5-Hydroxytryptamine induces transient Ca influx through Ni-insensitive Ca channels in rat vascular smooth muscle cells

The effects of Ni²⁺, a non-selective cation channel inhibitor, on 5-hydroxytryptamine (5-HT)- and angiotensin II (Ang II)-induced intracellular Ca²⁺ dynamics in rat aortic smooth muscle cells were investigated. Ni²⁺ (1 mM) significantly inhibited the transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by Ang II (100 nM) in aortic smooth muscle cells, as measured using fura-2. However, Ni²⁺ did not suppress the transient increase in Ca²⁺ influx induced by 5-HT (10 μM), while significantly suppressed the sustained increase. Ca²⁺ influx evoked by high KCl (80 mM), thapsigargin (TG) (1 μM) or depletion of intracellular Ca²⁺ store was almost completely suppressed by Ni²⁺. Ni²⁺ had no effect on 5-HT-induced inositol triphosphate production and Ca²⁺ release from the intracellular store(s). These results suggest that 5-HT, but not Ang II, induces transient Ca²⁺ influx through Ni²⁺-insensitive Ca²⁺ channels, which are distinguishable from the voltage-dependent or store-operated Ca²⁺ channels.     

Genistein potentiates activity of the cation channel TRPC5 independently of tyrosine kinases

Background and purpose:

TRPC5 is a Ca²⁺-permeable channel with multiple modes of activation. We have explored the effects of genistein, a plant-derived isoflavone, on TRPC5 activity, and the mechanism(s) involved.

Experimental approach:

Effects of genistein on TRPC5 channels were investigated in TRPC5-over-expressing human embryonic kidney 293 (HEK) cells and bovine aortic endothelial cells (BAECs) using fluorescent Ca²⁺ imaging and electrophysiological techniques.

Key results:

In TRPC5-over-expressing HEK cells, genistein stimulated TRPC5-mediated Ca²⁺ influx, concentration dependently (EC₅₀= 93 µM). Genistein and lanthanum activated TRPC5 channels synergistically. Effects of genistein on TRPC5 channels were mimicked by daidzein (100 µM), a genistein analogue inactive as a tyrosine kinase inhibitor, but not by known tyrosine kinase inhibitors herbimycin (2 µM), PP2 (20 µM) and lavendustin A (10 µM). Action of genistein on TRPC5 channels was not affected by an oestrogen receptor inhibitor ICI-182780 (50 µM) or a phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (10 µM), suggesting genistein did not act through oestrogen receptors or phospholipase C. In BAECs, genistein (100 µM) stimulated TRPC5-mediated Ca²⁺ influx. In patch clamp studies, both genistein (50 µM) and daidzein (50 µM) augmented TRPC5-mediated whole-cell cation current in TRPC5 over-expressing HEK cells. Genistein stimulated TRPC5 channel activity in excised inside-out membrane patch, suggesting that its action was relatively direct and did not require cytosolic factors.

Conclusions and implications:

The present study is the first to demonstrate stimulation of a TRP channel by isoflavones. Genistein is a lipophilic compound able to stimulate TRPC5 activity in TRPC5-over-expressing HEK cells and in native vascular endothelial cells.     

Riluzole activates TRPC5 channels independently of PLC activity

BACKGROUND AND PURPOSE

The transient receptor potential channel C5 (TRPC5) is a Ca²⁺-permeable cation channel, which is predominantly expressed in the brain. TRPC5 is activated in a PLC-dependent manner by, as yet, unidentified endogenous messengers. Recently, modulators of TRPC5, like Ca²⁺, pH and phospholipids, have been identified. However, the role of TRPC5 in vivo is only poorly understood. Novel specific modulators of TRPC5 might help to elucidate its function.

EXPERIMENTAL APPROACH

Novel modulators of TRPC5 were identified in a compound screening of approved drugs and natural compounds. The potency and selectivity of TRPC5-activating compounds were determined by fluorometric calcium imaging. The biophysical properties of channel activation by these compounds were analysed using electrophysiological measurements.

KEY RESULTS

Riluzole was identified as a novel activator of TRPC5 (EC₅₀ 9.2 ± 0.5 μM) and its mechanism of action was shown to be independent of G protein signalling and PLC activity. Riluzole-induced TRPC5 currents were potentiated by La³⁺ and, utilizing TRPC5 mutants that lack La³⁺ binding sites, it was confirmed that riluzole and La³⁺ activate TRPC5 by different mechanisms. Recordings of excised inside-out patches revealed a relatively direct effect of riluzole on TRPC5.

