Receptor-operated Cation Channels Research Articles

TRPC4 and GIRK channels underlie neuronal coding of firing patterns that reflect Gq/11–Gi/o coincidence signals of variable strengths

Transient receptor potential canonical 4 (TRPC4) is a receptor-operated cation channel codependent on both the Gq/11–phospholipase C signaling pathway and Gi/o proteins for activation. This makes TRPC4 an excellent coincidence sensor of neurotransmission through Gq/11- and Gi/o-coupled receptors. In whole-cell slice recordings of lateral septal neurons, TRPC4 mediates a strong depolarizing plateau that shuts down action potential firing, which may or may not be followed by a hyperpolarization that extends the firing pause to varying durations depending on the strength of Gi/o stimulation. We show that the depolarizing plateau is codependent on Gq/11-coupled group I metabotropic glutamate receptors and on Gi/o-coupled γ-aminobutyric acid type B receptors. The hyperpolarization is mediated by Gi/o activation of G protein–activated inwardly rectifying K+ (GIRK) channels. Moreover, the firing patterns, elicited by either electrical stimulation or receptor agonists, encode information about the relative strengths of Gq/11 and Gi/o inputs in the following fashion. Pure Gq/11 input produces weak depolarization accompanied by firing acceleration, whereas pure Gi/o input causes hyperpolarization that pauses firing. Although coincident Gq/11–Gi/o inputs also pause firing, the pause is preceded by a burst, and both the pause duration and firing recovery patterns reflect the relative strengths of Gq/11 versus Gi/o inputs. Computer simulations demonstrate that different combinations of TRPC4 and GIRK conductances are sufficient to produce the range of firing patterns observed experimentally. Thus, concurrent neurotransmission through the Gq/11 and Gi/o pathways is converted to discernible electrical responses by the joint actions of TRPC4 and GIRK for communication to downstream neurons.

Abstract MP240: Site-specific O-Glcnacylation Of Transient Receptor Potential Canonical Type 6 (trpc6) Channel Is Required For Normal Structure And Function

O-GlcNAcylation is a dynamic, reversible posttranslational modification (PTM) that regulates a multitude of biological processes. Fluctuations in O-GlcNAC of various calcium handling proteins impact their functionality in cardiomyocytes. Here, we show for the first time that TRPC6, a nonselective receptor-operated cation channel and mediator of hypertrophy and fibrosis, is constitutively O-GlcNAcylated in the ankyrin repeat domain (AR4), at Ser 14, Thr 70, and Thr 221 within the N-terminal cytoplasmic segment. Of these, only substitution of Thr 221 with alanine (T221A) results in a change of function, notably a hyperactive TRPC6 channel with 5X greater increase in consequent NFAT promoter activity, which is a marker of TRPC6 calcium signaling. Patch-clamp analysis of T221A mutant channels found a 75-80% increased conductance compared to WT. Myocardial injection of T221A in homozygous TRPC6 KO mice by AAV-9 mediated gene transfer results in systolic dysfunction, hypertrophy, and cardiac fibrosis, by loss of OGlcNAc modification at site T221. T221 is highly conserved across species and found in the AR4 domain, which forms the core structure of TRPC6 intracellular domain. Mutating the site in its closest homologs, TRPC3 and TRPC7, also activates channel activity. T221 O-GlcNAcylation also protects the nascent protein from premature proteasomal degradation. Molecular modeling from the crystal structure of human TRPC6 predicts that OGlcNACylation stabilizes electrostatic interactions with the 193-203 loop near AR4, and loop connecting AR4 to the linker helix 1 (LH1) at S199, E200, and E246. Mutating these sites to alanine also increases TRPC6-NFAT signaling similar to what was observed in the T221A mutant. In summary, this study highlights that O-GlcNAcylation of TRPC6 is an important PTM needed to stabilize channel function, and its decline results in gain-of-function related diseases.

What's New in Shock, February 2021?

What's New in Shock, February 2021?

