A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2+ channel and modulates its function.

In skeletal muscle, voltage-dependent potentiation of L-type Ca(2+) channel (Ca(V)1.1) activity requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A kinase-anchoring protein (AKAP15). However, the mechanism by which AKAP15 targets PKA to L-type Ca(2+) channels has not been elucidated. Here we report that AKAP15 directly interacts with the C-terminal domain of the alpha(1) subunit of Ca(V)1.1 via a leucine zipper (LZ) motif. Disruption of the LZ interaction effectively inhibits voltage-dependent potentiation of L-type Ca(2+) channels in skeletal muscle cells. Our results reveal a novel mechanism whereby anchoring of PKA to Ca(2+) channels via LZ interactions ensures rapid and efficient phosphorylation of Ca(2+) channels in response to local signals such as cAMP and depolarization.     

Voltage- and calcium-dependent inactivation in high voltage-gated Ca(2+) channels

Calcium influx into cardiac myocytes via voltage-gated Ca channels is a key step in initiating the contractile response. During prolonged depolarizations, toxic Ca(2+) overload is prevented by channel inactivation occurring through two different processes identified by their primary trigger: voltage or intracellular Ca(2+). In physiological situations, cardiac L-type (Ca(V)1.2) Ca(2+) channels inactivate primarily via Ca(2+)-dependent inactivation (CDI), while neuronal P/Q (Ca(V)2.1) Ca(2+) channels use preferentially voltage-dependent inactivation (VDI). In certain situations however, these two types of channels have been shown to be able to inactivate by both processes. From a structural view point, the rearrangement occurring during CDI and VDI is not precisely known, but functional studies have underlined the role played by at least 2 channel sequences: a C-terminal binding site for the Ca(2+) sensor calmodulin, essential for CDI, and the loop connecting domains I and II, essential for VDI. The conserved regulation of VDI and CDI by the auxiliary channel beta subunit strongly suggests that these two mechanisms may use a set of common protein-protein interactions that are influenced by the auxiliary subunit. We will review our current knowledge of these interactions. New data are presented on L-P/Q (Ca(V)1.2/Ca(V)2.1) channel chimera that confirm the role of the I-II loop in VDI and CDI, and reveal some of the essential steps in Ca(2+) channel inactivation.     

Competitive modulation of Ca2+ release-activated Ca2+ channel gating by STIM1 and 2-aminoethyldiphenyl borate

Activation of Ca(2+) release-activated Ca(2+) channels by depletion of intracellular Ca(2+) stores involves physical interactions between the endoplasmic reticulum Ca(2+) sensor, STIM1, and the channels composed of Orai subunits. Recent studies indicate that the Orai3 subtype, in addition to being store-operated, is also activated in a store-independent manner by 2-aminoethyldiphenyl borate (2-APB), a small molecule with complex pharmacology. However, it is unknown whether the store-dependent and -independent activation modes of Orai3 channels operate independently or whether there is cross-talk between these activation states. Here we report that in addition to causing direct activation, 2-APB also regulates store-operated gating of Orai3 channels, causing potentiation at low doses and inhibition at high doses. Inhibition of store-operated gating by 2-APB was accompanied by the suppression of several modes of Orai3 channel regulation that depend on STIM1, suggesting that high doses of 2-APB interrupt STIM1-Orai3 coupling. Conversely, STIM1-bound Orai3 (and Orai1) channels resisted direct gating by high doses of 2-APB. The rate of direct 2-APB activation of Orai3 channels increased linearly with the degree of STIM1-Orai3 uncoupling, suggesting that 2-APB has to first disengage STIM1 before it can directly gate Orai3 channels. Collectively, our results indicate that the store-dependent and -independent modes of Ca(2+) release-activated Ca(2+) channel activation are mutually exclusive: channels bound to STIM1 resist 2-APB gating, whereas 2-APB antagonizes STIM1 gating.     

Intracellular Ca2+ regulates the sensitivity of cyclic nucleotide-gated channels in olfactory receptor neurons.