CONCLUSIONS AND IMPLICATIONS

Riluzole can activate TRPC5 heterologously expressed in HEK293 cells as well as those endogenously expressed in the U-87 glioblastoma cell line. Riluzole does not activate any other member of the TRPC family and could, therefore, despite its action on other ion channels, be a useful pharmacological tool for identifying TRPC5-specific currents in immortalized cell lines or in acutely isolated primary cells.     

Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst₅ receptors (CHOsst₅ cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC₅₀ 7.10) and UTP (pEC₅₀ 5.14) caused concentration-dependent increases in [Ca²⁺]_i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca²⁺]_i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P₃ but the SRIF maximum was about 30% of that to UTP. Removal of [Ca²⁺ ]_e attenuated the SRIF-induced peak rise in [Ca²⁺]_i but had no effect on the peak increases in Ins(1,4,5)P₃. UTP-induced increases in [Ca²⁺]_i and Ins(1,4,5)P₃ were attenuated in the absence of [Ca²⁺]_e. Following pre-exposure to SRIF (1 μM) or UTP (100 μM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca²⁺]_e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca²⁺]_e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca²⁺]_i in CHOsst₅ cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca²⁺ and release from an Ins(1,4,5)P₃ sensitive Ca²⁺ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca²⁺]_i could be demonstrated in the presence or absence of extracellular Ca²⁺ respectively, and the latter appeared to involve depletion of a common intracellular Ca²⁺ store.     

Agonist-evoked calcium entry in vascular smooth muscle cells requires IP3 receptor-mediated activation of TRPC1

Transient receptor potential canonical (TRPC) proteins have been proposed to function as plasma membrane Ca2+ channels activated by store depletion and/or by receptor stimulation. However, their role in the increase in cytosolic Ca2+ activated by contractile agonists in vascular smooth muscle is not yet elucidated. The present study was designed to investigate the functional and molecular properties of the Ca2+ entry pathway activated by endothelin-1 in primary cultured aortic smooth muscle cells. Measurement of the Ca2+ signal in fura-2-loaded cells allowed to characterize endothelin-1-evoked Ca2+ entry, which was resistant to dihydropyridine, and was blocked by 2-aminoethoxydiphenylborate (2-APB) and micromolar concentration of Gd3+. It was not activated by store depletion, but was inhibited by the endothelin ETA receptor antagonist BQ-123, and by heparin. On the opposite, thapsigargin-induced store depletion activated a Ca2+ entry pathway that was not affected by 2-APB, BQ-123 or heparin, and was less sensitive to Gd3+ than was endothelin-1-evoked Ca2+ entry. Investigation of the gene expression of TRPC isoforms by real-time RT-PCR revealed that TRPC1 was the most abundant. In cells transfected with TRPC1 small interfering RNA sequence, TRPC1 mRNA and protein expression were decreased by 72+/-3% and 86+/-2%, respectively, while TRPC6 expression was unaffected. In TRPC1 knockdown cells, both endothelin-1-evoked Ca2+ entry and store-operated Ca2+ entry evoked by thapsigargin were blunted. These results indicate that in aortic smooth muscle cells, TRPC1 is not only involved in Ca2+ entry activated by store depletion but also in receptor-operated Ca2+ entry, which requires inositol (1,4,5) triphosphate receptor activation.     

Activation of apical CFTR and basolateral Ca(2+)-activated K+ channels by tetramethylpyrazine in Caco-2 cell line

