Expression of different connexin genes in rat uterus during decidualization and at term.

The expression of different connexin genes (cx26, cx32, cx37, cx43) that code for the protein subunits of gap junctions, was investigated in various uterine tissues during the estrous cycle of nonpregnant rats, in pregnant rats at decidualization and at term. Connexin gene expression was studied at the mRNA level by Northern blot hybridization and at the protein level by immunocytochemistry. In gap junctions from uterine epithelium, stroma, or myometrium, connexin 26 and/or connexin 43 are much more abundant than connexins 32 and 37. The expression of connexin 26 and 43 appears to be modulated by maternal steroid hormones. High expression of these connexins is found in developing decidual cells by day 7 to 8 post coitum; furthermore, coexpression of connexins 26 and 43 in myometrium is observed just before delivery on day 21 post coitum. In both the decidua and the myometrium, the connexin 26 protein appears to be distributed in lower abundance than connexin 43. In uterine epithelium only connexin 26 is expressed throughout all of the reproductive phases investigated. The enhanced expression of this gene correlates with higher levels of maternal estrogen both in the proestrus/estrus phase and at term. The distinct spatial and temporal pattern of expression of connexins 26 and 43 in different uterine tissues suggests a physiological role for these proteins during embryo implantation and subsequent contraction of the uterus at birth.     

Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes

Transient receptor potential canonical‐6 (TRPC6) ion channels, expressed at high levels in podocytes of the filtration barrier, are recently implicated in the pathogenesis of various forms of proteinuric kidney diseases. Indeed, inherited or acquired up‐regulation of TRPC6 activities are suggested to play a role in podocytopathies. Yet, we possess limited information about the regulation of TRPC6 in human podocytes. Therefore, in this study, we aimed at defining how the protein kinase C (PKC) system, one of the key intracellular signalling pathways, regulates TRPC6 function and expression. On human differentiated podocytes, we identified the molecular expressions of both TRPC6 and several PKC isoforms. We also showed that TRPC6 channels are functional since the TRPC6 activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) induced Ca²⁺‐influx to the cells. By assessing the regulatory roles of the PKCs, we found that inhibitors of the endogenous activities of classical and novel PKC isoforms markedly augmented TRPC6 activities. In contrast, activation of the PKC system by phorbol 12‐myristate 13‐acetate (PMA) exerted inhibitory actions on TRPC6 and suppressed its expression. Importantly, PMA treatment markedly down‐regulated the expression levels of PKCα, PKCβ, and PKCη reflecting their activation. Taken together, these results indicate that the PKC system exhibits a ‘tonic’ inhibition on TRPC6 activity in human podocytes suggesting that pathological conditions altering the expression and/or activation patterns of podocyte‐expressed PKCs may influence TRPC6 activity and hence podocyte functions. Therefore, it is proposed that targeted manipulation of certain PKC isoforms might be beneficial in certain proteinuric kidney diseases with altered TRPC6 functions.     

Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+) and G(q)/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 Ca(2+)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.     

Co-expression of non-selective cation channels of the transient receptor potential canonical family in central aminergic neurones

The mammalian transient receptor potential canonical (TRPC) group of channels is a family of Ca2+-permeable cation channels that are activated following receptor-mediated stimulation of different isoforms of phospholipase C. In vitro TRPC proteins can form hetero- or homo-oligomeric channels. We performed single-cell RT-PCR analysis to reveal the co-expression of seven TRPC channels in identified rat aminergic neurones. All serotonergic neurones of the dorsal raphe (DR), the majority of histaminergic (tuberomamillary nucleus; TMN) and dopaminergic cells of the ventral tegmental area (VTA), as well as some GABAergic neurones from the VTA, expressed at least one variant of TRPC channels. No TRPC channel expression was found in the locus coeruleus. In raphe neurones TRPC6 and TRPC5 mRNAs occurred most frequently. In VTA and TMN co-expression of TRPC4 with TRPC5 and TRPC6 with TRPC7 was not found in individual neurones (in contrast to the whole-brain regions). Their co-expression in non-neuronal cells could not be excluded. The neonatal TRPC3 subunit was rarely seen. In DR, but not in the other nuclei studied, the expression of orexin receptors correlated with the expression of TRPC channels. We conclude that several TRPC channel populations exist in individual neurones and that their subunit co-expression pattern is region and cell-type specific.     