In olfactory receptor neurons, odorants stimulate production of cAMP, which directly activates cyclic nucleotide-gated (CNG) channels. Olfactory adaptation is thought to result from a rise in intracellular Ca2+. To determine whether inhibition of CNG channels plays a role in adaptation, we have investigated the action of Ca2+ on these channels in inside-out "macro" patches from the dendrite and cilia of catfish olfactory neurons. Internal Ca2+, with a K1/2 of 3 microM, profoundly inhibits CNG channels by shifting the dose-response relationship to higher cAMP levels without altering the maximal response. The inhibition does not appear to result from a direct interaction of Ca2+ with the CNG channel. Thus, the inhibition washes out after excision of the inside-out patch, and Ca2+ does not inhibit the cloned catfish CNG channel expressed in Xenopus oocytes. Hence we propose that a regulatory Ca(2+)-binding protein, distinct from the CNG channel, controls the gain of signal transduction and contributes to olfactory adaptation by decreasing the sensitivity of the CNG channel to cAMP.     

Reconstituted slow muscarinic inhibition of neuronal (Ca(v)1.2c) L-type Ca2+ channels

Ca(2+) influx through L-type channels is critical for numerous physiological functions. Relatively little is known about modulation of neuronal L-type Ca(2+) channels. We studied modulation of neuronal Ca(V)1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. Galphaq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca(V)1.2c, whereas Galphai/o-coupled M2 and M4 receptors were ineffective. Channel inhibition through M1 receptors was studied in detail and was found to be kinetically slow, voltage-independent, and pertussis toxin-insensitive. Slow inhibition of Ca(V)1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against Galphaq/11. In contrast, inhibition was not reduced by coexpressing betaARK1ct or Galphat. These results indicate that slow inhibition required signaling by Galphaq/11, but not Gbetagamma, subunits. Slow inhibition did not require Ca(2+) transients or Ca(2+) influx through Ca(V)1.2c channels. Additionally, slow inhibition was insensitive to pharmacological inhibitors of phospholipases, protein kinases, and protein phosphatases. Intracellular BAPTA prevented slow inhibition via a mechanism other than Ca(2+) chelation. The cardiac splice-variant of Ca(V)1.2 (Ca(V)1.2a) and a splice-variant of the neuronal/neuroendocrine Ca(V)1.3 channel also appeared to undergo slow muscarinic inhibition. Thus, slow muscarinic inhibition may be a general characteristic of L-type channels having widespread physiological significance.     

Ca2+ dependence of the Ca2+-selective TRPV6 channel

Microfluorimetry and patch-clamp experiments were performed on TRPV6-expressing HEK cells to determine whether this Ca(2+)-sensing Ca(2+) channel is constitutively active. Intact cells loaded with fura-2 had an elevated intracellular free Ca(2+) concentration ([Ca(2+)](i)), which decreased to the same level such as in non-transfected cells if external Ca(2+) was chelated by EGTA. Whole cell recordings from non-transfected HEK cells and cells expressing human TRPV6 revealed the presence of a basal inward current in both types of cells when the internal solution contained 0.1 mm EGTA and 100 nm [Ca(2+)](i) or if the cytosolic Ca(2+) buffering remained undisturbed in perforated patch-clamp experiments. If recombinantly expressed TRPV6 forms open channels, one would expect Ca(2+)-induced current inhibition, because TRPV6 is negatively regulated by internal Ca(2+). However, dialyzing solutions with high [Ca(2+)] such as 1 microm into TRPV6-expressing cells did not block the basal inward current, which was not different from the recordings from non-transfected cells. In contrast, dialyzing 0.5 mm EGTA into TRPV6-expressing cells readily activated Ca(2+) inward currents, which were undetectable in non-transfected cells. Interestingly, monovalent cations permeated the TRPV6 channels under conditions where no Ca(2+) permeation was detectable, indicating that divalent cations block TRPV6 channels from the extracellular side. Like human TRPV6, the truncated human TRPV6(Delta695-725), which lacks the C-terminal domain required for Ca(2+)-calmodulin binding, does not form constitutive active channels, whereas the human TRPV6(D542A), carrying a point mutation in the presumed pore region, does not function as a channel. In summary, no constitutive open TRPV6 channels were detected in patch-clamp experiments from transfected HEK cells. However, channel activity is highly regulated by intracellular and extracellular divalent cations.     

