRPC1 expression is reduced in the kidney of diabetic ZDF- and STZ-treated rats. The present study aimed to evaluate the genetic and functional role of TRPC1 in the development of DN.
Genetic association study was performed with two independent cohorts, including 1,177 T1D European Americans with or without DN from GoKinD population and 850 African-American subjects with T2D-associated end-stage renal disease (ESRD), or with hypertensive (non-diabetic) ESRD, and nondiabetic controls. Seven tag SNP markers derived from HapMap data (phase II) were genotyped. TRPC1 gene expression was examined using real time RT-PCR.
No significant association of TRPC1 DNA polymorphisms with DN or ERSD was found in GoKinD and African-American populations. TRPC1 gene mRNA expression in kidney was found to be trendily reduced in 12-week and significantly in 26-week-old db/db mice.
TRPC1 genetic polymorphism may not fundamentally contribute to the development of DN, while reduction of the gene expression in kidney may be a late phenomenon of DN as seen in diabetic animal models.
PMCID: PMC2698220  PMID: 18802326
TRPC1 gene; Single-nucleotide polymorphism; Diabetic nephropathy; End-stage renal disease; Diabetes types 1 and 2
2.  Association of TRPC1 Gene Polymorphisms with Type 2 Diabetes and Diabetic Nephropathy in Han Chinese Population 
Endocrine Research  2013;38(2):59-68.
The recent genome-wide association studies reveal that chromosome 3q resides within the linkage region for diabetic nephropathy (DN) in type 1 and type 2 diabetes mellitus (T1D and T2D). The TRPC1 gene is on chromosome 3q22-24, and it has been demonstrated that TRPC1 expression is reduced in the kidney of diabetic animal models. Genetic association of TRPC1 polymorphism with T1D and DN has been reported in European Americans. However, there are no studies reporting the association of TRPC1 genetic polymorphism with T2D with and without DN in Chinese population. This study aimed to demonstrate the genetic role of TRPC1 in the development of T2D with and without DN in Chinese Han population. A genetic association study of TRPC1 was performed in T2D cases and in nondiabetic controls from Han population located in Northern Chinese areas. Six tag single nucleotide polymorphism (SNP) markers derived from HapMap data were genotyped. Among the six SNPs, only rs7638459 was suspected as risk factor of T2D without DN, fitting the log-additive model. The adjusted odds ratio (OR) for the CC genotyping was 2.39 (95% confidence interval (CI) = 1.00–5.68), compared with the TT genotyping. In addition, rs953239 was found to be a protective factor of getting DN in T2D, also fitting the log-additive model. When compared with the AA genotyping for SNP rs953239, the adjusted OR for CC genotyping was 0.63 (95% CI = 0.44–0.99). To summarize, this study shows that TRPC1 genetic polymorphisms are associated with T2D and DN in T2D in the Han Chinese population.
PMCID: PMC3619450  PMID: 23544998
TRPC1; Single nucleotide polymorphism; Type 2 diabetes mellitus; Diabetic nephropathy
3.  Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels 
Vasomotion describes oscillations of arterial vascular tone due to synchronized changes of intracellular calcium concentrations. Since increased calcium influx into vascular smooth muscle cells from spontaneously hypertensive rats (SHR) has been associated with variances of transient receptor potential canonical (TRPC) channels, in the present study we tested the hypothesis that increased vasomotion in hypertension is directly linked to increased TRPC expression. Using a small vessel myograph we observed significantly increased norepinephrine-induced vasomotion in mesenteric arterioles from SHR compared to normotensive Wistar–Kyoto (WKY) rats. Using immunoblottings we obtained significantly increased expression of TRPC1, TRPC3 and TRPC5 in mesenteric arterioles from SHR compared to WKY, whereas TRPC4 and TRPC6 showed no differences. Norepinephrine-induced vasomotion from SHR was significantly reduced in the presence of verapamil, SKF96365, 2-aminoethoxydiphenylborane (2-APB) or gadolinium. Pre-incubation of mesenteric arterioles with anti-TRPC1 and anti-TRPC3 antibodies significantly reduced norepinephrine-induced vasomotion and calcium influx. Control experiments with pre-incubation of TRPC antibodies plus their respective antigenic peptide or in the presence of anti-β-actin antibodies or random immunoglobulins not related to TRPC channels showed no inhibitory effects of norepinephrine-induced vasomotion and calcium influx. Administration of candesartan or telmisartan, but not amlodipine to SHR for 16 weeks significantly reduced either the expression of TRPC1, TRPC3 and TRPC5 as well as norepinephrine-induced vasomotion in mesenteric arterioles. In conclusion we gave experimental evidence that the increased TRPC1, TRPC3 and TRPC5 expression in mesenteric arterioles from SHR causes increased vasomotion in hypertension.
PMCID: PMC3823165  PMID: 19725917
vasomotion; transient receptor potential channels; angiotensin II 1 receptor blocker; calcium channel blocker; hypertension
4.  Decreased Anxiety-Like Behavior and Gαq/11-Dependent Responses in the Amygdala of Mice Lacking TRPC4 Channels 
The Journal of Neuroscience  2014;34(10):3653-3667.
