Capacitative Calcium Entry Channels Research Articles

Effect of spatial heterogeneity and colocalization of eNOS and capacitative calcium entry channels on shear stress-induced NO production by endothelial cells: A modeling approach.

Colocalization of endothelial nitric oxide synthase (eNOS) and capacitative Ca2+ entry (CCE) channels in microdomains such as cavaeolae in endothelial cells (ECs) has been shown to significantly affect intracellular Ca2+ dynamics and NO production, but the effect has not been well quantified. We developed a two-dimensional continuum model of an EC integrating shear stress-mediated ATP production, intracellular Ca2+ mobilization, and eNOS activation to investigate the effects of spatial colocalization of plasma membrane eNOS and CCE channels on Ca2+ dynamics and NO production in response to flow-induced shear stress. Our model examines the hypothesis that subcellular colocalization of cellular components can be critical for optimal coupling of NO production to blood flow. Our simulations predict that heterogeneity of CCE can result in formation of microdomains with significantly higher Ca2+ compared to the average cytosolic Ca2+. Ca2+ buffers with lower or no mobility further enhanced Ca2+ gradients relative to mobile buffers. Colocalization of eNOS to CCE channels significantly increased NO production. Our results provide quantitative understanding for the role of spatial heterogeneity and the compartmentalization of signals in regulation of shear stress-induced NO production.

Thymol-evoked Ca2+ Mobilization and Ion Currents in Pituitary GH3 Cells

In this study, an attempt was made to elucidate the effects of thymol, a monocyclic phenolic compound, on Ca2+ mobilization and ion currents in pituitary GH3 cells with the aid of fura-2 fluorimetry and the whole-cell voltage-clamp technique. Thymol increased intracellular Ca2+ concentrations ([Ca2+]i) in GH3 cells loaded with Ca2+-sensitive dye fura-2. Removing extracellular Ca2+ reduced the thymol-induced [Ca2+]i rise. In Ca2+ -free solution, thymol-evoked [Ca2+]i rise was unchanged by depleting the Ca2+ store with thapsigargin (1 microM), while the thapsigargin-induced [Ca2+]i rise was reduced by pretreatment with thymol. These results imply that the Ca2+ stores depleted by thymol comprise thapsigargin-sensitive and thapsigargin-insensitive pools. In addition, after depletion of the internal Ca2+ store with 100 microM thymol in Ca2+ -free solution, a subsequent application of Ca2+ greatly induced a [Ca2+]i increase. The results indicate that, similar to thapsigargin, 100 microM thymol may activate the capacitative calcium entry (CCE) channel. However, thymol (100 microM) had a slight depressant action in L-type calcium current (I(CaL)). The stimulatory actions of thymol on Ca2+ signaling may partly be responsible for the underlying cellular mechanisms through which it affects neuroendocrine functions.

Transport-dependent calcium signaling in spatially segregated cellular caveolar domains

We developed a two-dimensional model of transport-dependent intracellular calcium signaling in endothelial cells (ECs). Our purpose was to evaluate the effects of spatial colocalization of endothelial nitric oxide synthase (eNOS) and capacitative calcium entry (CCE) channels in caveolae on eNOS activation in response to ATP. Caveolae are specialized microdomains of the plasma membrane that contain a variety of signaling molecules to optimize their interactions and regulate their activity. In ECs, these molecules include CCE channels and eNOS. To achieve a quantitative understanding of the mechanisms of microdomain calcium signaling and the preferential sensitivity of eNOS to calcium entering the cell through CCE channels, we constructed a mathematical model incorporating the cell morphology and cellular physiological processes. The model predicts that the spatial segregation of calcium channels in ECs can create transport-dependent sharp gradients in calcium concentration within the cell. The calcium concentration gradient is affected by channel density and cell geometry. This transport-dependent calcium signaling specificity effect is enhanced in ECs by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how Ca2+, despite its many potential actions, can mediate selective activation of signaling pathways. We show that diffusion-limited calcium transport allows functional compartmentalization of signaling pathways based on the spatial arrangements of Ca2+ sources and targets.