TRPC6 deficiency causes increased body weight and glucose intolerance in mice fed a normal diet but does not amplify the obesogenic effect of a high fat diet

Transient receptor potential cation channel subfamily C member 6 (TPRC6) is a receptor‐operated cation channel that modulates cell Ca2+ influx. TRPC6 is expressed in many tissues including brain and adipose tissue. We previously showed that whole‐body TRPC6 deletion (TRPC6 KO) causes obesity, impaired glucose tolerance, and leptin resistance in mice fed a standard diet (SD). However, the metabolic consequences of feeding a high fat diet (HFD) to TRPC6 KO mice are still unknown. In this study, we investigated the impact of HFD (45% Cal from fat) from 6 to 18 weeks of age in male and female TRPC6 KO and B6/129s background control mice on body weight (BW), body fat/lean composition, food intake (FI), responses to acute leptin injection, and tolerance to an oral glucose load (OGTT). TRPC6 KO mice of both sexes were heavier than control mice at 6 weeks of age before starting HFD (27.3 ± 1.0 vs 24.2 ± 0.8 g in males and 24.1 ± 0.8 vs 19.1 ± 0.7 in females, n=7/sex, p<0.05). After 10 weeks on HFD, male and female TRPC6 KO mice gained less weight compared to control mice (BW gain: 8.5 ± 0.9 vs 15.2 ± 1.2 g in males and 9.4 ± 0.8 vs 12.2 ± 1.0 g in females from 6 to 16 weeks of age). EchoMRI scans showed that the percentage of lean mass was slightly lower while the percentage of fat mass was higher in TRPC6 KO mice compared to controls at the beginning of HFD. After 10 weeks on HFD, male and female TRPC6 KO mice gained less fat mass than control mice (fat mass gain: 4.2 ± 0.5 vs 11.6 ± 0.6 g in males and 5.7 ± 0.7 vs 8.8 ± 0.6 g in females from 6 to16 weeks of age), while lean mass increased similarly in both genotypes. Daily food intake in TRPC6 KO mice was significantly higher during the first 3 weeks of HDF (average daily FI: 3.5 ± 0.1 vs 2.8 ± 0.1 g in males, and 3.1 ± 0.1 vs 2.6 ± 0.1 in females from 6–8 weeks of age, n=7, p<0.05), after which FI was similar between groups (2.8 ± 0.1 vs 2.7 ± 0.1 g in males and 2.6 ± 0.1 vs 2.4 ± 0.1 in females from 9–16 weeks of age, n=7). After 12 weeks on a HFD, an acute leptin injection (5 mg/kg, i.p.) in fasted mice (n=7/sex/genotype) reduced 24‐hr FI in control mice by 0 % in males and 21% in females. In contrast, leptin reduced 24‐hr FI in TRPC6 KO mice by 15 % in males and 25% in females. An OGTT was performed after 12 weeks on a HFD and male and female TRPC6 KO mice exhibited impaired glucose tolerance compared to control mice (AUC: 19,198 ± 1,357 vs 9,450 ± 2,101 mg/dL × 120 min in males; and 17,117 ± 1,987 vs 5,470 ± 371 mg/dL × 120 min in females, n=7, p<0.05). Our results show that although TRPC6 KO mice have increased adiposity and BW on a SD, they appear to be resistant to HFD‐induced fat mass gain while exhibiting significant intolerance to glucose. Although TRPC6 may play an important role in the regulation of BW, FI, fat mass and glucose handling when fed a SD or HFD, the mechanisms involved in the different metabolic responses in RD and HFD are still unclear and may involve reduced sensitivity to leptin or differential roles of TRPC6 in brain.Support or Funding InformationNHLBI PO1HL51971, NIGMS P20GM104357 and U54GM115428, and NIDDK R00DK113280

Emerging Roles of Diacylglycerol-Sensitive TRPC4/5 Channels.