Transient receptor potential (TRP) channels are abundant in the brain where they regulate transmission of sensory signals. The expression patterns of different TRPC subunits (TRPC1, 4, and 5) are consistent with their potential role in fear-related behaviors. Accordingly, we found recently that mutant mice lacking a specific TRP channel subunit, TRPC5, exhibited decreased innate fear responses. Both TRPC5 and another member of the same subfamily, TRPC4, form heteromeric complexes with the TRPC1 subunit (TRPC1/5 and TRPC1/4, respectively). As TRP channels with specific subunit compositions may have different functional properties, we hypothesized that fear-related behaviors could be differentially controlled by TRPCs with distinct subunit arrangements. In this study, we focused on the analysis of mutant mice lacking the TRPC4 subunit, which, as we confirmed in experiments on control mice, is expressed in brain areas implicated in the control of fear and anxiety. In behavioral experiments, we found that constitutive ablation of TRPC4 was associated with diminished anxiety levels (innate fear). Furthermore, knockdown of TRPC4 protein in the lateral amygdala via lentiviral-mediated gene delivery of RNAi mimicked the behavioral phenotype of constitutive TRPC4-null (TRPC4−/−) mouse. Recordings in brain slices demonstrated that these behavioral modifications could stem from the lack of TRPC4 potentiation in neurons in the lateral nucleus of the amygdala through two Gαq/11 protein-coupled signaling pathways, activated via Group I metabotropic glutamate receptors and cholecystokinin 2 receptors, respectively. Thus, TRPC4 and the structurally and functionally related subunit, TRPC5, may both contribute to the mechanisms underlying regulation of innate fear responses.
PMCID: PMC3942581  PMID: 24599464
amygdala; anxiety; cholecystokinin 4; fear; TRP channel; TRPC4
5.  Corticolimbic Expression of TRPC4 and TRPC5 Channels in the Rodent Brain 
PLoS ONE  2007;2(6):e573.
The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca2+ and Gq/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6–9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2–6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca2+and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.
PMCID: PMC1892805  PMID: 17593972
6.  TRPC3 and TRPC6 associate with BKCa channels: Role in BKCa trafficking to the surface of cultured podocytes 
Molecular pharmacology  2008;75(3):466-477.
Large-conductance (BKCa-type) Ca2+-activated K+ channels encoded by the Slo1 gene and various TRPC channels are co-expressed in many cell types, including podocytes (visceral epithelial cells) of the renal glomerulus. In this study, we show by co-immunoprecipitation and GST pull-down assays that BKCa channels can associate with endogenous TRPC3 and TRPC6 channels in differentiated cells of a podocyte cell line. Both types of TRPC channels co-localize with Slo1 in podocytes, as well as in HEK293T cells transiently co-expressing the TRPC channels with Slo1. In HEK293T cells, co-expression of TRPC6 increased surface expression of a Slo1 subunit splice variant (Slo1VEDEC) that is typically retained in intracellular compartments, as assessed by cell-surface biotinylation assays and confocal microscopy. Corresponding currents through BKCa channels were also increased with TRPC6 co-expression, as assessed by whole-cell and excised inside-out patch recordings. By contrast, co-expression of TRPC3 had no effect on the surface expression of BKCa channels in HEK293T cells or on the amplitudes of currents in whole cells or excised patches. In podocytes, siRNA knockdown of endogenous TRPC6 reduced steady-state surface expression of endogenous Slo1 channels, but knockdown of TRPC3 had no effect. TRPC6, but not TRPC3 knockdown also reduced voltage-evoked outward current through podocyte BKCa channels. These data indicate that TRPC6 and TRPC3 channels can bind to Slo1, and this co-localization may allow them to serve as a source of Ca2+ for activation of BKCa channels. TRPC6 channels also play a role in regulation of surface expression of a subset of podocyte BKCa channels.
PMCID: PMC2645922  PMID: 19052171
7.  Tyrosine phosphorylation–dependent activation of TRPC6 regulated by PLC-γ1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis 
Molecular Biology of the Cell  2011;22(11):1824-1835.
The surface expression and channel activation of transient receptor potential canonical 6 (TRPC6) were regulated by tyrosine phosphorylation and resultant binding with stimulatory PLC-γ1 and inhibitory nephrin. Disease-causing mutations made the TRPC6s insensitive to nephrin suppression, suggesting that the cell-type–specific regulation of TRPC6 might be involved in the pathogenesis.