Calcium Inhibition and Calcium Potentiation of Orai1, Orai2, and Orai3 Calcium Release-activated Calcium Channels

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca(2+) sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca(2+) for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I(crac)), but Orai3 failed to produce any detectable Ca(2+)-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca(2+) and Ba(2+) or Mg(2+) solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na(+) currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na(+) currents in HEK293 cells, were all inhibited by extracellular Ca(2+) with a half-maximal concentration of approximately 20 mum. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca(2+), indicating similar affinities for Ca(2+) within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca(2+) depotentiation.

The Role of Canonical Transient Receptor Potential 7 in B-cell Receptor-activated Channels

Phospholipase C signaling stimulates Ca2+ entry across the plasma membrane through multiple mechanisms. Ca2+ store depletion stimulates store-operated Ca2+-selective channels, or alternatively, other phospholipase C-dependent events activate Ca2+-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca2+ entry. No difference in the store-operated current I(crac) was detected between TRPC7-/- and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTPgammaS, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7-/- cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels.

Capacitative calcium entry

A long-standing mystery in the cell biology of calcium channel regulation is the nature of the signal linking intracellular calcium stores to plasma membrane capacitative calcium entry channels. An RNAi-based screen of selected Drosophila genes has revealed that a calcium-binding protein, stromal interaction molecule (STIM), plays an essential role in the activation of these channels and may be the long sought sensor of calcium store content.

Phospholipase Cγ1 Is Required for Activation of Store-Operated Channels in Human Keratinocytes

Store-operated calcium entry depicts the movement of extracellular Ca2+ into cells through plasma membrane Ca2+ channels activated by depletion of intracellular Ca2+ stores. The members of the canonical subfamily of transient receptor potential channels (TRPC) have been implicated as the molecular bases for store-operated channels (SOC). Here we investigate the role of phospholipase C (PLC) in regulation of native SOC and the expression of endogenous TRPC in human epidermal keratinocytes. Calcium entry in response to store depletion with thapsigargin was reversibly blocked by 2-aminoethoxydiphenyl borane, an effective SOC inhibitor, and suppressed by the diacylglycerol analoge, 1-oleoyl-2-acetyl-sn-glycerol. Inhibition of PLC with U73122 or transfection of a PLCgamma1 antisense cDNA construct completely blocked SOC activity, indicating a requirement for PLC, especially PLCgamma1, in the activation of SOC. RT-PCR and immunoblotting analyses showed that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 are expressed in keratinocytes. Knockdown of the level of endogenous TRPC1 or TRPC4 inhibited store-operated calcium entry, indicating they are part of the native SOC. Co-immunoprecipitation studies demonstrated that TRPC1, but not TRPC4, interacts with PLCgamma1 and the inositol 1,4,5-trisphosphate receptor (IP3R). The association of TRPC1 with PLCgamma1 and IP3R decreased in keratinocytes with higher intracellular Ca2+, coinciding with a downregulation in SOC activity. Our results indicate that the activation of SOC in keratinocytes depends, at least partly, on the interaction of TRPC with PLCgamma1 and IP3R.

Protein biomarkers in the plasma of workers occupationally exposed to polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) form a chemical family containing several hundred compounds, including benzo[a]pyrene and pyrene. They are usually produced by the incomplete burning of coal, oil, gas, garbage, or other organic substances like tobacco or charbroiled meat. Exposure to PAH causes tumors, primarily in the lung, the bladder, and the skin. To investigate the differentially expressed proteins resultant from PAH exposure, the protein expression in human plasma was analyzed using two-dimensional electrophoresis (2-DE). The plasma exposed to PAH was obtained from 48 waste gas pollution measurers working at an automobile emission inspection center. The 1-hydroxypryene (1-OHP) level, which is the urinary PAH metabolite used for evaluation of PAH exposure, was 0.28 micromol/mol creatinine in PAH exposure groups, and 0.078 micromol/mol creatinine in unexposed groups (control, n = 33). A protein upregulated by PAH (putative capacitative calcium entry channel) and five overexpressed proteins (two fibrinogen gamma-A chain precursors, a hemopexin precursor, an albumin precursor, and T-cell receptor beta chain C region) were identified with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and confirmed with tandem MS (MS/MS) and Western blotting. The putative capacitative calcium entry channel was partially validated with a laboratory made antibody of a representative peptide fragment in PAH-exposed human plasma samples.

Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder.

We have evaluated the presence of capacitative Ca(2+) entry (CCE) in guinea pig gallbladder smooth muscle (GBSM), including a possible relation with activation of L-type Ca(2+) channels. Changes in cytosolic Ca(2+) concentration induced by Ca(2+) entry were assessed by digital microfluorometry in isolated, fura 2-loaded GBSM cells. Application of thapsigargin, a specific inhibitor of the Ca(2+) store pump, induced a transient Ca(2+) release followed by sustained entry of extracellular Ca(2+). Depletion of the stores with thapsigargin, cyclopiazonic acid, ryanodine and caffeine, high levels of the Ca(2+)-mobilizing hormone cholecystokinin octapeptide, or simple removal of external Ca(2+) resulted in a sustained increase in Ca(2+) entry on subsequent reapplication of Ca(2+). This entry was attenuated by 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockade, pinacidil, and Gd(3+). Accumulation of the voltage-sensitive dye 3,3'-dipentylcarbocyanine and direct intracellular recordings showed that depletion of the stores is sufficient for depolarization of the plasma membrane. Contractility studies in intact gallbladder muscle strips showed that CCE induced contractions. The CCE-evoked contraction was sensitive to 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockers, and Gd(3+). We conclude that, in GBSM, release of Ca(2+) from internal stores activates a CCE pathway and depolarizes plasma membrane, allowing coactivation of voltage-operated L-type Ca(2+) channels. This process may play a role in excitation-contraction coupling in GBSM.

Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle.

Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca2+ entry triggered by depletion of Ca2+ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca2+ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca2+ signaling in distal PASMCs, we measured [Ca2+]i by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca2+-free media containing cyclopiazonic acid (10 microM) and nifedipine (5 microM) to deplete ER Ca2+ stores and block voltage-dependent Ca2+ channels, restoration of extracellular Ca2+ (2.5 mM) caused marked increases in [Ca2+]i whereas MnCl2 (200 microM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl3, and NiCl2, blocked CCE at concentrations that did not alter Ca2+ responses to 60 mM KCl (IC50 6.3, 40.4, and 191 microM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, -4, and -6, but not TRPC2, -3, -5, or -7. Our results suggest that CCE through TRPC-encoded Ca2+ channels could contribute to Ca2+ signaling in myocytes from distal intrapulmonary arteries.

Capacitative calcium entry in the nervous system

Capacitative calcium entry is a process whereby the depletion of Ca 2+ from intracellular stores (likely endoplasmic or sarcoplasmic reticulum) activates plasma membrane Ca 2+ channels. Current research has focused on identification of capacitative calcium entry channels and the mechanism by which Ca 2+ store depletion activates the channels. Leading candidates for the channels are members of the transient receptor potential (TRP) superfamily, although no single gene or gene product has been definitively proven to mediate capacitative calcium entry. The mechanism for activation of the channels is not known; proposals fall into two general categories, either a diffusible signal released from the Ca 2+ stores when their Ca 2+ levels become depleted, or a more direct protein–protein interaction between constituents of the endoplasmic reticulum and the plasma membrane channels. Capacitative calcium entry is a major mechanism for regulated Ca 2+ influx in non-excitable cells, but recent research has indicated that this pathway plays an important role in the function of neuronal cells, and may be important in a number of neuropathological conditions. This review will summarize some of these more recent findings regarding the role of capacitative calcium entry in normal and pathological processes in the nervous system.

Enhanced capacitative calcium entry and TRPC channel gene expression in human LES smooth muscle.