Transient receptor potential classical or canonical 4 (TRPC4) and TRPC5 channels are members of the classical or canonical transient receptor potential (TRPC) channel family of non-selective cation channels. TRPC4 and TRPC5 channels are widely accepted as receptor-operated cation channels that are activated in a phospholipase C-dependent manner, following the Gq/11 protein-coupled receptor activation. However, their precise activation mechanism has remained largely elusive for a long time, as the TRPC4 and TRPC5 channels were considered as being insensitive to the second messenger diacylglycerol (DAG) in contrast to the other TRPC channels. Recent findings indicate that the C-terminal interactions with the scaffolding proteins Na+/H+ exchanger regulatory factor 1 and 2 (NHERF1 and NHERF2) dynamically regulate the DAG sensitivity of the TRPC4 and TRPC5 channels. Interestingly, the C-terminal NHERF binding suppresses, while the dissociation of NHERF enables, the DAG sensitivity of the TRPC4 and TRPC5 channels. This leads to the assumption that all of the TRPC channels are DAG sensitive. The identification of the regulatory function of the NHERF proteins in the TRPC4/5-NHERF protein complex offers a new starting point to get deeper insights into the molecular basis of TRPC channel activation. Future studies will have to unravel the physiological and pathophysiological functions of this multi-protein channel complex.

Abstract P192: Trpc6 Deficiency Causes Obesity, Impaired Glucose Tolerance and Leptin Resistance

Transient receptor potential cation channel subfamily C member 6 (TPRC6) is a receptor-operated cation channel that contributes to changes in cytosolic free Ca 2+ concentration. TRPCs have been suggested to play a role in the regulation of neurite outgrowth, synapse formation, and neuronal survival. However, the role of TRPC6 in controlling metabolic and cardiovascular functions is still unclear. We therefore investigated the impact of TRPC6 on regulation of energy balance, metabolic and cardiovascular functions and anorexic responses to leptin in male and female TRPC6 knockout mice and control B6/129 mice, including assessment of body weight (BW), food intake (FI), body fat/lean composition, responses to acute leptin injection, glucose tolerance tests (GTTs), and blood pressure (BP) and heart rate (HR). TRPC6 null mice of both sexes were heavier than control mice from 6 to 16 weeks of age when fed a standard diet (39.5 ± 1.5 vs 31.1 ± 1.2 g in males and 31.1 ± 1.2 vs 22.9 ± 1.0 in females at 16 weeks of age, n=7, p <0.05). EchoMRI scans showed that the higher body weight in TRPC6 null mice was mainly due to increased body fat compared to controls (30.0 % vs 22.6 % of fat mass/BW in males and 32.7 % vs 21.0 % in females at 16 weeks of age), and was associated with increased daily FI (3.4 ± 0.1 vs 2.7 ± 0.2 g in males and 2.7 ± 0.1 vs 2.0 ± 0.1 in females at 16 weeks of age, n=7, p <0.05). Acute leptin injections (5 mg/kg, i.p. at 18 weeks of age, n=7) significantly reduced 24-hr FI by 41 % and 32 % in male and female control mice, while only an 8 % and 18 % reduction in FI were observed in male and female TRPC6 null mice. At 20 weeks of age, male and female TRPC6 null mice also exhibited impaired glucose tolerance during GTT compared to controls (AUC: 16,138 ± 1,877 vs 8,016 ± 1,476 mg/dL x 120 min in males, and 6,152 ± 1,173 vs 1,975 ± 512 mg/dL x 120 min in females, n=7, p <0.05). BP and HR measured by telemetry for 5 consecutive days at 22 weeks of age were similar between groups. Our results indicate that TRPC6 plays an important role in normal control of food intake, body weight and glucose homeostasis as well as for leptin’s anorexic action. Although TRPC6 deficiency caused obesity and metabolic abnormalities, BP and HR did not increase.

Stop TRPC5 in Kidney Disease: A Game Changer for Renal Allograft Protection?

Stop TRPC5 in Kidney Disease: A Game Changer for Renal Allograft Protection?