Transient receptor potential canonicals (TRPCs) play important roles in the regulation of intracellular calcium concentration. Mutations in the TRPC6 gene are found in patients with focal segmental glomerulosclerosis (FSGS), a proteinuric disease characterized by dysregulated function of renal glomerular epithelial cells (podocytes). There is as yet no clear picture for the activation mechanism of TRPC6 at the molecular basis, however, and the association between its channel activity and pathogenesis remains unclear. We demonstrate here that tyrosine phosphorylation of TRPC6 induces a complex formation with phospholipase C (PLC)-γ1, which is prerequisite for TRPC6 surface expression. Furthermore, nephrin, an adhesion protein between the foot processes of podocytes, binds to phosphorylated TRPC6 via its cytoplasmic domain, competitively inhibiting TRPC6–PLC-γ1 complex formation, TRPC6 surface localization, and TRPC6 activation. Importantly, FSGS-associated mutations render the mutated TRPC6s insensitive to nephrin suppression, thereby promoting their surface expression and channel activation. These results delineate the mechanism of TRPC6 activation regulated by tyrosine phosphorylation, and imply the cell type–specific regulation, which correlates the FSGS mutations with deregulated TRPC6 channel activity.
PMCID: PMC3103399  PMID: 21471003
8.  TRPC6 Single Nucleotide Polymorphisms and Progression of Idiopathic Membranous Nephropathy 
PLoS ONE  2014;9(7):e102065.
Activating mutations in the Transient Receptor Potential channel C6 (TRPC6) cause autosomal dominant focal segmental glomerular sclerosis (FSGS). TRPC6 expression is upregulated in renal biopsies of patients with idiopathic membranous glomerulopathy (iMN) and animal models thereof. In iMN, disease progression is characterized by glomerulosclerosis. In addition, a context-dependent TRPC6 overexpression was recently suggested in complement-mediated podocyte injury in e.g. iMN. Hence, we hypothesized that genetic variants in TRPC6 might affect susceptibility to development or progression of iMN.
Methods & Results
Genomic DNA was isolated from blood samples of 101 iMN patients and 292 controls. By direct sequencing of the entire TRPC6 gene, 13 single nucleotide polymorphisms (SNPs) were identified in the iMN cohort, two of which were causing an amino acid substitution (rs3802829; Pro15Ser and rs36111323, Ala404Val). No statistically significant differences in genotypes or allele frequencies between patients and controls were observed. Clinical outcome in patients was determined (remission n = 26, renal failure n = 46, persistent proteinuria n = 29, follow-up median 80 months {range 51–166}). The 13 identified SNPs showed no association with remission or renal failure. There were no differences in genotypes or allele frequencies between patients in remission and progressors.
Our data suggest that TRPC6 polymorphisms do not affect susceptibility to iMN, or clinical outcome in iMN.
PMCID: PMC4096511  PMID: 25019165
9.  An intergenic region on chromosome 13q33.3 is associated with the susceptibility to kidney disease in type 1 and 2 diabetes 
Kidney international  2011;80(1):105-111.
A genome-wide association (GWA) scan of the Genetics of Kidneys in Diabetes (GoKinD) collections identified four novel susceptibility loci, located on chromosomes 7p14.3, 9q21.32, 11p15.4, and 13q33.3 that were associated with nephropathy in type 1 diabetes. The recent examination of these loci in Japanese patients with type 2 diabetes further supported associations at the chromosome 13q33.3 locus. To follow up these findings, we focused on these same four loci and examined whether single nucleotide polymorphisms (SNPs) at these susceptibility loci were associated with diabetic nephropathy in the Joslin Study of Genetics of Nephropathy in Type 2 Diabetes collection. A total of six SNPs across these loci were genotyped in 646 normoalbuminuric controls and 743 nephropathy cases of European ancestry. A significant association was identified at the 13q33.3 locus (rs9521445: OR=1.25, P=4.4×10−3). At this same locus, rs1411766 was also associated with type 2 diabetic nephropathy in this collection (OR=1.19, P=0.03). A meta-analysis combining this data with that from the Japanese and GoKinD collections significantly improved the strength of this association (OR=1.29 P=9.7×10−9). Additionally, we also observed an association at the 11p15.4 locus (rs451041: OR=1.21, P=0.02). Our analysis increases support that associations identified in the GoKinD collections on chromosomes 11p15.4 (near the CARS gene) and 13q33.3 (within an intergenic region between MYO16 and IRS2) are true diabetic nephropathy susceptibility loci common to both type 1 and type 2 diabetes.
PMCID: PMC3774030  PMID: 21412220
10.  Association of polymorphisms in the klotho gene with severity of non-diabetic ESRD in African Americans 
Nephrology Dialysis Transplantation  2010;25(10):3348-3355.
Background. Non-diabetic forms of nephropathy commonly lead to end-stage renal disease (non-DM ESRD). Previous studies have demonstrated that African Americans are more susceptible to non-DM ESRD compared to other ethnic groups, and this risk has a strong genetic component. A genome-wide scan for ESRD in African American families enriched for non-DM ESRD showed evidence for linkage in chromosome 13q33.3, and a candidate gene in this region, klotho, was selected for a detailed analysis in a follow-up case-control association study.
Methods. Thirty-four single-nucleotide polymorphisms (SNPs) in the klotho gene were genotyped in 317 unrelated African American non-DM ESRD cases and 354 non-nephropathy controls, including 12 SNPs identified by re-sequencing a region around exon 4.
