The Role of TRPM4 in Cardiac Electrophysiology and Arrhythmogenesis.

The transient receptor potential melastatin 4 (TRPM4) channel is a non-selective cation channel that activates in response to increased intracellular Ca2+ levels but does not allow Ca2+ to pass through directly. It plays a crucial role in regulating diverse cellular functions associated with intracellular Ca2+ homeostasis/dynamics. TRPM4 is widely expressed in the heart and is involved in various physiological and pathological processes therein. Specifically, it has a significant impact on the electrical activity of cardiomyocytes by depolarizing the membrane, presumably via Na+ loading. The TRPM4 channel likely contributes to the development of cardiac arrhythmias associated with specific genetic backgrounds and cardiac remodeling. This short review aims to overview what is known so far about the TRPM4 channel in cardiac electrophysiology and arrhythmogenesis, highlighting its potential as a novel therapeutic target to effectively prevent and treat cardiac arrhythmias.

TRP channels in cardiac and intestinal fibrosis.

It is now widely accepted that advanced fibrosis underlies many chronic inflammatory disorders and is the main cause of morbidity and mortality of the modern world. The pathogenic mechanism of advanced fibrosis involves diverse and intricate interplays between numerous extracellular and intracellular signaling molecules, among which the non-trivial roles of a stress-responsive Ca2+/Na+-permeable cation channel superfamily, the transient receptor potential (TRP) protein, are receiving growing attention. Available evidence suggests that several TRP channels such as TRPC3, TRPC6, TRPV1, TRPV3, TRPV4, TRPA1, TRPM6 and TRPM7 may play central roles in the progression and/or prevention of fibroproliferative disorders in vital visceral organs such as lung, heart, liver, kidney, and bowel as well as brain, blood vessels and skin, and may contribute to both acute and chronic inflammatory processes involved therein. This short paper overviews the current knowledge accumulated in this rapidly growing field, with particular focus on cardiac and intestinal fibrosis, which are tightly associated with the pathogenesis of atrial fibrillation and inflammatory bowel diseases such as Crohn's disease.

Pathological activation of CaMKII induces arrhythmogenicity through TRPM4 overactivation.

TRPM4 is a Ca2+-activated nonselective cation channel involved in cardiovascular physiology and pathophysiology. Based on cellular experiments and numerical simulations, the present study aimed to explore the potential arrhythmogenicity of CaMKII-mediated TRPM4 channel overactivation linked to Ca2+ dysregulation in the heart. The confocal immunofluorescence microscopy, western blot, and proximity ligation assay (PLA) in HL-1 atrial cardiomyocytes and/or TRPM4-expressing TSA201 cells suggested that TRPM4 and CaMKII proteins are closely localized. Co-expression of TRPM4 and CaMKIIδ or a FRET-based sensor Camui in HEK293 cells showed that the extent of TRPM4 channel activation was correlated with that of CaMKII activity, suggesting their functional interaction. Both expressions and interaction of the two proteins were greatly enhanced by angiotensin II treatment, which induced early afterdepolarizations (EADs) at the repolarization phase of action potentials (APs) recorded from HL-1 cells by the current clamp mode of patch clamp technique. This arrhythmic change disappeared after treatment with the TRPM4 channel blocker 9-phenanthrol or CaMKII inhibitor KN-62. In order to quantitatively assess how CaMKII modulates the gating behavior of TRPM4 channel, the ionomycin-permeabilized cell-attached recording was employed to obtain the voltage-dependent parameters such as steady-state open probability and time constants for activation/deactivation at different [Ca2+]i. Numerical simulations incorporating these kinetic data into a modified HL-1 model indicated that > 3-fold increase in TRPM4 current density induces EADs at the late repolarization phase and CaMKII inhibition (by KN-62) completely eliminates them. These results collectively suggest a novel arrhythmogenic mechanism involving excessive CaMKII activity that causes TRPM4 overactivation in the stressed heart.

Theoretical Investigation of the Mechanism by which A Gain-of-Function Mutation of the TRPM4 Channel Causes Conduction Block.