C60 fullerenes disrupt cellular signalling leading to TRPC4 and TRPC6 channels opening by the activation of muscarinic receptors and G-proteins in small intestinal smooth muscles

The effect of water-soluble pristine C60 fullerene nanoparticles (C60NPs) on receptor-operated cation channels formed by TRPC4/C6 proteins in ileal smooth muscle cells was investigated for the first time. Activation of these channels subsequent to acetylcholine binding to the expressed in these cells M2 and M3 muscarinic receptors represents the key event in the parasympathetic control of gastrointestinal smooth muscle motility and cholinergic excitation-contraction coupling. Experiments were performed on single collagenase-dispersed mouse ileal myocytes using patch-clamp techniques with symmetrical 125mM Cs+ solutions and [Ca2+]i ‘clamped’ at 100nM in order to isolate the muscarinic cation current (mICAT). The current was induced by intracellular infusion of 200μM GTPγS, which activates G-proteins directly, i.e. bypassing the muscarinic receptors. C60NPs applied at 10−6M at peak response to activation of G-proteins caused mICAT inhibition by 47.0±3.5% (n=9). The inhibition developed rather slowly, with the time constant of 119±16s, was voltage-independent and irreversible. Thus, C60NPs are unlikely to cause any direct block of TRPC4/C6 channels; rather, they may accumulate in the membrane and disrupt G-protein signalling leading to mICAT generation. C60NPs may represent a novel class of biocompatible molecules for the treatment of disorders associated with enhanced gastrointestinal motility.

SARAF modulates TRPC1, but not TRPC6, channel function in a STIM1-independent manner.

Canonical transient receptor potential-1 (TRPC1) is an almost ubiquitously expressed channel that plays a relevant role in cell function. As other TRPC members, TRPC1 forms receptor-operated cation channels that exhibit both STIM1-dependent and store-independent behaviour. The STIM1 inhibitor SARAF (for store-operated Ca2+ entry (SOCE)-associated regulatory factor) modulates SOCE by interaction with the STIM1 region responsible for Orai1 activation (SOAR). Furthermore, SARAF modulates Ca2+ entry through the arachidonate-regulated Ca2+ (ARC) channels, consisting of Orai1 and Orai3 heteropentamers and plasma membrane-resident STIM1. While a role for STIM1-Orai1-mediated signals has been demonstrated, the possible role of SARAF in TRPC1 function remains unknown. Here, we provide evidence for the interaction of SARAF with TRPC1, independently of STIM1 both in STIM1-deficient NG115-401L cells and SH-SY5Y cells endogenously expressing STIM1. Silencing of SARAF expression in STIM1-deficient cells demonstrated that SARAF plays a negative regulatory role in TRPC1-mediated Ca2+ entry. The interaction of SARAF with TRPC1 in STIM1-deficient cells, as well as with the TRPC1 pool not associated with STIM1 in STIM1-expressing cells was enhanced by stimulation with the physiological agonist ATP. In contrast with TRPC1, we found that the interaction between SARAF and TRPC6 was constitutive rather than inducible by agonist stimulation. Furthermore, we found that SARAF expression silencing was without effect on Ca2+ entry evoked by agonists in TRPC6 overexpressing cells, as well as in Ca2+ influx evoked by the TRPC6 activator Hyp9. These findings provide evidence for a new regulator of TRPC1 channel function and highlight the relevance of SARAF in intracellular Ca2+ homeostasis.

Species-Related Differences in the Properties of TRPC4 Channels in Intestinal Myocytes of Rodents