Results. Two SNPs demonstrated modest admixture-adjusted evidence of association with non-DM ESRD, rs650439 (P = 0.013, recessive model) and rs643780 (P = 0.017, recessive model), while rs17643698 approached significance (P = 0.0953, two degrees of freedom test). Eight of the most significant SNPs were tested for replication in a second case-control collection (557 African American non-DM ESRD cases and 187 controls), and there was no evidence of association in replicate cases and controls; nor when the samples were combined for a total of 874 non-DM cases and 541 controls. Cox proportional hazards models were computed to test for association between polymorphisms in klotho and age at onset of ESRD. A three-SNP haplotype, rs526906, rs525014 and rs571118 (T/T/A), was associated with age of onset of ESRD [P = 0.007, recessive model; hazard ratio (HR) = 0.70]. Subjects homozygous for this haplotype had a mean 4 years later onset of ESRD, suggesting a slower disease progression. HapMap subjects homozygous for this haplotype had increased expression of klotho, further supporting a protective role of this variant in ESRD.
Conclusion. We conclude that three SNPs in intron 1 of the klotho gene are associated with delayed age at onset of non-DM ESRD in African Americans.
PMCID: PMC2948839  PMID: 20466664
genetics; klotho; non-diabetic ESRD
11.  Expression of trpC1 and trpC6 orthologs in zebrafish 
Gene expression patterns : GEP  2008;8(5):291-296.
Transient receptor potential (TRP) genes encode subunits that form cation-selective ion channels in a variety of organisms and cell types. TRP channels serve diverse functions ranging from thermal, tactile, taste, and osmolar sensing to fluid flow sensing. TRPC1 and TRPC6 belong to the TRPC subfamily, members of which are thought to contribute to several cellular events such as regulated migration of neuronal dendrites, contractile responses of smooth muscle cells and maintenance of the structural integrity of kidney podocytes. Pathogenic roles have been suggested for TRPC1 in asthma and chronic obstructive pulmonary disease, and TRPC6 dysfunction was recently linked to proteinuric kidney disease. To explore the potential roles for TRPC channels in zebrafish organ function, we cloned zebrafish trpC1 and trpC6 cDNAs, and investigated their expression during zebrafish development. We detected trpC1 expression in the head, in cells surrounding the outflow tract of the heart, and in the ganglion cells as well as the inner nuclear layer of the eye. trpC6 expression was detected in the head, pectoral fins, aortic endothelial cells, and gastrointestinal smooth muscle cells. Our results point to roles of TRPC channels in several tissues during zebrafish development, and suggest that the zebrafish may be a suitable model system to study the pathophysiology of TRPC1 and TRPC6 in specific cell types.
PMCID: PMC2431112  PMID: 18378501
Transient receptor potential; ion channel; smooth muscle; in situ hybridization
12.  Identification of TRPC6 as a possible candidate target gene within an amplicon at 11q21-q22.2 for migratory capacity in head and neck squamous cell carcinomas 
BMC Cancer  2013;13:116.
Cytogenetic and gene expression analyses in head and neck squamous cell carcinomas (HNSCC) have allowed identification of genomic aberrations that may contribute to cancer pathophysiology. Nevertheless, the molecular consequences of numerous genetic alterations still remain unclear.
To identify novel genes implicated in HNSCC pathogenesis, we analyzed the genomic alterations present in five HNSCC-derived cell lines by array CGH, and compared high level focal gene amplifications with gene expression levels to identify genes whose expression is directly impacted by these genetic events. Next, we knocked down TRPC6, one of the most highly amplified and over-expressed genes, to characterize the biological roles of TRPC6 in carcinogenesis. Finally, real time PCR was performed to determine TRPC6 gene dosage and mRNA levels in normal mucosa and human HNSCC tissues.
The data showed that the HNSCC-derived cell lines carry most of the recurrent genomic abnormalities previously described in primary tumors. High-level genomic amplifications were found at four chromosomal sites (11q21-q22.2, 18p11.31-p11.21, 19p13.2-p13.13, and 21q11) with associated gene expression changes in selective candidate genes suggesting that they may play an important role in the malignant behavior of HNSCC. One of the most dramatic alterations of gene transcription involved the TRPC6 gene (located at 11q21-q22.2) which has been recently implicated in tumour invasiveness. siRNA-induced knockdown of TRPC6 expression in HNSCC-derived cells dramatically inhibited HNSCC-cell invasion but did not significantly alter cell proliferation. Importantly, amplification and concomitant overexpression of TRPC6 was also found in HNSCC tumour samples.
Altogether, these data show that TRPC6 is likely to be a target for 11q21–22.2 amplification that confers enhanced invasive behavior to HNSCC cells. Therefore, TRPC6 may be a promising therapeutic target in the treatment of HNSCC.
PMCID: PMC3606258  PMID: 23497198
Head and neck squamous cell carcinoma; TRPC6; Invasion; Gene amplification
13.  Local Ca2+ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca2+ Signals Required for Specific Cell Functions 
PLoS Biology  2011;9(3):e1001025.
Store-operated Ca2+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca2+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent ISOC, activated in response to Ca2+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated ICRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca2+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd3+, removal of extracellular Ca2+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca2+-containing, but not Ca2+-free, medium. Consistent with this, ICRAC is activated in cells pretreated with thapsigargin in Ca2+-free medium while ISOC is activated in cells pretreated in Ca2+-containing medium. Significantly, TRPC1 function is required for sustained KCa activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca2+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca2+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.