Expression and modulation of CD44 variant isoforms in humans

CD44 is a ubiquitous surface molecule that exists as a number of isoforms, generated by alternative splicing of 10 "variant" exons. Little is known about the expression and function of the variant isoforms, except that certain isoforms may play a role in cancer metastasis. We produced mAbs against CD44 variant regions encoded by exons 4v, 6v, and 9v, by immunizing mice with a fusion protein spanning variant exons 3v to 10v. A comprehensive analysis of human tissues revealed that CD44 variant isoforms were expressed widely throughout the body, principally by epithelial cells. However there was differential expression of CD44 variant exons by different epithelia. Most epithelia expressed exon 9v, but much fewer expressed 6v or 4v. The regions of epithelia that expressed the highest levels of the variant isoforms were the generative cells, particularly the basal cells of stratified squamous epithelium, and of glandular epithelium. CD44 variant isoforms were also expressed differentially by leukocytes, with CD44-9v expressed at very low levels and CD44-6v and 4v virtually absent. However, CD44-9v and CD44-6v were the main variants that were transiently upregulated on T cells after mitogenic stimulation and on myelomonocytic cell lines by TNF alpha and IFN gamma treatment. Some epithelial cell lines could preferentially upregulate CD44-6v upon IFN gamma incubation. These results show that CD44 variant isoforms are expressed much more widely than first appreciated, and that expression of the variant isoforms on some cell types can be modulated by particular cytokines.     

Heterogeneous distribution of TRPC proteins in the embryonic cortex

The present study was initiated to gain some information about the tissue distribution of transient receptor potential proteins of C-type (TRPC), a family of voltage-independent cation channels, at the beginning of neurogenesis in the telencephalon of embryonic mice. The mRNAs of all known TRPCs (TRPC1–TRPC7) could be found in the cortex at E13. TRPC1, TRPC3 and TRPC5 were the main isoforms, whereas the mRNAs for TRPC2, TRPC4, TRPC6 and TRPC7 were less abundant. The distribution throughout the cortical wall of TRPC1, TRPC3 and TRPC6 was studied by means of immuno-histochemistry. The data collected pointed to a heterogeneous expression of the channels. Three groups were identified. The first one comprises TRPC1, specifically found in the preplate but only in some post-mitotic neurons. It was mainly observed in a subset of cells distinct from the Cajal-Retzius cells. The second group is composed of TRPC3. It was found in non-neuronal cells and also in dividing (5-bromo-2′-deoxyuridine-positive) cells, indicating that TRPC3 is present in precursor cells. The third group contains TRPC6 detected in neuronal and in dividing non-neuronal cells. Double immunostaining experiments showed that TRPC3-positive cells also express TRPC6. Collectively, this report highlights a specific TRPC expression pattern in the immature cortical wall. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. S. Boisseau and C. Kunert-Keil have contributed equally to this work.     

Canonical transient receptor potential TRPC7 can function as both a receptor- and store-operated channel in HEK-293 cells

Previous studies on the activation mechanism of canonical transient receptor potential (TRPC) channels have often produced conflicting conclusions. All seven have been shown to be activated by phospholipase C (PLC)-coupled receptors, but TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC7 have also been proposed to function as store-operated channels.¹ In the case of TRPC3, the expression environment and the expression level appear to determine the mode of regulation. Evidence of a close structural relative of TRPC3, TRPC7, has been presented that this channel is activated by receptor activation or by store depletion. On the basis of previous findings for TRPC3, we reasoned that subtle differences in structure or expression conditions might account for the apparent distinct gating mechanisms of TRPC7. To reexamine the mode of activation of TRPC7, we stably and transiently transfected human embryonic kidney (HEK)-293 cells with cDNA encoding for human TRPC7. We examined the ability of a PLC-activating agonist and an intracellular Ca²⁺ store-depleting agent to activate these channels. Our findings demonstrate that when transiently expressed in HEK-293 cells, TRPC7 forms channels that are activated by PLC-stimulating agonists, but not by Ca²⁺ store depletion. However, when stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca²⁺ store depletion or PLC activation. To our knowledge, this is the first demonstration of a channel protein that can be activated by both receptor- and store-operated modes in the same cell. In addition, the results reconcile the apparently conflicting findings of other laboratories regarding TRPC7 regulation. calcium signaling; nonselective cation channels     