TRPC4 proteins form receptor-operated cation channels that are activated in synergy by M2 and M3 ACh receptors coupled to Gq/11 and Gi/o proteins, respectively. These channels are widely expressed in the brain and smooth muscles where they perform a number of important functions, including control of GABA release from the dendrites and cholinergic excitation of smooth muscles. The biophysical properties of TRPC4 currents directly activated by GTPγS in mouse cells remain mostly unknown. We, thus, aimed to investigate these channels in mouse ileal myocytes where a prominent TRPC4-mediated cation current termed mICAT is observed, and to compare the behavior of this current to that of the better studied mICAT in guinea-pig myocytes. Although cation current responses to carbachol at –50 mV (i.e., at the value close to the normal resting potential in these cells) were highly similar, mICAT in the mouse lacked the permissive action of intracellular Ca2+ on channel opening. The slope factor of the muscarinic cation conductance, which is a defining property of voltage-dependent behavior, was identical in both species. There were differences in the potential at which the current peaked at negative potentials, but not in the maximal current densities. Major differences were found in the kinetics of mICAT voltagedependent relaxations, which were much faster in the mouse. The above rodent species employ two different strategies for the open probability increase by activated G-proteins; the mean open time was shorter in the mouse compared to that in the guinea-pig (15.1 ± 5.2 msec, n = 8, vs. 80.0 ± 19.7 msec, n = 9; P < 0.01). Correspondingly, the instantaneous frequency of channel opening was much higher in the mouse (154.1 ± 18.8 sec–1 vs. 70.2 ± 7.3 sec–1 in the guinea-pig; P < 0.001). These functional differences are discussed based on structural differences found in the corresponding TRPC4 amino acid sequences of the two rodent species, which are mainly clustered in the cytosolic C-terminus of TRPC4 protein.

Muscarinic Receptor Induced Contractions of the Detrusor are Mediated by Activation of TRPC4 Channels

Muscarinic receptor mediated contractions of the detrusor rely on Ca2+ influx through voltage-gated Ca2+ channels but to our knowledge the mechanism linking stimulation of M3Rs to the activation of voltage dependent Ca2+ channels has not been established. TRPC4 channels are receptor operated cation channels that couple muscarinic receptor activation to depolarization of intestinal smooth muscle cells, voltage-activated Ca2+ influx and contraction. We investigated whether TRPC4 channels are involved in cholinergic mediated contractions of the detrusor. Isometric tension recordings were made on strips of murine detrusor and intracellular Ca2+ measurements were made on isolated detrusor myocytes using confocal microscopy. Transcriptional expression of TRPC and IP3R subtypes in intact detrusor strips and isolated detrusor myocytes was assessed using reverse transcriptase-polymerase chain reaction. Cholinergic stimulation of the detrusor induced by electrical field stimulation or exogenous application of carbachol or neostigmine evoked contractions consisting of a transient plus a tonic response, which was blocked by ML204, an inhibitor of TRPC4 channels. A phasic oscillatory component was blocked by the IP3R inhibitor 2-APB. Carbachol evoked reproducible Ca2+ responses in isolated detrusor myocytes, consisting of an initial Ca2+ transient followed by Ca2+ oscillations. ML204 inhibited the initial Ca2+ transient whereas 2-APB inhibited the Ca2+ oscillations. Reverse transcriptase-polymerase chain reaction experiments showed that TRPC4β, TRPC6 and IP3R1 were selectively expressed in isolated detrusor myocytes. Control experiments demonstrated that ML204 did not affect L-type Ca2+ or BK current amplitude, caffeine induced Ca2+ transients or KCl induced contractions of the detrusor. Muscarinic receptor mediated contractions of the detrusor involve the activation of TRPC4β channels.

Increased expression of TRPC5 in cortical lesions of the focal cortical dysplasia.

Focal cortical dysplasias (FCDs) are frequently associated with the medical refractory epilepsy in both children and adults. Transient receptor potential canonical channel 5 (TRPC5), a receptor-operated cation channel, has been well recognized as a regulator in the central nervous system. Here, we examined the expression and cellular distribution of TRPC5 in the specimens from patients with FCDIa (n = 14), FCDIIa (n = 12), and FCDIIb (n = 12) compared with the age-matched control cortex (CTX). TRPC5 mRNA and protein levels were significantly higher in FCDs compared with CTX. Immunohistochemical data showed that TRPC5 was strongly expressed in the misshapen cells, particularly in neuronal microcolumns, dysmorphic neurons, and balloon cells. Moreover, the double-label immunofluorescence analyses demonstrated that TRPC5 localized on NeuN-positive neurons. In addition, its co-localization with glutamate and gamma-aminobutyric acid (GABA) indicated that TRPC5 was distributed on both glutamatergic and GABAergic neurons. Taken together, these results suggested that increased expression of TRPC5 in FCDs and the cell-specific distribution patterns of TRPC5 in the misshapen neurons in FCDs could potentially contribute to the epileptogenesis of FCDs.