Author Summary
Store-operated Ca2+ entry is present in all cell types and determines sustained cytosolic [Ca2+] increases that are critical for regulating a wide variety of physiological functions. This Ca2+ entry mechanism is activated in response to depletion of Ca2+ in the endoplasmic reticulum (ER). When ER [Ca2+] is decreased, the Ca2+-sensor protein STIM1 aggregates in the ER membrane and moves to regions in the periphery of the cells where it interacts with and activates two major types of channels that contribute to store-operated Ca2+ entry: CRAC and SOC. While gating of Orai1 by STIM1 is sufficient for CRAC channel activity, both Orai1 and transient receptor potential channel 1 (TRPC1) contribute to SOC channel function. The molecular composition of SOC channels and the critical role of Orai1 in activation of TRPC1 have not yet been established. In this study, we demonstrate that TRPC1 and Orai1 are components of distinct channels, both of which are regulated by STIM1. Importantly, we show that Orai1-mediated Ca2+ entry triggers plasma membrane insertion of TRPC1 which is then gated by STIM1. Ca2+ entry via functional TRPC1-STIM1 channels provides additional increase in cytosolic [Ca2+] that is required for regulation of specific cell functions such as KCa activation. Together, our findings elucidate the critical role of Orai1 in TRPC1 channel function. We suggest that the regulation of TRPC1 trafficking provides a mechanism for rapidly modulating cytosolic [Ca2+] following Ca2+ store depletion.
PMCID: PMC3050638  PMID: 21408196
14.  Trpc2 Depletion Protects RBC from Oxidative Stress-Induced Hemolysis 
Experimental hematology  2011;40(1):71-83.
Transient receptor potential channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca2+]i) through TRPC2 and TRPC3. Because modulation of [Ca2+]i is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild type littermate controls. Although the erythropoietin induced increase in [Ca2+]i was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenyhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. While erythropoietin induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.
PMCID: PMC3237850  PMID: 21924222
TRP Channels; Trpc2; Trpc3; erythropoietin; oxidative stress
15.  Association Analysis of the Ephrin-B2 Gene in African-Americans with End-Stage Renal Disease 
American Journal of Nephrology  2008;28(6):914-920.
Genome scans in African-Americans with end-stage renal disease (ESRD) identified linkage on chromosome 13q33 in the region containing the ephrin-B2 ligand (EFNB2) genes. Interactions between the ephrin-B2 receptor and ephrin-B2 ligand play essential roles in renal angiogenesis, blood vessel maturation, and kidney disease.
The EFNB2 gene was evaluated as a positional candidate for non-diabetic and diabetic ESRD susceptibility in 1,071 unrelated African-American subjects; 316 with non-diabetic etiologies of ESRD, 394 with type 2 diabetes-associated ESRD and 361 healthy controls. Single nucleotide polymorphism (SNP) genotyping was performed on the Sequenom Mass Array System. Statistical analyses were computed using Dandelion version 1.26, Snpaddmix version 1.4 and Haploview version 3.32.
Twenty-eight HapMap tag SNPs were genotyped spanning the 39 kilobases (kb) of the EFNB2 coding region, with average spacing of 1.43 kb. Analysis of 710 ESRD patient samples and 361 controls provided no evidence of single SNP associations in either diabetic or non-diabetic ESRD; although nominal evidence of association with all-cause ESRD was observed with a two SNP (p = 0.022) and three SNP (p = 0.023) haplotype, both containing SNPs rs7490924 and rs2391335 in intron 1.
Although an attractive positional candidate gene, polymorphisms in the EFNB2 gene do not appear to contribute in a substantial way to non-diabetic, diabetic or all-cause ESRD susceptibility in African-Americans. Additional genes within the chromosome 13q33 linkage interval are likely contributors to African-American non-diabetic ESRD.
PMCID: PMC2786015  PMID: 18580054
African-Americans; End-stage renal disease; Ephrin; Genetics; Hypertensive nephrosclerosis
16.  Caveolin-1 Contributes to Assembly of Store-operated Ca2+ Influx Channels by Regulating Plasma Membrane Localization of TRPC1 
The Journal of biological chemistry  2003;278(29):27208-27215.