We have previously demonstrated that tetramethylpyrazine (TMP) could stimulate colonic and pancreatic anion secretion. The present study investigated the signaling pathways and cellular mechanisms underlying the effect of TMP using human colonic Caco-2 cells, with permeabilized apical or basolateral membranes, in conjunction with Ussing chamber technique, intracellular cAMP and Ca²⁺ measurements as well as competitive RT-PCR for mRNA expression of cystic fibrosis transmembrane conductance regulator (CFTR) and Ca²⁺-dependent Cl⁻ channels (CACC). Basolateral addition of TMP induced a short circuit current (I_SC) response, which could be mimicked by forskolin and 3-isobutyl-1-methylxanthine (IBMX). Adenylate cyclase inhibitor, MDL12330A, and intracellular Ca²⁺ chelator, BAPTA-AM, significantly inhibited the TMP-induced I_SC. In basolateral membrane-permeabilized cells, TMP, as well as forskolin and IBMX, induced an I_SC response, which was sensitive to MDL-12330A, H₈₉, and specific channel blocker CFTR_inh-172, but insensitive to apical application of 4-4′-didsothiocyanostilbene-2, 2′-disulfonic acid (DIDS) and basolateral pretreatment with BAPTA-AM. In apical membrane-permeabilized cells, TMP, similar to forskolin and IBMX, produced a very small current increase, which was sensitive to K⁺ channel blockers, BaCl₂ and tetraethylammonium (TEA), but not Chromanol 293B and charybdotoxin (ChTX), alone or combined. However, in intact Caco-2 monolayers, the TMP-induced I_SC could be partially inhibited by ChTX. TMP (5 mM) could stimulate intracellular cAMP production. Intracellular Ca²⁺ was also increased by TMP (5 mM) in both Ca²⁺-containing and Ca²⁺-free bathing solutions. RT-PCR showed that the expression of CFTR in Caco-2 cells was 5.2 fold higher than that of Ca²⁺-activated Cl⁻ channel (CACC). In conclusion, TMP stimulates Cl⁻ secretion by activating cAMP and [Ca²⁺]_i signaling pathways leading to subsequent activation of apical CFTR and basolateral K⁺ channels.     

CGP37157 modulates mitochondrial Ca homeostasis in cultured rat dorsal root ganglion neurons

The effects of 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-on (CGP37157), an inhibitor of mitochondrial Na⁺/Ca²⁺ exchange, on depolarization-induced intracellular free Ca²⁺ concentration ([Ca²⁺]_i) transients were studied in cultured rat dorsal root ganglion neurons with indo-1-based microfluorimetry. A characteristic plateau in the recovery phase of the [Ca²⁺]_i transient resulted from mitochondrion-mediated [Ca²⁺]_i buffering. It was blocked by metabolic poisons and was not dependent on extracellular Ca²⁺. CGP37157 produced a concentration-dependent decrease in the amplitude of the mitochondrion-mediated plateau phase (IC₅₀=4±1 μM). This decrease in [Ca²⁺]_i was followed by an increase in [Ca²⁺]_i upon removal of the drug, suggesting that Ca²⁺ trapped in the matrix was released when the CGP37157 was removed from the bath. CGP37157 also inhibited depolarization-induced Ca²⁺ influx at the concentrations required to see effects on [Ca²⁺]_i buffering. Thus, CGP37157 inhibits mitochondrial Na⁺/Ca²⁺ exchange and directly inhibits voltage-gated Ca²⁺ channels, suggesting caution in its use to study [Ca²⁺]_i regulation in intact cells.     

Effects of Ca channel blockers on Ca loading induced by metabolic inhibition and hyperkalemia in cardiomyocytes

The effects of the L-type (nifedipine and verapamil) and the T-type (mibefradil) Ca²⁺ channel blockers on the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by NaCN metabolic inhibition and hyperkalemia were examined in chicken cardiomyocytes using fluorescence imaging with Fura-2. NaCN induced a slow and sustained rise in [Ca²⁺]_i, which was not affected by pretreating the cells for 5 min with nifedipine, verapamil, or mibefradil at 100 nM or 10 μM. Pretreatment of the cells with 10 μM nifedipine, verapamil, or mibefradil for 5 min remarkably inhibited the K⁺-induced increase in [Ca²⁺]_i. These inhibitory effects diminished after 48-h pretreatment with nifedipine or verapamil but not with mibefradil. Ryanodine also induces an increase in [Ca²⁺]_i, and this effect was enhanced by 48-h pretreatment of the cells with 10 μM verapamil but not with 10 μM mibefradil. We conclude that the NaCN-induced increase in [Ca²⁺]_i is independent of the Ca²⁺ influx though the L-type or T-type Ca²⁺ channels. Chronic inhibition of the L-type Ca²⁺ channels but not T-type channels may enhance the ryanodine receptor-mediated Ca²⁺ release, which may be responsible for the development of tolerance to their inhibitory effects on K⁺-induced increase in [Ca²⁺]_i.     