Evidence for M2 and M3 Muscarinic Receptor Involvement in Cholinergic Excitatory Junction Potentials Through Synergistic Activation of Cation Channels in the Longitudinal Muscle of Mouse Ileum

Cholinergic nerve–mediated excitatory junction potentials (EJPs) in the longitudinal muscle of mouse ileum were characterized by using M2 or M3 muscarinic receptor–knockout (KO) mice and 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) and pertussis toxin (PTX). EJPs evoked by electrical field stimulation (EFS) in wild-type preparations, initially determined to be cholinergic in origin using tetrodotoxin, atropine, and eserine, were profoundly depressed after SK&F 96365 treatment known to block muscarinic receptor–operated cation channels. A similar depression of the EJPs was also observed by PTX treatment, which is predicted to disrupt M2-mediated pathways linked to cation channel activation. In M2-KO mouse preparations, cholinergic EJPs were evoked by EFS with their relative amplitude of 20% – 30% to the wild-type EJP and strongly inhibited by SK&F 96365. No cholinergic EJP was seen in M3-KO as well as M2/M3 double-KO preparations. The results suggest that the wild-type cholinergic EJP is not a simple mixture of M2 and M3 responses, but due to synergistic activation of cation channels by both M2 and M3 receptors in the murine ileal longitudinal muscle.

Manganese inhibits ATP-induced calcium entry through the transient receptor potential channel TRPC3 in astrocytes

Chronic exposure to elevated levels of manganese (Mn2+) causes neuronal injury and inflammatory activation of glia. Astrocytes selectively accumulate Mn2+, which inhibits mitochondrial respiration and increases production of reactive oxygen species. We previously reported that sub-acute exposure to low micromolar levels of Mn2+ in primary astrocytes inhibited ATP-induced calcium (Ca2+) signaling, associated with decreased levels of endoplasmic reticulum Ca2+ and increased mitochondrial Ca2+ loads. In the present studies, we postulated that the mechanism underlying the capacity of Mn2+ to inhibit these purinergic signals in astrocytes could be due to competition with Ca2+ for entry through a plasma membrane channel. These data demonstrate that acutely applied Mn2+ rapidly inhibited ATP-induced Ca2+ waves and transients in primary striatal astrocytes. Mn2+ also decreased influx of extracellular Ca2+ induced by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a direct activator of the transient receptor potential channel, TRPC3. The TRPC3 inhibitor, pyrazole-3, prevented ATP- and OAG-dependent transport of Mn2+ from extracellular stores, demonstrated by a dramatic reduction in the rate of fluorescence quenching of Fura-2. These data indicate that Mn2+ can acutely inhibit ATP-dependent Ca2+ signaling in astrocytes by blocking Ca2+ entry through the receptor-operated cation channel, TRPC3. Loss of normal astrocytic responses to purinergic signals due to accumulation of Mn2+ could therefore comprise critical homeostatic functions necessary for metabolic and trophic support of neurons.

Heteromeric Canonical Transient Receptor Potential 1 and 4 Channels Play a Critical Role in Epileptiform Burst Firing and Seizure-Induced Neurodegeneration

Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity.

Cilostazol Suppresses Angiotensin II–Induced Vasoconstriction via Protein Kinase A–Mediated Phosphorylation of the Transient Receptor Potential Canonical 6 Channel