In the heart, TRPM4 is most abundantly distributed in the conduction system. Previously, a single mutation, 'E7K', was identified in its distal N-terminus to cause conduction disorder because of enhanced cell-surface expression. It remains, however, unclear how this expression increase leads to conduction failure rather than abnormally enhanced cardiac excitability. To address this issue theoretically, we mathematically formulated the gating kinetics of the E7K-mutant TRPM4 channel by a combined use of voltage jump analysis and ionomycin-perforated cell-attached recording technique and incorporated the resultant rate constants of opening and closing into a human Purkinje fiber single-cell action potential (AP) model (Trovato model) to perform 1D-cable simulations. The results from TRPM4 expressing HEK293 cells showed that as compared with the wild-type, the open state is much preferred in the E7K mutant with increased voltage-and Ca2+-sensitivities. These theoretical predictions were confirmed by power spectrum and single channel analyses of expressed wild-type and E7K-mutant TRPM4 channels. In our modified Trovato model, the facilitated opening of the E7K mutant channel markedly prolonged AP duration with concomitant depolarizing shifts of the resting membrane potential in a manner dependent on the channel density (or maximal activity). This was, however, little evident in the wild-type TRPM4 channel. Moreover, 1D-cable simulations with the modified Trovato model revealed that increasing the density of E7K (but not of wild-type) TRPM4 channels progressively reduced AP conduction velocity eventually culminating in complete conduction block. These results clearly suggest the brady-arrhythmogenicity of the E7K mutant channel which likely results from its pathologically enhanced activity.

Eicosapentaenoic acid ameliorates pulmonary hypertension via inhibition of tyrosine kinase Fyn.

Pulmonary arterial hypertension (PAH) is a multifactorial disease characterized by pulmonary arterial vasoconstriction and remodeling. Src family tyrosine kinases, including Fyn, play critical roles in vascular remodeling via the inhibition of STAT3 signaling. EPA is known to inhibit Fyn kinase activity. This study investigated the therapeutic potential and underlying mechanisms of EPA and its metabolite, resolvin E1 (RvE1), to treat PAH using monocrotaline-induced PAH model rats (MCT-PAH), human pulmonary artery endothelial cells (HPAECs), and human pulmonary artery smooth muscle cells (HPASMCs). Administration of EPA 1 and 2 weeks after MCT injection both ameliorated right ventricular hypertrophy, remodeling and dysfunction, and medial wall thickening of the pulmonary arteries and prolonged survival in MCT-PAH rats. EPA attenuated the enhanced contractile response to 5-hydroxytryptamine in isolated pulmonary arteries of MCT-PAH rats. Mechanistically, the treatment with EPA and RvE1 or the introduction of dominant-negative Fyn prevented TGF-ß2-induced endothelial-to-mesenchymal transition and IL-6-induced phosphorylation of STAT3 in cultured HPAECs. EPA and RvE1 suppressed Src family kinases' activity as evaluated by their phosphorylation status in cultured HPAECs and HPASMCs. EPA and RvE1 suppressed vasocontraction of rat and human PA. Furthermore, EPA and RvE1 inhibited the enhanced proliferation and activity of Src family kinases in HPASMCs derived from patients with idiopathic PAH. EPA ameliorated PAH's pathophysiology by mitigating vascular remodeling and vasoconstriction, probably inhibiting Src family kinases, especially Fyn. Thus, EPA is considered a potent therapeutic agent for the treatment of PAH.

SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation.

Cancer is currently one of the major causes of death in patients with type 2 diabetes mellitus. We previously reported the beneficial effects of the glucagon-like peptide-1 receptor agonist exendin-4 against prostate and breast cancer. In the present study, we examined the anti-cancer effect of the sodium-glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin using a breast cancer model. In human breast cancer MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. Ipragliflozin at 1-50 µM significantly and dose-dependently suppressed the growth of MCF-7 cells. BrdU assay also revealed that ipragliflozin attenuated the proliferation of MCF-7 cells in a dose-dependent manner. Because the effect of ipragliflozin against breast cancer cells was completely canceled by knocking down SGLT2, ipragliflozin could act via inhibiting SGLT2. We next measured membrane potential and whole-cell current using the patch clamp technique. When we treated MCF-7 cells with ipragliflozin or glucose-free medium, membrane hyperpolarization was observed. In addition, glucose-free medium and knockdown of SGLT2 by siRNA suppressed the glucose-induced whole-cell current of MCF-7 cells, suggesting that ipragliflozin inhibits sodium and glucose cotransport through SGLT2. Furthermore, JC-1 green fluorescence was significantly increased by ipragliflozin, suggesting the change of mitochondrial membrane potential. These findings suggest that the SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation via membrane hyperpolarization and mitochondrial membrane instability.

Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes.

BACKGROUND: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved. METHODS: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes. RESULTS: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton. CONCLUSIONS: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.

Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.

The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.

Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds.

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a new class of synthetic neurotrophic drugs.