Mapping the interacting domains of STIM1 and Orai1 in Ca2+ release-activated Ca2+ channel activation

STIM1 and Orai1 are essential components of Ca(2+) release-activated Ca(2+) channels (CRACs). After endoplasmic reticulum Ca(2+) store depletion, STIM1 in the endoplasmic reticulum aggregates and migrates toward the cell periphery to co-localize with Orai1 on the opposing plasma membrane. Little is known about the roles of different domains of STIM1 and Orai1 in protein clustering, migration, interaction, and, ultimately, opening CRAC channels. Here we demonstrate that the coiled-coil domain in the C terminus of STIM1 is crucial for its aggregation. Amino acids 425-671 of STIM1, which contain a serine-proline-rich region, are important for the correct targeting of the STIM1 cluster to the cell periphery after calcium store depletion. The polycationic region in the C-terminal tail of STIM1 also helps STIM1 targeting but is not essential for CRAC channel activation. The cytoplasmic C terminus but not the N terminus of Orai1 is required for its interaction with STIM1. We further identify a highly conserved region in the N terminus of Orai1 (amino acids 74-90) that is necessary for CRAC channel opening. Finally, we show that the transmembrane domain of Orai1 participates in Orai1-Orai1 interactions.     

Dose-dependent and isoform-specific modulation of Ca2+ channels by RGK GTPases

Although inhibition of voltage-gated calcium channels by RGK GTPases (RGKs) represents an important mode of regulation to control Ca(2+) influx in excitable cells, their exact mechanism of inhibition remains controversial. This has prevented an understanding of how RGK regulation can be significant in a physiological context. Here we show that RGKs-Gem, Rem, and Rem2-decreased Ca(V)1.2 Ca(2+) current amplitude in a dose-dependent manner. Moreover, Rem2, but not Rem or Gem, produced dose-dependent alterations on gating kinetics, uncovering a new mode by which certain RGKs can precisely modulate Ca(2+) currents and affect Ca(2+) influx during action potentials. To explore how RGKs influence gating kinetics, we separated the roles mediated by the Ca(2+) channel accessory beta subunit's interaction with its high affinity binding site in the pore-forming alpha(1C) subunit (AID) from its other putative contact sites by utilizing an alpha(1C)*beta3 concatemer in which the AID was mutated to prevent beta subunit interaction. This mutant concatemer generated currents with all the hallmarks of beta subunit modulation, demonstrating that AID-beta-independent interactions are sufficient for beta subunit modulation. Using this construct we found that although inhibition of current amplitude was still partially sensitive to RGKs, Rem2 no longer altered gating kinetics, implicating different determinants for this specific mode of Rem2-mediated regulation. Together, these results offer new insights into the molecular mechanism of RGK-mediated Ca(2+) channel current modulation.     

N-type Ca2+ channels as scaffold proteins in the assembly of signaling molecules for GABAB receptor effects

An emerging concept in signal transduction is the organization of neuronal receptors and channels into microdomains in which signaling proteins are brought together to regulate functional responses. With the multiplicity of potential protein-protein interactions arises the need for the regulation and timing of these interactions. We have identified N-type Ca(2+) channel-signaling molecule complexes formed at different times upon activation of gamma-aminobutyric acid, type B, receptors. The first type of interaction involves pre-association of signaling proteins such as Src kinase with the Ca(2+) channel, because it is rapidly activated by the receptors and regulates the magnitude of the inhibition of the Ca(2+) channel. The second type of interaction involves signaling molecules that are recruited to the channel by receptor activation and control the rate of the channel response. Recruitment of members of the Ras pathway has two effects as follows: 1) modulation of the rate of onset of the gamma-aminobutyric acid-mediated inhibition of Ca(2+) current, and 2) activation of MAP kinase. Our results suggest that the Ca(2+) channel alpha(1) subunit functions as a dynamic scaffold allowing assembly of intracellular signaling components that alter channel activity and route signals to the MAP kinase pathway.     