Post-transcriptional silencing of TRPC1 ion channel gene by RNA interference upregulates TRPC6 expression and store-operated Ca entry in A7r5 vascular smooth muscle cells

This study investigates functional consequences of TRPC1 ion channel downregulation observed in aging rat aorta by employing RNA interference in cultured vascular smooth muscle cells. For this purpose, A7r5 aortic smooth muscle cells were used in quantitative gene and protein expression as well as in functional analyses. According to quantitative RT-PCR results, TRPC3, TRPC4 and TRPC5 mRNAs were not at detectable levels. In siTRPC1-transfected cells, TRPC1 mRNA and protein levels were decreased by 40% and 64%; however, those of TRPC6 were drastically increased by 100% and 200%, respectively. In fura-2-loaded TRPC1 knockdown cells, despite the decreased TRPC1 levels, cyclopiazonic acid-induced Ca²⁺ entry and store-operated Ca²⁺ entry following Ca²⁺ addition were elevated by 77% and 135%, respectively. Results suggest that decrease in TRPC1 may be compensated by upregulated TRPC6 that possibly takes part in store-operated Ca²⁺ entry in vascular smooth muscle cells.     

Stim, ORAI and TRPC channels in the control of calcium entry signals in smooth muscle

Ca2+ entry signals are crucial in the control of smooth muscle contraction. Smooth muscle cells are unusual in containing plasma membrane (PM) Ca2+ entry channels that respond to voltage changes, receptor activation and Ca2+ store depletion. Activation of these channel subtypes is highly coordinated. The TRPC6 channel, widely expressed in most smooth muscle cell types, is largely non-selective to cations and is activated by diacylglycerol arising from receptor-induced phosholipase C activation. Receptor activation results largely in Na+ ion movement through TRPC6 channels, depolarization and subsequent activation of voltage-dependent L-type Ca2+ channels. The TRPC6 channels also appear to be activated by mechanical stretch, resulting again in depolarization and L-type Ca2+ channel activation. Such a coupling may be crucial in mediating the myogenic tone response in vascular smooth muscle. The emptying of stores mediated by inositol 1,4,5-trisphosphate receptors triggers the endoplasmic reticulum (ER) Ca2+ sensing protein stromal-interacting molecule (STIM) 1 to translocate into defined ER-PM junctional areas in which coupling occurs to Orai proteins, which serve as highly Ca2+-selective low-conductance Ca2+ entry channels. These ER-PM junctional domains may serve as crucial sites of interaction and integration between the function of store-operated, receptor-operated and voltage-operated Ca2+ channels. The STIM, Orai and TRPC channels represent highly promising new pharmacological targets through which such control may be induced.     

Receptor-operated cation channels formed by TRPC4 and TRPC5

TRPC4 and TRPC5 form cation channels that contribute to phospholipase C-dependent Ca²⁺ entry following stimulation of G-protein-coupled receptors or receptor tyrosine kinases. Surprisingly, in different studies, TRPC4 and TRPC5 have been shown to form either store-operated channels with a relatively high Ca²⁺ permeability, or nonselective cation channels activated independently of store depletion. In this review, we summarize and discuss data on the regulation and permeability properties of TRPC4 and TRPC5, and data on native channels that might be composed of these isoforms.     

Mechanism of inhibition of TRPC cation channels by 2-aminoethoxydiphenylborane

We investigated the actions of the organoborane, 2-aminoethoxydiphenylborane (2APB), on Ca2+ signaling in wild-type human embryonic kidney (HEK) 293 cells and in HEK293 cells stably expressing canonical transient receptor potential (TRPC) channels. Previous reports have suggested that 2APB inhibits agonist activation of TRPC channels because of its ability to act as a membrane-permeant inhibitor of inositol 1,4,5-trisphosphate (IP3) receptors. 2APB was specifically said to inhibit TRPC3 channels when activated through a phospholipase C-linked receptor but not when activated more directly by a synthetic diacylglycerol, oleyl-acetyl-glycerol (OAG) [Science (Wash DC) 287:1647-1651, 2000]. However, we subsequently reported that IP3 does not activate TRPC3; rather the mechanism of activation by phospholipase C-linked receptors seemed to result from diacylglycerol [J Biol Chem 278:16244-16252, 2003]. Thus, the current study was carried out to address the mechanism of action of 2APB in inhibiting TRPC channels. We found that, although the release of Ca2+ by a muscarinic agonist was reduced by high concentrations of 2APB, this effect was indistinguishable from that seen when stores were discharged by thapsigargin, which does not involve IP3 receptors. This indicates that 2APB is incapable of significant inhibition of IP3 receptors when applied to intact cells. We found that 2APB partially inhibits divalent cation entry in cells expressing TRPC3, TRPC6, or TRPC7 and that this partial inhibition was observed whether the channels were activated by a muscarinic agonist or by OAG. Thus, as concluded for store-operated channels, 2APB seems to inhibit TRPC channels by a direct mechanism not involving IP3 receptors.     