The goal of this study was to determine whether inhibition of transient receptor potential canonical (TRPC) channels underlies attenuation of angiotensin II (Ang II)-induced vasoconstriction by phosphodiesterase (PDE) 3 inhibition. Pretreatment of rat thoracic aorta with cilostazol, a selective PDE3 inhibitor, suppressed vasoconstriction induced by Ang II but not that induced by KCl. The Ang II-induced contraction was largely dependent on Ca(2+) influx via receptor-operated cation channels. Cilostazol specifically suppressed diacylglycerol-activated TRPC channels (TRPC3/TRPC6/TRPC7) through protein kinase A (PKA)-dependent phosphorylation of TRPC channels in HEK293 cells. In contrast, we found that phosphorylation of TRPC6 at Thr69 was essential for the suppression of Ang II-induced Ca(2+) influx by PDE3 inhibition in rat aortic smooth muscle cells (RAoSMCs). Cilostazol specifically induced phosphorylation of endogenous TRPC6 at Thr69. The endogenous TRPC6, but not TRPC3, formed a ternary complex with PDE3 and PKA in RAoSMCs, suggesting the specificity of TRPC6 phosphorylation by PDE3 inhibition. Furthermore, inhibition of PDE3 suppressed the Ang II-induced contraction of reconstituted ring with RAoSMCs, which were abolished by the expression of a phosphorylation-deficient mutant of TRPC6. PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II.

Attenuation of store‐operated Ca2+ entry and enhanced expression of TRPC channels in caudal artery smooth muscle from Type 2 diabetic Goto‐Kakizaki rats

1. Previously, we found that Ca(2+) entry from the extracellular space via alpha(1)-adrenoceptor-activated, Ca(2+)-permeable channels, but not voltage-gated Ca(2+) channels, is impaired in endothelium-denuded caudal artery smooth muscle from Type 2 diabetic Goto-Kakizaki (GK) rats. In the present study, we investigated the impairment of Ca(2+) entry mechanisms via Ca(2+)-permeable channels from the extracellular space in response to alpha(1)-adrenoceptor stimulation (cirazoline) in endothelium-denuded caudal artery strips isolated from GK rats. 2. The contraction of caudal artery strips from GK rats in response to the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (10 micromol/L), which causes depletion of Ca(2+) stores and subsequent store-operated Ca(2+) (SOC) entry, was significantly depressed compared with that of Wistar rats (maximal force 0.023 +/- 0.004 vs 0.058 +/- 0.005 mN/mg tissue wet weight, respectively). These results suggest that receptor-activated Ca(2+) entry through SOC channels is impaired in caudal artery smooth muscle in GK rats. 3. The classic transient receptor potential (TRPC) channels, which constitute SOC and receptor-operated cation channels, play an important role in Ca(2+) regulation. Therefore, we investigated the mRNA and protein expression of TRPC channels in caudal artery smooth muscle from Wistar and GK rats using reverse transcription-polymerase chain reaction and immunoblotting. 4. Expression of TRPC1, TRPC3 and TRPC6 mRNA and protein was found in Wistar rats. However, in GK rats, in addition to the expression of these TRPC channels, mRNA and protein expression of TRPC4 was found. The expression of TRPC1 and TRPC6, but not TRPC3, was increased approximately twofold in GK rats compared with Wistar rats. 5. These results suggest that changes in TRPC channel expression may be responsible, in part, for the dysfunction of receptor-mediated Ca(2+) entry in caudal artery smooth muscle of GK rats.

Characterization of receptor‐ and store‐operated cation currents in rat pulmonary arterial smooth muscle cells (PASMCs)

Store‐ and receptor‐operated cation channels (SOC and ROC) in PASMCs have been implicated to play important roles in many physiological functions, including hypoxia‐ and agonist‐induced vasoconstriction, and pulmonary hypertension. Here we characterized the SOC and ROC currents in rat intralobar PASMCs. PASMCs were whole‐cell voltage‐clamped. K+ and Ca2+ currents were eliminated by substituting K+ with Cs+, and by nifedipine. Depletion of SR Ca2+ stores with cyclopiazonic acid activated a small inward current at −60 mV. The current was elicited with a time‐delay, exhibited typical bimodal dependence to extracellular Ca2+ due to the effect of anomalous mole fraction, and was blocked by La3+ or substitution of Na+ with N‐methyl‐D‐glucamine. I‐V relationship of La3+‐sensitive SOC current was quasi‐linear with a reverse potential close to 0 mM, indicative of its non‐selective nature. ROC agonist 1‐oleoyl‐2‐acetyl‐sn‐glycerol also activated a La3+ (1 mM) sensitive non‐selective cation current with slight outward rectification. Moreover, ROC currents recorded in PASMCs of chronic hypoxic rats (10% O2, 4 weeks) were significantly larger than those of normoxic rats. These results are consistent with the notion that functional SOC and ROC are present in PASMCs and their activities are up‐regulated in hypoxic pulmonary hypertension. (Supported by NIH R01HL074134, NSFC 30670772 and NSF(Fujian) C0620002)