Transient receptor potential channel (TRPC) genes encode Ca(2+)-permeable channels mediating capacitative Ca(2+) entry (CCE), which maintains intracellular Ca(2+) stores. We compared TRPC gene expression and CCE in human esophageal body (EB) and lower esophageal sphincter (LES), because these smooth muscles have distinct contractile functions that are likely associated with different Ca(2+) regulatory mechanisms. Circular layer smooth muscle cells were grown in primary culture. Transcriptional expression of TRPC genes was compared by semiquantitative RT-PCR. CCE was measured by fura 2 Ca(2+) fluorescence after blockade of sarcoplasmic reticulum Ca(2+)-ATPase with thapsigargin. mRNA for TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 was identified in EB and LES. TRPC3 and TRPC4 were more abundant in LES than EB. Basal concentration of free intracellular Ca(2+) ([Ca(2+)](i)) was similar in cells from LES (138 +/- 8 nmol/l) and EB (110 +/- 6 nmol/l) and increased with ACh (10 micromol/l; 650 +/- 28 and 590 +/- 21 nmol/l, respectively). With zero Ca(2+) in bath, thapsigargin (2 micromol/l) increased [Ca(2+)](i) more in LES (550 +/- 22 nmol/l) than EB (250 +/- 15 nmol/l, P < 0.001). Subsequent external application of 1 mmol/l Ca(2+) increased [Ca(2+)](i) more in LES (585 +/- 35 nmol/l) than EB (295 +/- 21 nmol/l, P < 0.001), indicating enhanced CCE in LES. This demonstrates CCE and TRPC transcriptional expression in human esophageal smooth muscle. In LES cells, enhanced CCE and expression of TRPC3 and TRPC4 may contribute to the physiological characteristics that distinguish LES from EB.

Cloning and Functional Expression of a Novel Splice Variant of Rat TRPC4.

Transient receptor potential protein 4 (TRPC4) has been identified as a candidate for the capacitative calcium entry (CCE) channels, but its functional role is still controversial. Using a RT-PCR technique, a novel isoform of TRPC4, designated rTRPC4gamma, was isolated. It was nearly identical to full-length rTRPC4 (rTRPC4alpha), except that it lacked 53 nucleotides that correspond to the predicted linker between the second and third transmembrane domain of rTRPC4alpha, and its mRNA was expressed in brain and heart. This splice variant encoded a potential protein of 400 residues that consists of an amino-terminal cytoplasmic domain and 2 transmembrane domains by a frameshift mutation. When rTRPC4gamma cDNA was transiently transfected to HEK-293 cells, thapsigargin (TG)-induced Ca2+ entry was suppressed significantly. By contrast, expression of rTRPC4 a did not affect TG-induced Ca2+ entry. To investigate the subcellular localization, plasmids were constructed with green fluorescence protein (GFP) as an amino-terminal fusion to rTRPC4 variants. GFP-rTRPC4gamma fusion protein, unlike GFP-rTRPC4alpha, was localized to the cytoplasm as well as plasma membrane. These results suggest that rTRPC4gamma may play a modulatory role in CCE channel activity in the brain and heart.

Mutual Antagonism of Calcium Entry by Capacitative and Arachidonic Acid-mediated Calcium Entry Pathways

In nonexcitable cells, the predominant mechanism for regulated entry of Ca(2+) is capacitative calcium entry, whereby depletion of intracellular Ca(2+) stores signals the activation of plasma membrane calcium channels. A number of other regulated Ca(2+) entry pathways occur in specific cell types, however, and it is not know to what degree the different pathways interact when present in the same cell. In this study, we have examined the interaction between capacitative calcium entry and arachidonic acid-activated calcium entry, which co-exist in HEK293 cells. These two pathways exhibit mutual antagonism. That is, capacitative calcium entry is potently inhibited by arachidonic acid, and arachidonic acid-activated entry is inhibited by the pre-activation of capacitative calcium entry with thapsigargin. In the latter case, the inhibition does not seem to result from a direct action of thapsigargin, inhibition of endoplasmic reticulum Ca(2+) pumps, depletion of Ca(2+) stores, or entry of Ca(2+) through capacitative calcium entry channels. Rather, it seems that a discrete step in the pathway signaling capacitative calcium entry interacts with and inhibits the arachidonic acid pathway. The findings reveal a novel process of mutual antagonism between two distinct calcium entry pathways. This mutual antagonism may provide an important protective mechanism for the cell, guarding against toxic Ca(2+) overload.

Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3

Mammalian homologues of the Drosophila transient receptor potential (TRP) protein have been proposed to function as ion channels, and in some cases as store-operated or capacitative calcium entry channels. However, for each of the mammalian TRP proteins, different laboratories have reported distinct modes of cellular regulation. In the present study we describe the cloning and functional expression of the human form of TRP4 (hTRP4), and compare its activity with another well studied protein, hTRP3. When hTRP4 was transiently expressed in human embryonic kidney (HEK)-293 cells, basal bivalent cation permeability (barium) was increased. Whole-cell patch-clamp studies of hTRP4 expressed in Chinese hamster ovary cells revealed a constitutively active non-selective cation current which probably underlies the increased bivalent cation entry. Barium entry into hTRP4-transfected HEK-293 cells was not further increased by phospholipase C (PLC)-linked receptor activation, by intracellular calcium store depletion with thapsigargin, or by a synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In contrast, transient expression of hTRP3 resulted in a bivalent cation influx that was markedly increased by PLC-linked receptor activation and by OAG, but not by thapsigargin. Despite the apparent differences in regulation of these two putative channel proteins, green fluorescent protein fusions of both molecules localized similarly to the plasma-membrane, notably in discrete punctate regions suggestive of specialized signalling complexes. Our findings indicate that while both hTRP4 and hTRP3 can apparently function as cation channels, their putative roles as components of capacitative calcium entry channels are not readily demonstrable by examining their behaviour when exogenously expressed in cells.

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.

Intimate Plasma Membrane–ER Interactions Underlie Capacitative Calcium Entry: “Kissin' Cousins”

Intimate Plasma Membrane–ER Interactions Underlie Capacitative Calcium Entry: “Kissin' Cousins”

Capacitative calcium entry channels.

In the phospholipase C signaling system, Ca(2+) is mobilized from intracellular stores by an action of inositol 1,4,5-trisphosphate. The depletion of intracellular calcium stores activates a calcium entry mechanism at the plasma membrane called capacitative calcium entry. The signal for activating the entry is unknown but likely involves either the generation or release, or both, from the endoplasmic reticulum of some diffusible signal. Recent research has focused on mammalian homologues of the Drosophila TRP protein as potential candidates for capacitative calcium entry channels. This review summarizes current knowledge about the nature of capacitative calcium entry signals, as well as the potential role of mammalian TRP proteins as capacitative calcium entry channel molecules.

Capacitative calcium entry channels

Capacitative calcium entry channels

A novel capacitative calcium entry channel expressed in excitable cells.

In addition to voltage-gated calcium influx, capacitative calcium entry (CCE) represents a major pathway for calcium entry into the cell. Here we report the structure, expression and functional properties of a novel CCE channel, TRP5. This channel is a member of a new subfamily of mammalian homologues of the Drosophila transient receptor potential (TRP) protein, now comprising TRP5 (also CCE2) and the structurally related CCE1 (also TRP4). Like TRP4, TRP5 forms ion channels mainly permeable for Ca2+ which are not active under resting conditions but can be activated by manoeuvres known to deplete intracellular calcium stores. Accordingly, dialysis of TRP5-expressing cells with inositol-(1,4,5)-trisphosphate evokes inward rectifying currents which reversed polarity at potentials more positive than +30 mV. Ca2+ store depletion with thapsigargin induced TRP5-mediated calcium entry dependent on the concentration of extracellular calcium, as seen by dual wavelength fura-2 fluorescence ratio measurements. TRP5 transcripts are expressed almost exclusively in brain, where they are present in mitral cells of the olfactory bulb, in lateral cerebellar nuclei and, together with TRP4 transcripts, in CA1 pyramidal neurons of the hippocampus, indicating the presence of CCE channels in excitable cells and their participation in neuronal calcium homeostasis.