Block of TRPC5 channels by 2-aminoethoxydiphenyl borate: a differential, extracellular and voltage-dependent effect

1 2-aminoethoxydiphenyl borate (2-APB) has been widely used to examine the roles of inositol 1,4,5-trisphosphate receptors (IP3Rs) and store-operated Ca2+ entry and is an emerging modulator of cationic channels encoded by transient receptor potential (TRP) genes. 2 Using Ca2+-indicator dye and patch-clamp recording we first examined the blocking effect of 2-APB on human TRPC5 channels expressed in HEK-293 cells. 3 The concentration-response curve has an IC50 of 20 microM and slope close to 1.0, suggesting one 2-APB molecule binds per channel. The blocking effect is not shared by other Ca2+ channel blockers including methoxyverapamil, nifedipine, N-propargylnitrendipine, or berberine. 4 In whole-cell and excised membrane patch recordings, 2-APB acts from the extracellular but not intracellular face of the membrane. 5 Block of TRPC5 by 2-APB is less at positive voltages, suggesting that it enters the electric field or acts by modulating channel gating. 6 2-APB also blocks TRPC6 and TRPM3 expressed in HEK-293 cells, but not TRPM2. 7 Block of TRP channels by 2-APB may be relevant to cell proliferation because 2-APB has a greater inhibitory effect on proliferation in cells overexpressing TRPC5. 8 Our data indicate a specific and functionally important binding site on TRPC5 that enables block by 2-APB. The site is only available via an extracellular route and the block shows mild voltage-dependence.     

TRPC1 is involved in Ca influx and cytotoxicity following Pb exposure in human embryonic kidney cells

Lead (Pb²⁺) is a divalent heavy metal ion which causes severe damage to almost all life forms and is therefore considered a notorious toxicant. Exposure to Pb²⁺ is associated with poor cognitive development in children at relatively low levels that previously were thought to be safe. The mechanism through which Pb²⁺ enters cells, however, is unclear. Previous studies have showed that Ca²⁺ release-activated Ca²⁺ protein 1 (Orai1), a component of store-operated Ca²⁺ channels (SOCs), contributes to Pb²⁺ cellular entry. Canonical transient receptor potential (TRPC1) channel 1 is a transient receptor potential (TRP) channel which is sometimes referred to as a SOC. The present study was designed to investigate the role of TRPC1 in Pb²⁺ entry and toxicity in human embryonic kidney cells (HEK293). Additionally, changes in intracellular Ca²⁺ concentration were determined through Fluo-4 and Mag-fluo-4 fluorescent Ca²⁺ imaging. Following Pb²⁺ exposure, there was a dose-dependent decrease in cell viability. Overexpression of TRPC1 increased Pb²⁺-induced cell death, while knockdown of this channel attenuated cell death. There was increased entry of Pb²⁺, as measured by inductively coupled plasma mass spectrometry (ICP-MS), following overexpression of TRPC1. Conversely, knockdown of TRPC1 led to a decrease in Pb²⁺ influx. Down-regulation of STIM1 by RNA interference attenuated the Pb²⁺ influx, and transfection with a mutant STIM1, which could not gate TRPC1, had a similar effect. Co-transfection of mutant STIM1 and mutant TRPC1, which restore the electrostatic interaction between STIM1 and TRPC1, resumed Pb²⁺ entry in HEK293 cells. Down-regulation of TRPC1 by RNA interference decreased Ca²⁺ influx whilst its overexpression increased Ca²⁺ entry in HEK293 cells. These results suggest that TRPC1 is involved in the cytotoxicity and entry of Pb²⁺ through molecular interactions with STIM1 and subsequent Ca²⁺ influx in HEK293 cells.     

Stereo-selective inhibition of transient receptor potential TRPC5 cation channels by neuroactive steroids

BACKGROUND AND PURPOSE

Transient receptor potential canonical 5 (TRPC5) channels are widely expressed, including in the CNS, where they potentiate fear responses. They also contribute to other non-selective cation channels that are stimulated by G-protein-coupled receptor agonists and lipid and redox factors. Steroids are known to modulate fear and anxiety states, and we therefore investigated whether TRPC5 exhibited sensitivity to steroids.