Distinct pharmacological and molecular properties of the acid-sensitive outwardly rectifying (ASOR) anion channel from those of the volume-sensitive outwardly rectifying (VSOR) anion channel.

Expressed by many cell types, acid-sensitive outwardly rectifying (ASOR) anion channels are known to be activated by extracellular acidification and involved in acidotoxic necrotic cell death. In contrast, ubiquitously expressed volume-sensitive outwardly rectifying (VSOR) anion channels are activated by osmotic cell swelling and involved in cell volume regulation and apoptotic cell death. Distinct inhibitors to distinguish ASOR from VSOR anion channels have not been identified. Although leucine-rich repeats containing 8A (LRRC8A) was recently found to be an essential component of VSOR anion channels, the possibility of an LRRC8 family member serving as a component of ASOR anion channels has not been examined. In this study, we explored the effects of 12 known VSOR channel inhibitors and small interfering RNA (siRNA)-mediated knockdown of LRRC8 family members on ASOR and VSOR currents in HeLa cells. Among these inhibitors, eight putative VSOR blockers, including 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), were totally ineffective at blocking ASOR channel activity, whereas suramin, R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] acetic acid (DIOA), arachidonic acid, and niflumic acid were found to be effective ASOR anion channel antagonists. In addition, gene-silencing studies showed that no LRRC8 family members are essentially involved in ASOR anion channel activity, whereas LRRC8A is involved in VSOR anion channel activity in HeLa cells.

Brain-derived neurotrophic factor (BDNF) induces sustained intracellular Ca2+ elevation through the up-regulation of surface transient receptor potential 3 (TRPC3) channels in rodent microglia.

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca(2+) elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.

New experimental trends for phosphoinositides research on ion transporter/channel regulation.

Phosphoinositides(4,5)-bisphosphates [PI(4,5)P2] critically controls membrane excitability, the disruption of which leads to pathophysiological states. PI(4,5)P2 plays a primary role in regulating the conduction and gating properties of ion channels/transporters, through electrostatic and hydrophobic interactions that allow direct associations. In recent years, the development of many molecular tools have brought deep insights into the mechanisms underlying PI(4,5)P2-mediated regulation. This review summarizes the methods currently available to manipulate the cell membrane PI(4,5)P2 level including pharmacological interventions as well as newly designed molecular tools. We concisely introduce materials and experimental designs suitable for the study of PI(4,5)P2-mediated regulation of ion-conducting molecules, in order to assist researchers who are interested in this area. It is our further hope that the knowledge introduced in this review will help to promote our understanding about the pathology of diseases such as cardiac arrhythmias, bipolar disorders, and Alzheimer's disease which are somehow associated with a disruption of PI(4,5)P2 metabolism.

Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II.

The molecular mechanism underlying Ca(2+)/calmodulin (CaM)-dependent kinase II (CaMKII)-mediated regulation of the mouse transient receptor potential channel TRPC6 was explored by chimera, deletion and site-directed mutagenesis approaches. Induction of currents (ICCh) in TRPC6-expressing HEK293 cells by a muscarinic agonist carbachol (CCh; 100 µm) was strongly attenuated by a CaMKII-specific peptide, autocamtide-2-related inhibitory peptide (AIP; 10 µm). TRPC6/C7 chimera experiments showed that the TRPC6 C-terminal sequence is indispensable for ICCh to be sensitive to AIP-induced CaMKII inhibition. Further, deletion of a distal region (Gln(855)-Glu(877)) of the C-terminal CaM/inositol-1,4,5-trisphosphate receptor binding domain (CIRB) of TRPC6 was sufficient to abolish ICCh. Systematic alanine scanning for potential CaMKII phosphorylation sites revealed that Thr(487) was solely responsible for the activation of the TRPC6 channel by receptor stimulation. The abrogating effect of the alanine mutation of Thr(487) (T487A) was reproduced with other non-polar amino acids, namely glutamine or asparagine, while being partially rescued by phosphomimetic mutations with glutamate or aspartate. The cellular expression and distribution of TRPC6 channels did not significantly change with these mutations. Electrophysiological and immunocytochemical data with the Myc-tagged TRPC6 channel indicated that Thr(487) is most likely located at the intracellular side of the cell membrane. Overexpression of T487A caused significant reduction of endogenous TRPC6-like current induced by Arg(8)-vasopressin in A7r5 aortic myocytes. Based on these results, we propose that the optimal spatial arrangement of a C-terminal domain (presumably the distal CIRB region) around a single CaMKII phosphorylation site Thr(487) may be essential for CaMKII-mediated regulation of TRPC6 channels. This mechanism may be of physiological significance in a native environment such as in vascular smooth muscle cells.