Crystal structure of the CaV2 IQ domain in complex with Ca2+/calmodulin: high-resolution mechanistic implications for channel regulation by Ca2+

Calmodulin (CaM) regulation of Ca(2+) channels is central to Ca(2+) signaling. Ca(V)1 versus Ca(V)2 classes of these channels exhibit divergent forms of regulation, potentially relating to customized CaM/IQ interactions among different channels. Here we report the crystal structures for the Ca(2+)/CaM IQ domains of both Ca(V)2.1 and Ca(V)2.3 channels. These highly similar structures emphasize that major CaM contacts with the IQ domain extend well upstream of traditional consensus residues. Surprisingly, upstream mutations strongly diminished Ca(V)2.1 regulation, whereas downstream perturbations had limited effects. Furthermore, our Ca(V)2 structures closely resemble published Ca(2+)/CaM-Ca(V)1.2 IQ structures, arguing against Ca(V)1/2 regulatory differences based solely on contrasting CaM/IQ conformations. Instead, alanine scanning of the Ca(V)2.1 IQ domain, combined with structure-based molecular simulation of corresponding CaM/IQ binding energy perturbations, suggests that the C lobe of CaM partially dislodges from the IQ element during channel regulation, allowing exposed IQ residues to trigger regulation via isoform-specific interactions with alternative channel regions.     

Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel

Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular "pore gate" has also been proposed. We have used the voltage dependence of block by extracellular Y(3+) as a tool to locate the activation gate of the alpha1G (Ca(V)3.1) T-type calcium channel. Y(3+) block exhibited no clear voltage dependence from -40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y(3+) at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca(2+) was replaced with Ba(2+), the rate of open block by Y(3+) was unaffected, but closed block was threefold faster than in Ca(2+), suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.     

CaV2.2 and CaV2.3 (N- and R-type) Ca2+ channels in depolarization-evoked entry of Ca2+ into mouse sperm

As sperm prepare for fertilization, surface Ca(2+) channels must open to initiate required, Ca(2+)-mediated events. However, the molecular identity and functional properties of sperm Ca(2+) channels remain uncertain. Here, we use rapid local perfusion and single-cell photometry to examine the kinetics of calcium responses of mouse sperm to depolarizing stimuli. The linear rise of intracellular [Ca(2+)] evoked by approximately 10-s applications of an alkaline high [K(+)] medium directly reports activity of voltage-gated Ca(2+) channels. Little response occurs if external Ca(2+) is removed or if external or internal pH is elevated without depolarization. Responses are inhibited 30-40% by 30-100 micrometer Ni(2+) and more completely by 100-300 micrometer Cd(2+). They resist the dihydropyridines nitrendipine and PN200-110, but 1-10 micrometer mibefradil inhibits reversibly. They also resist the venom toxins calciseptine, omega-conotoxin MVIIC, and kurtoxin, but omega-conotoxin GVIA (5 micrometer) inhibits approximately 50%. GVIA also partially blocks transient, low voltage activated Ca(2+) currents of patch-clamped spermatids. Differential sensitivity of sperm responses to Ni(2+) and Cd(2+) and partial blockade by GVIA indicate that depolarization opens at least two types of voltage-gated Ca(2+) channels in epididymal sperm examined prior to capacitation. Involvement of a previously undetected Ca(V)2.2 (N-type) channel, suggested by the action of GVIA, is substantiated by immunodetection of Ca(2+) channel alpha(1B) subunits in sperm and sperm extracts. Resistance to dihydropyridines, calciseptine, MVIIC, and kurtoxin indicates that Ca(V)1, Ca(V)2.1, and Ca(V)3 (L-, P/Q-, and T-type) channels contribute little to this evoked response. Partial sensitivity to 1 micrometer mibefradil and an enhanced sensitivity of the GVIA-resistant component of response to Ni(2+) suggest participation of a Ca(V)2.3 (R-type) channel specified by previously found alpha(1E) subunits. Our examination of depolarization-evoked Ca(2+) entry indicates that mature sperm possess a larger palette of voltage-gated Ca(2+) channels than previously thought. Such diversity may permit specific responses to multiple cues encountered on the path to fertilization.     