A TRPC1/TRPC3-mediated increase in store-operated calcium entry is required for differentiation of H19-7 hippocampal neuronal cells

Store-operated calcium entry (SOCE) and TRPC protein expression were investigated in the rat-derived hippocampal H19-7 cell line. Thapsigargin-stimulated Ba2+ entry and the expression of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 mRNA and protein were observed in proliferating H19-7 cells. When cells were placed under differentiating conditions, a change in TRPC homolog expression profile occurred. The expression of TRPC1 and TRPC3 mRNA and protein dramatically increased, while the expression of TRPC4 and TRPC7 mRNA and protein dramatically decreased; in parallel a 3.4-fold increase in the level of thapsigargin-stimulated Ba2+ entry was observed and found to be inhibited by 2-aminoethoxydiphenylborane. The selective suppression of TRPC protein levels by small interfering RNA (siRNA) approaches indicated that TRPC1 and TRPC3 are involved in mediating SOCE in proliferating H19-7 cells. Although TRPC4 and TRPC7 are expressed at much higher levels than TRPC1 and TRPC3 in proliferating cells, they do not appear to mediate SOCE. The co-expression of siRNA specific for TRPC1 and TRPC3 in proliferating cells inhibited approximately the same amount of SOCE as observed with expression of either siRNA alone, suggesting that TRPC1 and TRPC3 work in tandem to mediate SOCE. Under differentiating conditions, co-expression of siRNA for TRPC1 and TRPC3 blocked the normal 3.4-fold increase in SOCE and in turn blocked the differentiation of H19-7 cells. This study suggests that placing H19-7 cells under differentiating conditions significantly alters TRPC gene expression and increases the level of SOCE and that this increase in SOCE is necessary for cell differentiation.     

Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.     

Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal transduction.     

Expression level of the canonical transient receptor potential 3 (TRPC3) channel determines its mechanism of activation

Studies on the mechanism of activation of canonical transient receptor potential (TRPC) channels have often yielded conflicting results. In the current study, we have investigated the influence of expression level on the mode of regulation of TRPC3 channels. At relatively low levels of expression in DT40 chicken B-lymphocytes, TRPC3 was activated by the depletion of Ca2+ stores. Expression was increased by either transfecting with a 10-fold greater concentration of plasmid or transfecting with TRPC3 under control of a more efficient avian beta-actin promoter. At higher levels of expression, TRPC3 was no longer store-operated but could be activated through receptor-coupled phospholipase C. Under these expression conditions, TRPC3 was efficiently activated in DT40 cells lacking inositol 1,4,5-trisphosphate receptors. The Ca2+ store-operated channels formed upon expression of TRPC3 at limited levels were blocked by gadolinium; the receptor-activated channels formed upon expression of higher levels of TRPC3 were insensitive to gadolinium. These findings indicate that a single ion channel protein can form or contribute to the formation of channels regulated in two very distinct ways, i.e. either by phospholipase C-derived messengers or Ca2+ store-depletion. The mechanism of regulation of the channels depends on their level of expression.     

Activation of the cytosolic calcium-independent phospholipase A2 β isoform contributes to TRPC6 externalization via release of arachidonic acid

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A₂ (PLA₂), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA₂ in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA₂ enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA₂ involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA₂α, cPLA₂γ, iPLA₂β, or iPLA₂γ. Downregulation of the β isoform of iPLA₂ blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.     