TRPC1, a component of store-operated Ca2+ entry (SOCE) channels, is assembled in a complex with caveolin- 1 (Cav1) and key Ca2+ signaling proteins. This study examines the role of Cav1 in the function of TRPC1. TRPC1 and Cav1 were colocalized in the plasma membrane region of human submandibular gland and Madin-Darby canine kidney cells. Full-length Cav1 bound to both the N and C termini of TRPC1. Amino acids 271–349, which includes a Cav1 binding motif (amino acids 322–349), was identified as the Cav1 binding domain in the TRPC1 N terminus. Deletion of amino acids 271–349 or 322–349 prevented plasma membrane localization of TRPC1. Importantly, TRPC1Δ271–349 induced a dominant suppression of SOCE and was associated with wild-type TRPC1. Although the role of the C-terminal Cav1 binding domain is not known, its deletion did not affect localization of TRPC1 (Singh, B. B., Liu, X., and Ambudkar, I. S. (2000) J. Biol. Chem. 275, 36483–36486). Further, expression of a truncated Cav1 (Cav1Δ51–169), but not full-length Cav1, similarly disrupted plasma membrane localization of endogenously and exogenously expressed TRPC1 in human Submandibular gland and Madin-Darby canine kidney cells. Cav1Δ51–169 also suppressed thapsigargin- and carbachol-stimulated Ca2+ influx and increased the detergent solubility of TRPC1, although plasma membrane lipid raft domains were not disrupted. These data demonstrate that plasma membrane localization of TRPC1 depends on an interaction between its N terminus and Cav1. Thus, our data suggest that Cav1 has an important role in the assembly of SOCE channel(s).
PMCID: PMC3621139  PMID: 12732636
17.  Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity 
Cardiovascular Research  2012;95(4):439-447.
Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery.
Methods and results
TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SKCa (apamin) or IKCa (TRAM-34) block, Pyr3 abolished the residual respective IKCa- and SKCa-dependent EDH-mediated vasodilation.
The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SKCa and IKCa activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.
PMCID: PMC3422079  PMID: 22721989
Endothelium; Calcium channel; Potassium channel; Signalling microdomain; Smooth muscle; Vasodilation
18.  Transient Receptor Potential Canonical-3 Channel–Dependent Fibroblast Regulation in Atrial Fibrillation 
Circulation  2012;126(17):2051-2064.
Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)–promoting remodeling. Here, we investigated fibroblast regulation by Ca2+-permeable transient receptor potential canonical-3 (TRPC3) channels.
Methods and Results
Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (INSC) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 μmol/L). Pyrazole-3 suppressed angiotensin II-induced Ca2+ influx, proliferation, and α-smooth muscle actin protein expression in fibroblasts. Ca2+ removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, α-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5′ promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression.
TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca2+ influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.
PMCID: PMC3675169  PMID: 22992321
arrhythmia; calcium; ion channels; fibrillation; remodeling
19.  Molecular and clinical analysis of TRPC6 and AGTR1 genes in patients with pulmonary arterial hypertension 
Pulmonary arterial hypertension (PAH) is a rare and progressive vascular disorder characterized by increased pulmonary vascular resistance and right heart failure. The aim of this study was to analyze 5′UTR region in canonical transient receptor potential isoform 6 (TRPC6) and 3′UTR region in Angiotensin II type I receptor (AGTR1) genes in patients with idiopathic and associated PAH. Correlation among mutations and clinical and functional parameters was further analyzed.
Analysis of TRPC6 and AGTR1 genes was performed by polymerase chain reaction (PCR) and direct sequencing. We used a non-parametric test to determine if significant differences were found between the groups studied and chi-square test to compare clinical and hemodynamic variables among genotypes.
Fifty five patients and fifty two controls were included in this study. We found statistically significant differences for c.1-361A > T (p = 0.0077), c.1-254C > G (p < 0.0001) and c.1-218C > T (p = 0.0021) in TRPC6 gene and c.1166A > C (p < 0.001) in AGTR1 gene, between patients and controls. Idiopathic PAH patients (IPAH) and controls presented significant differences for all 3 TRPC6 polymorphisms (p = 0.020), (p = 0.002) and (p = 0.008) respectively, and also showed differences for AGTR1 gene (p < 0.001). In associated PAH (APAH) patients we found statistical differences for c.1-254C > G (p < 0.001) and c.1-218C > T (p = 0.001) in TRPC6 gene and c.1166A > C (p = 0.001) in AGTR1 gene. Several clinical and hemodynamic parameters showed significant differences between carriers and non-carriers of these single nucleotide polymorphisms (SNPs). Nineteen patients were carriers of all 3 SNPs in TRPC6 gene and presented a more severe phenotype with differences in mean pulmonary arterial pressure (p = 0.016), systolic pulmonary arterial pressure (p = 0.040), cardiac index (p < 0.001) and 6 minute walking test (p = 0.049). 16 of these patients harbored the SNP in AGTR1 gene. These patients showed differences in age at diagnosis (p = 0.049), mean pulmonary arterial pressure (p = 0.033), cardiac index (p = 0.002) and 6 minute walking test (p = 0.039).
PAH is a rare disease with pulmonary vascular remodeling caused in part by a heterogeneous constellation of genetic arrangements. This study seems to suggest that c.1-361A > T, c.1-254C > G and c.1-218C > T polymorphisms in TRPC6 gene and c.1166A > C polymorphism in AGTR1 could have a role in the development of this disease.
PMCID: PMC4307182  PMID: 25603901
Pulmonary Arterial Hypertension; TRPC6; AGTR1; Polymorphism; Correlation genotype/phenotype
20.  Insights to the Genetics of Diabetic Nephropathy through a Genome-wide Association Study of the GoKinD Collection 
Seminars in nephrology  2010;30(2):126-140.