Interactions of calmodulin with two peptides derived from the c-terminal cytoplasmic domain of the Ca(v)1.2 Ca2+ channel provide evidence for a molecular switch involved in Ca2+-induced inactivation

When opened by depolarization, L-type calcium channels are rapidly inactivated by the elevation of Ca(2+) concentration on the cytoplasmic side. Recent studies have shown that the interaction of calmodulin with the proximal part of the cytoplasmic C-terminal tail of the channel plays a prominent role in this modulation. Two motifs interacting with calmodulin in a Ca(2+)-dependent manner have been described: the IQ sequence and more recently the neighboring CB sequence. Here, using synthetic peptides and fusion proteins derived from the Ca(v)1.2 channel combined with biochemical techniques, we show that these two peptides are the only motifs of the cytoplasmic tail susceptible to interact with calmodulin. We determined the K(d) of the CB interaction with calmodulin to be 12 nm, i.e. below the K(d) of IQ-calmodulin, thereby precluding a competitive displacement of CB by IQ in the presence of Ca(2+). In place, we demonstrated that a ternary complex is formed at high Ca(2+) concentration, provided that calmodulin and the peptides are initially allowed to interact at a low Ca(2+) concentration. These results provide evidence that CB and IQ motifs interacting together with calmodulin constitute a minimal molecular switch leading to Ca(2+)-induced inactivation. In addition, we suggest that they could also be the tethering site of calmodulin.     

Molecular determinants of Gem protein inhibition of P/Q-type Ca2+ channels

The RGK family of monomeric GTP-binding proteins potently inhibits high voltage-activated Ca(2+) channels. The molecular mechanisms of this inhibition are largely unclear. In Xenopus oocytes, Gem suppresses the activity of P/Q-type Ca(2+) channels on the plasma membrane. This is presumed to occur through direct interactions of one or more Gem inhibitory sites and the pore-forming Ca(v)2.1 subunit in a manner dependent on the Ca(2+) channel subunit β (Ca(v)β). In this study we investigated the molecular determinants in Gem that are critical for this inhibition. Like other RGK proteins, Gem contains a conserved Ras-like core and extended N and C termini. A 12-amino acid fragment in the C terminus was found to be crucial for and sufficient to produce Ca(v)β-dependent inhibition, suggesting that this region forms an inhibitory site. A three-amino acid motif in the core was also found to be critical, possibly forming another inhibitory site. Mutating either site individually did not hamper Gem inhibition, but mutating both sites together completely abolished Gem inhibition without affecting Gem protein expression level or disrupting Gem interaction with Ca(v)2.1 or Ca(v)β. Mutating Gem residues that are crucial for interactions with previously demonstrated RGK modulators such as calmodulin, 14-3-3, and phosphatidylinositol lipids did not significantly affect Gem inhibition. These results suggest that Gem contains two candidate inhibitory sites, each capable of producing full inhibition of P/Q-type Ca(2+) channels.     