A self-limiting regulation of vasoconstrictor-activated TRPC3/C6/C7 channels coupled to PI(4,5)P2-diacylglycerol signalling.

Activation of transient receptor potential (TRP) canonical TRPC3/C6/C7 channels by diacylglycerol (DAG) upon stimulation of phospholipase C (PLC)-coupled receptors results in the breakdown of phosphoinositides (PIPs). The critical importance of PIPs to various ion-transporting molecules is well documented, but their function in relation to TRPC3/C6/C7 channels remains controversial. By using an ectopic voltage-sensing PIP phosphatase (DrVSP), we found that dephosphorylation of PIPs robustly inhibits currents induced by carbachol (CCh), 1-oleolyl-2-acetyl-sn-glycerol (OAG) or RHC80267 in TRPC3, TRPC6 and TRPC7 channels, though the strength of the DrVSP-mediated inhibition (VMI) varied among the channels with a rank order of C7>C6>C3. Pharmacological and molecular interventions suggest that depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is most likely the critical event for VMI in all three channels.When the PLC catalytic signal was vigorously activated through overexpression of the muscarinic type-I receptor (M1R), the inactivation of macroscopic TRPC currents was greatly accelerated in the same rank order as the VMI, and VMI of these currents was attenuated or lost. VMI was also rarely detected in vasopressin-induced TRPC6-like currents inA7r5 vascular smooth muscle cells, indicating that the inactivation by PI(4,5)P2 depletion underlies the physiological condition. Simultaneous fluorescence resonance energy transfer (FRET)-based measurement of PI(4,5)P2 levels and TRPC6 currents confirmed that VMI magnitude reflects the degree of PI(4,5)P2 depletion. These results demonstrate that TRPC3/C6/C7 channels are differentially regulated by depletion of PI(4,5)P2, and that the bimodal signal produced by PLC activation controls these channels in a self-limiting manner.

BKCa and KV channels limit conducted vasomotor responses in rat mesenteric terminal arterioles.

Intracellular Ca(2+) signals underlying conducted vasoconstriction to local application of a brief depolarizing KCl stimulus was investigated in rat mesenteric terminal arterioles (<40 µm). Using a computer model of an arteriole segment comprised of coupled endothelial cells (EC) and vascular smooth muscle cells (VSMC) simulations of both membrane potential and intracellular [Ca(2+)] were performed. The "characteristic" length constant, λ, was approximated using a modified cable equation in both experiments and simulations. We hypothesized that K(+) conductance in the arteriolar wall limit the electrotonic spread of a local depolarization along arterioles by current dissipation across the VSMC plasma membrane. Thus, we anticipated an increased λ by inhibition of voltage-activated K(+) channels. Application of the BK(Ca) channel blocker iberiotoxin (100 nM) onto mesenteric arterioles in vitro and inhibition of BK(Ca) channel current in silico increased λ by 34% and 32%, respectively. Similarly, inhibition of K(V) channels in vitro (4-aminopyridine, 1 mM) or in silico increased λ by 41% and 21%, respectively. Immunofluorescence microscopy demonstrated expression of BK(Ca), Kv1.5, Kv2.1, but not Kv1.2, in VSMCs of rat mesenteric terminal arterioles. Our results demonstrate that inhibition of voltage-activated K(+) channels enhance vascular-conducted responses to local depolarization in terminal arterioles by increasing the membrane resistance of VSMCs. These data contribute to our understanding of how differential expression patterns of voltage-activated K(+) channels may influence conducted vasoconstriction in small arteriolar networks. This finding is potentially relevant to understanding the compromised microcirculatory blood flow in systemic vascular diseases such as diabetes mellitus and hypertension.

Ca2+ influx and protein scaffolding via TRPC3 sustain PKCbeta and ERK activation in B cells.

Ca(2+) signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca(2+) entry activated by Ins(1,4,5)P(3)-induced Ca(2+) release from endoplasmic reticulum, the importance of DAG-activated Ca(2+) entry remains elusive. Here, we describe the physiological role of DAG-activated Ca(2+) entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cbeta (PKCbeta) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCbeta that maintained localization of PKCbeta at the PM. Thus, TRPC3 functions as both a Ca(2+)-permeable channel and a protein scaffold at the PM for downstream PKCbeta activation in B cells.