Enhancement of receptor-operated cation current and TRPC6 expression in arterial smooth muscle cells of deoxycorticosterone acetate-salt hypertensive rats

In deoxycorticosterone acetate (DOCA)-salt hypertensive rats, altered reactivity of blood vessels to vasoactive agonists is frequently associated with an elevation in blood pressure. Canonical transient receptor potential (TRPC) channels are believed to encode receptor-operated cation channels (ROC), the activation of which is involved in smooth muscle depolarization and vasoconstriction. The aims of the present study were to investigate whether the ROC current is increased in DOCA-hypertensive rats and determine whether aldosterone directly enhances the expression of TRPC. The nystatin-perforated patch-clamp technique was used for the recording of receptor-stimulated ion currents in mesenteric arterial smooth muscle cells, which were enzymatically dispersed from sham-operated and DOCA-salt hypertensive rats. Expressions of TRPCs were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blot analysis. Receptor-stimulated currents activated by 5-hydroxytryptamine (serotonin) and norepinephrine were increased significantly in the mesenteric arterial smooth muscle cells of DOCA-salt hypertensive rats compared to sham-operated rats. Ion-substitution experiments revealed that the enhanced currents were cation currents (ROC currents). Enhanced expression of TRPC6 in mesenteric arteries from DOCA-salt hypertensive rats was demonstrated by real-time RT-PCR. Up-regulation of TRPC6 by aldosterone treatment in vitro was also observed in A7r5 cells by RT-PCR and in western blots. These results suggest that aldosterone enhances TRPC6 expression and ROC currents in vascular smooth muscle cells, and that this may in turn contribute to altered vascular reactivity and to hypertension.

The reverse mode of the Na+/Ca2+ exchanger provides a source of Ca2+ for store refilling following agonist-induced Ca2+ mobilization

Agonist-induced contraction of airway smooth muscle (ASM) can be triggered by an elevation in the intracellular Ca(2+) concentration, primarily through the release of Ca(2+) from the sarcoplasmic reticulum (SR). The refilling of the SR is integral for subsequent contractions. It has been suggested that Ca(2+) entry via store-operated cation (SOC) and receptor-operated cation channels may facilitate refilling of the SR. Indeed, depletion of the SR activates substantial inward SOC currents in ASM that are composed of both Ca(2+) and Na(+). Accumulation of Na(+) within the cell may regulate Ca(2+) handling in ASM by forcing the Na(+)/Ca(2+) exchanger (NCX) into the reverse mode, leading to the influx of Ca(2+) from the extracellular domain. Since depletion of the SR activates substantial inward Na(+) current, it is conceivable that the reverse mode of the NCX may contribute to the intracellular Ca(2+) pool from which the SR is refilled. Indeed, successive contractions of bovine ASM, evoked by various agonists (ACh, histamine, 5-HT, caffeine) were significantly reduced upon removal of extracellular Na(+); whereas contractions evoked by KCl were unchanged by Na(+) depletion. Ouabain, a selective inhibitor of the Na(+)/K(+) pump, had no effect on the reductions observed under normal and zero-Na(+) conditions. KB-R7943, a selective inhibitor of the reverse mode of the NCX, significantly reduced successive contractions induced by all agonists without altering KCl responses. Furthermore, KB-R7943 abolished successive caffeine-induced Ca(2+) transients in single ASM cells. Together, these data suggest a role for the reverse mode of the NCX in refilling the SR in ASM following Ca(2+) mobilization.