Plasma membrane targeting is essential for Rem-mediated Ca2+ channel inhibition

The small GTPase Rem is a potent negative regulator of high voltage-activated Ca(2+) channels and a known interacting partner for Ca(2+) channel accessory beta subunits. The mechanism for Rem-mediated channel inhibition remains controversial, although it has been proposed that Ca(V)beta association is required. Previous work has shown that a C-terminal truncation of Rem (Rem-(1-265)) displays reduced in vivo binding to membrane-localized beta 2a and lacks channel regulatory function. In this paper, we describe a role for the Rem C terminus in plasma membrane localization through association with phosphatidylinositol lipids. Moreover, Rem-(1-265) can associate with beta 2a in vitro and beta 1b in vivo, suggesting that the C terminus does not directly participate in Ca(V)beta association. Despite demonstrated beta 1b binding, Rem-(1-265) was not capable of regulating a Ca(V)1.2-beta 1b channel complex, indicating that beta subunit binding is not sufficient for channel regulation. However, fusion of the CAAX domain from K-Ras4B or H-Ras to the Rem-(1-265) C terminus restored membrane localization and Ca(2+) channel regulation, suggesting that beta binding and membrane localization are independent events required for channel inhibition.     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

High extracellular Ca2+ stimulates Ca2+-activated Cl- currents in frog parathyroid cells through the mediation of arachidonic acid cascade

Elevation of extracellular Ca(2+) concentration induces intracellular Ca(2+) signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca(2+) pathways, but the direct mechanism responsible for the rise of intracellular Ca(2+) concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca(2+) signaling in frog parathyroid cells and show that Ca(2+)-activated Cl(-) channels are activated by intracellular Ca(2+) increase through an inositol 1,4,5-trisphophate (IP(3))-independent pathway. High extracellular Ca(2+) induced an outwardly-rectifying conductance in a dose-dependent manner (EC(50) ～6 mM). The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca(2+)-induced and Ca(2+) dialysis-induced currents reversed at the equilibrium potential of Cl(-) and were inhibited by niflumic acid (a specific blocker of Ca(2+)-activated Cl(-) channel). Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca(2+)-induced current, suggesting the change of intracellular Cl(-) concentration in a few minutes. Extracellular Ca(2+)-induced currents displayed a moderate dependency on guanosine triphosphate (GTP). All blockers for phospholipase C, diacylglycerol (DAG) lipase, monoacylglycerol (MAG) lipase and lipoxygenase inhibited extracellular Ca(2+)-induced current. IP(3) dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG), arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HPETE) dialysis increased the conductance identical to extracellular Ca(2+)-induced conductance. These results indicate that high extracellular Ca(2+) raises intracellular Ca(2+) concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl(-) conductance.     

Expression of polycystin-1 C-terminal fragment enhances the ATP-induced Ca2+ release in human kidney cells

Polycystin-1 (PC1) is a membrane protein expressed in tubular epithelia of developing kidneys and in other ductal structures. Recent studies indicate this protein to be putatively important in regulating intracellular Ca(2+) levels in various cell types, but little evidence exists for kidney epithelial cells. Here we examined the role of the PC1 cytoplasmic tail on the activity of store operated Ca(2+) channels in human kidney epithelial HEK-293 cell line. Cells were transiently transfected with chimeric proteins containing 1-226 or 26-226 aa of the PC1 cytoplasmic tail fused to the transmembrane domain of the human Trk-A receptor: TrkPC1 wild-type and control Trk truncated peptides were expressed at comparable levels and localized at the plasma membrane. Ca(2+) measurements were performed in cells co-transfected with PC1 chimeras and the cytoplasmic Ca(2+)-sensitive photoprotein aequorin, upon activation of the phosphoinositide pathway by ATP, that, via purinoceptors, is coupled to the release of Ca(2+) from intracellular stores. The expression of TrkPC1 peptide, but not of its truncated form, enhanced the ATP-evoked cytosolic Ca(2+) concentrations. When Ca(2+) assays were performed in HeLa cells characterized by Ca(2+) stores greater than those of HEK-293 cells, the histamine-evoked cytosolic Ca(2+) increase was enhanced by TrkPC1 expression, even in absence of external Ca(2+). These observations indicate that the C-terminal tail of PC1 in kidney and other epithelial cells upregulates a Ca(2+) channel activity also involved in the release of intracellular stores.