EXPERIMENTAL APPROACH

Human TRPC5 channels were conditionally expressed in HEK293 cells and studied using intracellular Ca²⁺ measurement, whole-cell voltage-clamp and excised patch techniques. For comparison, control experiments were performed with cells lacking TRPC5 channels or expressing another TRP channel, TRPM2. Native TRPC channel activity was recorded from vascular smooth muscle cells.

KEY RESULTS

Extracellular application of pregnenolone sulphate, pregnanolone sulphate, pregnanolone, progesterone or dihydrotestosterone inhibited TRPC5 activity within 1–2 min. Dehydroepiandrosterone sulphate or 17β-oestradiol had weak inhibitory effects. Pregnenolone, and allopregnanolone, a progesterone metabolite and stereo-isomer of pregnanolone, all had no effects. Progesterone was the most potent of the steroids, especially against TRPC5 channel activity evoked by sphingosine-1-phosphate. In outside-out patch recordings, bath-applied progesterone and dihydrotestosterone had strong and reversible effects, suggesting relatively direct mechanisms of action. Progesterone inhibited native TRPC5-containing channel activity, evoked by oxidized phospholipid.

CONCLUSIONS AND IMPLICATIONS

Our data suggest that TRPC5 channels are susceptible to relatively direct and rapid stereo-selective steroid modulation, leading to channel inhibition. The study adds to growing appreciation of TRP channels as non-genomic steroid sensors.     

TRPC1 Ca(2+)-permeable channels in animal cells

The full-length transient receptor (TRPC)1 polypeptide is composed of about 790 amino acids, and several splice variants are known. The predicted structure and topology is of an integral membrane protein composed of six transmembrane domains, and a cytoplasmic C- and N-terminal domain. The N-terminal domain includes three ankyrin repeat motifs. Antibodies which recognise TRPC1 have been developed, but it has been difficult to obtain antibodies which have high affinity and specificity for TRPC1. This has made studies of the cellular functions of TRPC1 somewhat difficult. The TRPC1 protein is widely expressed in different types of animal cells, and within a given cell is found at the plasma membrane and at intracellular sites. TRPC1 interacts with calmodulin, caveolin-1, the InsP3 receptor, Homer, phospholipase C and several other proteins. Investigations of the biological roles and mechanisms of action of TRPC1 have employed ectopic (over-expression or heterologous expression) of the polypeptide in addition to studies of endogenous TRPC1. Both approaches have encountered difficulties. TRPC1 forms heterotetramers with other TRPC polypeptides resulting in cation channels which are non-selective. TRPC1 may be: a component of the pore of store-operated Ca2+ channels (SOCs); a subsidiary protein in the pathway of activation of SOCs; activated by interaction with InsP3R; and/or activated by stretch. Further experiments are required to resolve the exact roles and mechanisms of activation of TRPC1. Cation entry through the TRPC1 channel is feed-back inhibited by Ca2+ through interaction with calmodulin, and is inhibited by Gd3+, La3+, SKF96365 and 2-APB, and by antibodies targeted to the external mouth of the TRPC1 pore. Activation of TRPC1 leads to the entry to the cytoplasmic space of substantial amounts of Na+ as well as Ca2+. A requirement for TRPC1 is implicated in numerous downstream cellular pathways. The most clearly described roles are in the regulation of growth cone turning in neurons. It is concluded that TRPC1 is a most interesting protein because of the apparent wide variety of its roles and functions and the challenges posed to those attempting to elucidate its primary intracellular functions and mechanisms of action.     

Effects of cytosolic ATP and other nucleotides on Ca-activated K channels in cultured bovine adrenal chromaffin cells

The effects of cytosolic ATP on Ca²⁺-dependent K⁺ (K_Ca) channel activation in cultured bovine adrenal chromaffin cells were investigated by using single-channel recording patch-clamp techniques. Application of ATP to the intracellular surface of excised inside-out patches activated K_Ca channels in a dose-dependent manner at 30 μM to 10 mM. The K_Ca channels also were activated by 3 mM of adenosine 5′-O-(3′-thiotriphosphate) (ATPγS), a non-hydrolyzable analogue of ATP, but not by 5′-adenylylimidodiphosphate (AMP-PNP) (from 300 μM to 3 mM). Furthermore, other nucleotides also activated K_Ca channels in inside-out patches. This modulation took place without addition of exogenous protein kinase and was dependent on the presence of Mg²⁺ in the bathing solution. Staurosporine, a non-specific kinase inhibitor, or H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), a cAMP-dependent protein kinase inhibitor, was unable to alter ATP-mediated K_Ca channel activation. Following complete removal of Mg²⁺, a higher concentration of ATP (10 mM) and other nucleotides was required to activate K_Ca channels; however, Mg²⁺ was ineffective in altering the activation of K_Ca channels by itself. It is concluded that intracellular ATP and other nucleotides activate K_Ca channels directly. These nucleotides may regulate catecholamine release by changing the cell membrane potential in adrenal chromaffin cells.     