Inhibition of TRPC6 channel activity contributes to the antihypertrophic effects of natriuretic peptides-guanylyl cyclase-A signaling in the heart.

RATIONALE: Atrial and brain natriuretic peptides (ANP and BNP, respectively) exert antihypertrophic effects in the heart via their common receptor, guanylyl cyclase (GC)-A, which catalyzes the synthesis of cGMP, leading to activation of protein kinase (PK)G. Still, much of the network of molecular mediators via which ANP/BNP-GC-A signaling inhibit cardiac hypertrophy remains to be characterized. OBJECTIVE: We investigated the effect of ANP-GC-A signaling on transient receptor potential subfamily C (TRPC)6, a receptor-operated Ca(2+) channel known to positively regulate prohypertrophic calcineurin-nuclear factor of activated T cells (NFAT) signaling. METHODS AND RESULTS: In cardiac myocytes, ANP induced phosphorylation of TRPC6 at threonine 69, the PKG phosphorylation site, and significantly inhibited agonist-evoked NFAT activation and Ca(2+) influx, whereas in HEK293 cells, it dramatically inhibited agonist-evoked TRPC6 channel activity. These inhibitory effects of ANP were abolished in the presence of specific PKG inhibitors or by substituting an alanine for threonine 69 in TRPC6. In model mice lacking GC-A, the calcineurin-NFAT pathway is constitutively activated, and BTP2, a selective TRPC channel blocker, significantly attenuated the cardiac hypertrophy otherwise seen. Conversely, overexpression of TRPC6 in mice lacking GC-A exacerbated cardiac hypertrophy. BTP2 also significantly inhibited angiotensin II-induced cardiac hypertrophy in mice. CONCLUSIONS: Collectively, these findings suggest that TRPC6 is a critical target of antihypertrophic effects elicited via the cardiac ANP/BNP-GC-A pathway and suggest TRPC6 blockade could be an effective therapeutic strategy for preventing pathological cardiac remodeling.

Cilostazol suppresses angiotensin II-induced vasoconstriction via protein kinase A-mediated phosphorylation of the transient receptor potential canonical 6 channel.

OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II.

Potential of the TRPM7 channel as a novel therapeutic target for pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is an intractable vascular disease characterized by a progressive increase in pulmonary vascular resistance caused by pulmonary vascular remodeling, which ultimately leads to right-sided heart failure. PAH remains incurable, despite the development of PAH-targeted therapeutics centered on pulmonary artery relaxants. It is necessary to identify the target molecules that contribute to pulmonary artery remodeling. Transient receptor potential (TRP) channels have been suggested to modulate pulmonary artery remodeling. Our study focused on the transient receptor potential ion channel subfamily M, member 7, or the TRPM7 channel, which modulates endothelial-to-mesenchymal transition and smooth muscle proliferation in the pulmonary artery. In this review, we summarize the role and expression profile of TRPM7 channels in PAH progression and discuss TRPM7 channels as possible therapeutic targets. In addition, we discuss the therapeutic effect of a Chinese herbal medicine, Ophiocordyceps sinensis (OCS), on PAH progression, which partly involves TRPM7 inhibition.

Critical contributions of pre-S1 shoulder and distal TRP box in DAG-activated TRPC6 channel by PIP2 regulation.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) regulates the activities of numerous membrane proteins, including diacylglycerol(DAG)-activated TRPC3/6/7 channels. Although PIP2 binding is known to support DAG-activated TRP channel activity, its binding site remains unknown. We screened for PIP2 binding sites within TRPC6 channels through extensive mutagenesis. Using voltage-sensitive phosphatase (DrVSP), we found that Arg437 and Lys442, located in the channel's pre-S1 domain/shoulder, are crucial for interaction with PIP2. To gain structural insights, we conducted computer protein-ligand docking simulations with the pre-S1 domain/shoulder of TRPC6 channels. Further, the functional significance of PIP2 binding to the pre-S1 shoulder was assessed for receptor-operated channel functions, cross-reactivity to DAG activation, and the kinetic model simulation. These results revealed that basic residues in the pre-S1 domain/shoulder play a central role in the regulation of PIP2-dependent gating. In addition, neutralizing mutation of K771 in the distal TRP box reversed the effect of PIP2 depletion from inhibiting to potentiating channel activity. A similar effect was seen in TRPV1 channels, which suggests that TRPC6 possesses a common but robust polarity switch mediating the PIP2-dependent effect. Overall, these mutagenesis studies reveal functional and structural insights for how basic residues and channel segments in TRP channels are controlled through phosphoinositides recognition.