The Genetics of Kidneys in Diabetes (GoKinD) study was initiated to facilitate research aimed at identifying genes involved in diabetic nephropathy (DN) in type 1 diabetes (T1D). In this review, we present on overview of this study and the various reports that have utilized its collection. At the forefront of these efforts is the recent genome-wide association (GWA) scan implemented on the GoKinD collection. We highlight the results from our analysis of these data and describe compelling evidence from animal models that further support the potential role of associated loci in the susceptibility of DN. To enhance our analysis of genetic associations in GoKinD, using genome-wide imputation (GWI), we expanded our analysis of this collection to include genotype data from more than 2.4 million common SNPs. We illustrate the added utility of this enhanced dataset through the comprehensive fine-mapping of candidate genomic regions previously linked with DN and the targeted investigation of genes involved in candidate pathway implicated in its pathogenesis. Collectively, GWA and GWI data from the GoKinD collection will serve as a springboard for future investigations into the genetic basis of DN in T1D.
PMCID: PMC2847588  PMID: 20347642
genome-wide association; diabetic nephropathy; type 1 diabetes; imputation
21.  A Functional Single-Nucleotide Polymorphism in the TRPC6 Gene Promoter Associated With Idiopathic Pulmonary Arterial Hypertension 
Circulation  2009;119(17):2313-2322.
Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role in the development of idiopathic pulmonary arterial hypertension (IPAH), whereas a rise in cytosolic Ca2+ concentration triggers PASMC contraction and stimulates PASMC proliferation. Recently, we demonstrated that upregulation of the TRPC6 channel contributes to proliferation of PASMCs isolated from IPAH patients. This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC6 gene promoter that are associated with IPAH and have functional significance in regulating TRPC6 activity in PASMCs.
Methods and Results
Genomic DNA was isolated from blood samples of 237 normal subjects and 268 IPAH patients. Three biallelic SNPs, −361 (A/T), −254(C/G), and −218 (C/T), were identified in the 2000-bp sequence upstream of the transcriptional start site of TRPC6. Although the allele frequencies of the −361 and −218 SNPs were not different between the groups, the allele frequency of the −254(C→G) SNP in IPAH patients (12%) was significantly higher than in normal subjects (6%; P<0.01). Genotype data showed that the percentage of −254G/G homozygotes in IPAH patients was 2.85 times that of normal subjects. Moreover, the −254(C→G) SNP creates a binding sequence for nuclear factor-κB. Functional analyses revealed that the −254(C→G) SNP enhanced nuclear factor-κB–mediated promoter activity and stimulated TRPC6 expression in PASMCs. Inhibition of nuclear factor-κB activity attenuated TRPC6 expression and decreased agonist-activated Ca2+ influx in PASMCs of IPAH patients harboring the −254G allele.
These results suggest that the −254(C→G) SNP may predispose individuals to an increased risk of IPAH by linking abnormal TRPC6 transcription to nuclear factor-κB, an inflammatory transcription factor.
PMCID: PMC2749566  PMID: 19380626
calcium; hypertension; pulmonary; ion channels; muscle, smooth; NF-kappa B
22.  Gq signaling causes glomerular injury by activating TRPC6 
The Journal of Clinical Investigation  2015;125(5):1913-1926.
Familial forms of focal segmental glomerulosclerosis (FSGS) have been linked to gain-of-function mutations in the gene encoding the transient receptor potential channel C6 (TRPC6). GPCRs coupled to Gq signaling activate TRPC6, suggesting that Gq-dependent TRPC6 activation underlies glomerular diseases. Here, we developed a murine model in which a constitutively active Gq α subunit (GqQ209L, referred to herein as GqQ>L) is specifically expressed in podocytes and examined the effects of this mutation in response to puromycin aminonucleoside (PAN) nephrosis. We found that compared with control animals, animals expressing GqQ>L exhibited robust albuminuria, structural features of FSGS, and reduced numbers of glomerular podocytes. Gq activation stimulated calcineurin (CN) activity, resulting in CN-dependent upregulation of TRPC6 in murine kidneys. Deletion of TRPC6 in GqQ>L-expressing mice prevented FSGS development and inhibited both tubular damage and podocyte loss induced by PAN nephrosis. Similarly, administration of the CN inhibitor FK506 reduced proteinuria and tubular injury but had more modest effects on glomerular pathology and podocyte numbers in animals with constitutive Gq activation. Moreover, these Gq-dependent effects on podocyte injury were generalizable to diabetic kidney disease, as expression of GqQ>L promoted albuminuria, mesangial expansion, and increased glomerular basement membrane width in diabetic mice. Together, these results suggest that targeting Gq/TRPC6 signaling may have therapeutic benefits for the treatment of glomerular diseases.
PMCID: PMC4463190  PMID: 25844902
23.  Confirmation of Genetic Associations at ELMO1 in the GoKinD Collection Supports Its Role as a Susceptibility Gene in Diabetic Nephropathy 
Diabetes  2009;58(11):2698-2702.