Regulation of the cellular localization and function of human transient receptor potential channel 1 by other members of the TRPC family

Members of the Canonical Transient Receptor Potential (TRPC) family of ionic channels are able to form homo- and heterotetrameric channels. Depending on the study, TRPC1 has been detected on both the surface and inside the cell, probably in the endoplasmic reticulum (ER). Likewise, TRPC1 has been described both as a store-operated channel and as one unable to function when forming a homotetramer. It is possible that the apparent differences in the expression and function of TRPC1 are due to its association with other proteins, possibly from the same TRPC family. In the present study we used confocal microscopy and a fluorescently tagged TRPC1 to examine the localization of this protein when co-expressed with other members of the TRPC family. Whole-cell and single channel electrophysiological recordings were conducted to study the function of TRPC1 expressed alone or co-expressed with other members of the TRPC family. A FRET-based calcium sensor fused to TRPC1 was used to assess the functionality of the intracellular TRPC1. Our results showed that TRPC4 and TRPC5 were able to increase the amount of membrane-expressed TRPC1 as evaluated by confocal microscopy and patch clamp recordings. The FRET-based calcium sensor fused to TRPC1 strongly suggests that this protein forms ER-expressed functional homotetrameric channels activated by agonists coupled to the IP(3) cascade. These results indicate that TRPC1 is a multifunctional protein able to form intracellular calcium release channels when expressed alone, and plasma membrane channels when co-expressed with TRPC4 or TRPC5, but not TRPC3 or TRPC6. Both (ER and plasma membrane) forms of the channel are activated upon addition of agonists coupled to the IP(3) cascade.     

Selective association of TRPC channel subunits in rat brain synaptosomes

TRPC genes encode a ubiquitous family of ion channel proteins responsible for Ca(2+) influx following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. These channels may be localized to large multimeric signaling complexes via association with PDZ-containing scaffolding proteins. Based on sequence homology, the TRPC channel family can be divided into two major subgroups: TRPC1, -C4, and -C5 and TRPC3, -C6, and -C7. Although TRPC channels are thought to be tetramers, the actual subunit composition remains unknown. To determine subunit arrangement, individual TRPC channel pairs were heterologously expressed in Sf9 insect cells and immunoprecipitated using affinity-purified rabbit polyclonal antibodies specific for each channel subtype. Reciprocal co-immunoprecipitations showed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate but that cross-association between the two major subgroups does not occur. Additionally, the interaction between each TRPC channel and the PDZ-containing protein, INAD (protein responsible for the inactivation-no-after-potential Drosophila mutant), was examined. TRPC1, -C4, and -C5 co-immunoprecipitated with INAD, whereas TRPC3, -C6, and -C7 did not. To define channel subunit interactions in vivo, immunoprecipitations were performed from isolated rat brain synaptosomal preparations. The results revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur. These results demonstrate that TRPC channels are present in nerve terminals and provide the first direct evidence for selective assembly of channel subunits in vivo.     

Interaction between TRPC channel subunits in endothelial cells

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.     

Interaction Between TRPC Channel Subunits in Endothelial Cells

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.     

TRPC3 ion channel subunit immunolocalization in the cochlea

Canonical transient receptor potential (TRPC) subunits assemble as tetramers to form ion channels with high calcium (Ca²⁺) permeability. Here, we investigated the possibility that TRPC3 ion channels are broadly expressed in the adult guinea pig and mouse cochleae. Using immunofluorescence, pronounced labeling occurred in the spiral ganglion (SG) neurons, inner hair cells (IHC), outer hair cells (OHC) and epithelial cells lining scala media. TRPC3 expression was homogeneous in the SG throughout the cochlea. In contrast, there was marked spatial variation in the immunolabeling in the cochlear hair cells with respect to location. This likely relates to the tonotopy of these cells. TRPC3 immunolabeling was more pronounced in the IHC than OHC. Both basal region IHC and OHC had higher TRPC3 expression levels than the corresponding cells from the apical region of the cochlea. These data suggest that TRPC3 ion channels contribute to Ca²⁺ homeostasis associated with the hair cells, with higher ion fluxes in more basal regions of the cochlea, and may also be a significant pathway for Ca²⁺ entry associated with auditory neurotransmission via the SG neurons. TRPC3 expression was also identified within the spiral limbus region, inner and outer sulcus, but without evidence for spatial variation in expression level. Expression in these gap junction-coupled epithelial cells lining scala media is indicative of a contribution of TRPC3 channels to cochlear electrochemical homeostasis.