Heptachlor epoxide induces a non-capacitative type of Ca2+ entry and immediate early gene expression in mouse hepatoma cells

The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE) and a related non-tumor promoting OC, delta-hexachlorocyclohexane (δ-HCH), on the dynamics of intracellular calcium (Ca²⁺) were investigated in mouse 1c1c7 hepatoma cells. HE induced a non-capacitative, Ca²⁺ entry-like phenomenon, which was transient and concentration-dependent with 10 and 50 μM HE. The plasma membrane Ca²⁺ channel blocker SKF-96365 antagonized this HE-induced Ca²⁺ entry. δ-HCH failed to induce Ca²⁺ entry, rather it antagonized the HE-induced Ca²⁺ entry. Both HE and δ-HCH induced Ca²⁺ release from endoplasmic reticulum (ER) at treatment concentrations as low as 10 μM; at 50 μM, the former induced 5× as much Ca²⁺ release as the latter. The HE-induced Ca²⁺ release from the ER was antagonized using the IP₃ receptor/channel blocker xestospongin C, suggesting that HE induces ER Ca²⁺ release through the IP₃ receptor/channel pore. These results show that the effect of HE on cellular Ca²⁺ mimics that of mitogens such as epidermal and hepatocyte growth factors. They also provide insight into the similarities and differences between tumorigenic and non-tumorigenic OCs, in terms of the mechanisms and the extent of the [Ca²⁺]_i increased by these agents.     

Inhibition of voltage-gated ca channel activity in small cell lung carcinoma by the ca/calmodulin-dependent protein kinase inhibitor KN-62 (1-[n,o-bis(5-isoquinolinesulfonyl)-n-methyl-L-tyrosyl]-4-phenylpiperazine)

Although small cell lung carcinoma (SCLC) cells express both voltage-gated Ca²⁺ channels (VGCC) and second messenger-operated Ca²⁺ channels (SMOCC), little is known about the factors that regulate the activity of these channels in SCLC cells. Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) type II has been implicated recently in regulating Ca²⁺ channel activity in other cell types. Because of this, we investigated the effects of the specific CaM kinase antagonist 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tryosyl]-4-phenylpiperazine (KN-62) on Ca²⁺ channel activity in SCLC cells. Incubation with 10 μM KN-62 for 20 min inhibited depolarization-dependent ⁴⁵Ca²⁺ influx by 96.1 ± 2.1% in four independent SCLC cell lines, and by 42.2 ± 6.8% in the NCI-H146 SCLC cell line. Similar inhibitory effects of KN-62 were observed when Fura-2 was used to measure depolarization-dependent Ca²⁺ influx. These results indicate that KN-62 potently inhibits VGCC activity in SCLC cells. In contrast, KN-62 (10 μM, 20 min) did not inhibit significantly Ca²⁺ mobilization induced by muscarinic acetylcholine receptor (mAChR) activation in SCLC cells. This indicates that SMOCC are less susceptible than VGCC to inhibition by KN-62 in SCLC cells. Because mAChR activation also inhibits VGCC activity in SCLC cells, we examined the effects of KN-62 on the mAChR-mediated inhibition of VGCC activity. To do this, we measured depolarization-dependent ⁴⁵Ca²⁺ influx in SCLC cells incubated with submaximal concentrations of KN-62 and the mAChR agonist carbachol. Treatment of cells with both drugs resulted in almost twice as much inhibition of VGCC activity as in cells treated with only one of the drugs. This indicates that inactivation of CaM kinase with KN-62 does not suppress the ability of mAChR agonists to inhibit VGCC activity.     

Phospholipase C not protein kinase C is required for the activation of TRPC5 channels by cholecystokinin

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to G(q/11) proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca(2+). The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP(3) receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca(2+) stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.