To examine the association between single nucleotide polymorphisms (SNPs) in the engulfment and cell motility 1 (ELMO1) gene, a locus previously shown to be associated with diabetic nephropathy in two ethnically distinct type 2 diabetic populations, and the risk of nephropathy in type 1 diabetes.
Genotypic data from a genome-wide association scan (GWAS) of the Genetics of Kidneys in Diabetes (GoKinD) study collection were analyzed for associations across the ELMO1 locus. In total, genetic associations were assessed using 118 SNPs and 1,705 individuals of European ancestry with type 1 diabetes (885 normoalbuminuric control subjects and 820 advanced diabetic nephropathy case subjects).
The strongest associations in ELMO1 occurred at rs11769038 (odds ratio [OR] 1.24; P = 1.7 × 10−3) and rs1882080 (OR 1.23; P = 3.2 × 10−3) located in intron 16. Two additional SNPs, located in introns 18 and 20, respectively, were also associated with diabetic nephropathy. No evidence of association for variants previously reported in type 2 diabetes was observed in our collection.
Using GWAS data from the GoKinD collection, we comprehensively examined evidence of association across the ELMO1 locus. Our investigation marks the third report of associations in ELMO1 with diabetic nephropathy, further establishing its role in the susceptibility of this disease. There is evidence of allelic heterogeneity, contributed by the diverse genetic backgrounds of the different ethnic groups examined. Further investigation of SNPs at this locus is necessary to fully understand the commonality of these associations and the mechanism(s) underlying their role in diabetic nephropathy.
PMCID: PMC2768169  PMID: 19651817
24.  Tissue-specific expression of TRP channel genes in the mouse and its variation in three different mouse strains 
BMC Genomics  2006;7:159.
The purpose of this work was to study the gene expression of transient receptor potential (TRP) channels in the mouse. The application of a standardized and quantitative technique, TaqMan RT-PCR, should give information about the pattern and relative importance of TRP channels for murine tissues and cell types. To verify data sets with an independent method, we studied the occurrence of some of the transcripts by in situ hybridization.
We have characterized the mRNA expression of 22 TRP channels in the mouse with a focus on nerve and muscle tissues. This is the first study to describe the expression profiles of all channel isoforms of the four related Group 1 subfamilies (TRPC, TRPV, TRPM and TRPA) with a standardized and quantitative technique. Comparisons of transcript abundance showed a consistent dominance of TRPM7 and TRPC3 in most tissues. We further observed characteristic patterns and differences in gene expression of individual channels ranging over three orders of magnitude. The overall level of TRP channel mRNAs was highest in brain areas followed by kidney, lung, reproductive organs and muscle. In brain TRPM3 and TRPM7 dominated and 19 other isoforms were detected. In lung and kidney TRPV4, TRPV5 and TRPM7 were found in highest levels. TRPM7, TRPC3, TRPC6 and TRPM3 mRNAs were characteristically present in all tested muscle tissues. Most data obtained with the C57Bl/10 mouse strain were confirmed with Balb/c and NOD mice. However, TRPC3, C6, TRPM7, M3, TRPV2 and V4 expression showed marked differences in the three tested mouse strains. In situ hybridization revealed co-expression of transcripts on the cellular level and widely confirmed the data obtained with RT-PCR.
Transcripts coding for members of the TRPC, TRPV, TRPM and TRPA subfamilies of TRP cation channels are present in a broad spectrum of murine tissues. Several channel isoforms often coexist in a specific tissue or cell type. TRP channel expression does not show typical tissue specific dominance of individual members as is known from other ion channel families. Mouse strain specific variations of TRP channel expression indicate that genetic background or physiological requirements considerably influence expression levels.
PMCID: PMC1557673  PMID: 16787531
25.  Genetics of Kidneys in Diabetes (GoKinD) Study: A Genetics Collection Available for Identifying Genetic Susceptibility Factors for Diabetic Nephropathy in Type 1 Diabetes 
The Genetics of Kidneys in Diabetes (GoKinD) study is an initiative that aims to identify genes that are involved in diabetic nephropathy. A large number of individuals with type 1 diabetes were screened to identify two subsets, one with clear-cut kidney disease and another with normal renal status despite long-term diabetes. Those who met additional entry criteria and consented to participate were enrolled. When possible, both parents also were enrolled to form family trios. As of November 2005, GoKinD included 3075 participants who comprise 671 case singletons, 623 control singletons, 272 case trios, and 323 control trios. Interested investigators may request the DNA collection and corresponding clinical data for GoKinD participants using the instructions and application form that are available at Participating scientists will have access to three data sets, each with distinct advantages. The set of 1294 singletons has adequate power to detect a wide range of genetic effects, even those of modest size. The set of case trios, which has adequate power to detect effects of moderate size, is not susceptible to false-positive results because of population substructure. The set of control trios is critical for excluding certain false-positive results that can occur in case trios and may be particularly useful for testing gene—environment interactions. Integration of the evidence from these three components into a single, unified analysis presents a challenge. This overview of the GoKinD study examines in detail the power of each study component and discusses analytic challenges that investigators will face in using this resource.
PMCID: PMC2770870  PMID: 16775037

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