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Key points • Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) plays pivotal roles in diverse Ca2+-mediated cellular functions including the physiology/pathophysiology of the cardiovascular system, through modulation of a variety of Ca2+-permeable channels such as a non-voltage-gated Ca2+ channel TRPC6. • In this study, we investigated the molecular mechanism underlying its positive regulation by CaMKII with chimera, deletion and site-directed mutagenesis approaches. • The results indicate that two spatially separated sites of TRPC6 channel, i.e. a distal part of the C-terminal inositol-1,4,5-trisphosphate receptor/CaM binding domain and Thr487 located on the putative first intracellular loop, are crucial for the CaMKII-mediated regulation of TRPC6 channels. • This mechanism may serve as an effective positive feedback regulation of Ca2+ influx through TRPC6 channels, in concert with intracellular and transmembrane Ca2+ mobilization upon phospholipase C-coupled receptor stimulation by neurohormonal factors, thereby fine-tuning the cardiovascular functions. • Disruption of these could lead to pathological states such as cardiac hypertrophy and arrhythmia, hypertension and atherosclerosis. Abstract The molecular mechanism underlying Ca2+/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 (Gln855–Glu877) 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 Thr487 was solely responsible for the activation of the TRPC6 channel by receptor stimulation. The abrogating effect of the alanine mutation of Thr487 (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 Thr487 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 Arg8-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 Thr487 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.

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.

We investigated the inhibitory role of the nitric oxide (NO)–cGMP–protein kinase G (PKG) pathway on receptor-activated TRPC6 channels in both a heterologous expression system (HEK293 cells) and A7r5 vascular myocytes. Cationic currents due to TRPC6 expression were strongly suppressed (by ∼70%) by a NO donor SNAP (100 μm) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 μm) or after G-protein activation with intracellular perfusion of GTPγS (100 μm). A similar extent of suppression was also observed with a membrane-permeable analogue of cGMP, 8Br-cGMP (100 μm). The inhibitory effects of SNAP and 8Br-cGMP on TRPC6 channel currents were strongly attenuated by the presence of inhibitors for guanylyl cyclase and PKG such as ODQ, KT5823 and DT3. Alanine substitution for the PKG phosphorylation candidate site at T69 but not at other sites (T14A, S28A, T193A, S321A) of TRPC6 similarly attenuated the inhibitory effects of SNAP and 8Br-cGMP. SNAP also significantly reduced single TRPC6 channel activity recorded in the inside-out configuration in a PKG-dependent manner. SNAP-induced PKG activation stimulated the incorporation of 32P into wild-type and S321A-mutant TRPC6 proteins immunoprecipitated by TRPC6-specific antibody, but this was greatly attenuated in the T69A mutant. SNAP or 8Br-cGMP strongly suppressed TRPC6-like cation currents and membrane depolarization evoked by Arg8-vasopressin in A7r5 myocytes. These results strongly suggest that TRPC6 channels can be negatively regulated by the NO–cGMP–PKG pathway, probably via T69 phosphorylation of the N-terminal. This mechanism may be physiologically important in vascular tissues where NO is constantly released from vascular endothelial cells or nitrergic nerves.

We investigated the receptor-mediated regulation of nifedipine-insensitive, high voltage-activated Ca(2+) currents in guinea-pig terminal mesenteric arterioles (I(mVDCC)) using the whole-cell clamp technique. Screening of various vasoactive substances revealed that ATP, histamine and substance P exert modulatory effects on I(mVDCC). The effects of ATP on I(mVDCC) after complete P2X receptor desensitization exhibited a complex concentration dependence. With 5 mM Ba(2+), ATP potentiated I(mVDCC) at low concentrations (approximately 1-100 microM), but inhibited it at higher concentrations (100 microM). The potentiating effects of ATP were abolished by suramin (100 microM) and PPADS (10 microM) and by intracellular application of GDPbetaS (500 microM), whereas a substantial part of I(mVDCC) inhibition by milimolar concentrations of ATP remained unaffected; due probably to its divalent cation chelating actions. In divalent cation-free solution, I(mVDCC) was enlarged and underwent biphasic effects by ATPgammaS and ADP, while 2-methylthio ATP (2MeSATP) exerted only inhibition, and pyrimidines such as UTP and UDP were ineffective. ATP-induced I(mVDCC) potentiation was selectively inhibited by anti-Galpha(s) antibodies or protein kinase A (PKA) inhibitory peptides and mimicked by dibutyryl cAMP. In contrast, ATP-induced inhibition was selectively inhibited by Galpha(q/11) antibodies or protein kinase C (PKC) inhibitory peptides and mimicked by PDBu. Pretreatment with pertussis toxin was ineffective. The apparent efficacy for I(mVDCC) potentiation with PKC inhibitors was: ATPgammaSATP/=ADP and for inhibition with PKA inhibitors was: 2MeSATPATPgammaSATPADP. Neither I(mVDCC) potentiation nor inhibition showed voltage dependence. These results suggest that I(mVDCC) is multi-phasically regulated by external ATP via P2Y(11)-resembling receptor/G(s)/PKA pathway, P2Y(1)-like receptor/G(q/11)/PKC pathway, and metal chelation.

1. The effects of NO donors on Ca2+-dependent Cl- currents (ICl(Ca)) were investigated in freshly isolated cat tracheal myocytes using the whole-cell patch clamp technique. 2. With nystatin-perforated whole-cell recording, carbachol (CCh, >/= 1 microM) induced a transient inward current (ICCh) with a reversal potential of about -20 mV. Activation of ICCh probably occurred through the M3 muscarinic receptor, since nanomolar concentrations of 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) greatly inhibited this current, while 11-(2-(diethylamino)methyl)-1-piperidinylacetyl)-5, 11-dihydro-6H-pyrido (2,3beta) (1,4)benzodiazepine-6-one (AF-DX 116) or pirenzepine at concentrations of up to 1 microM were almost ineffective. 3. Chloride channel/transporter blockers such as DIDS (100 microM), anthracene-9-carboxylic acid (9-AC, 100 microM) and niflumic acid (100 microM) greatly inhibited ICCh, but cation channel blockers, such as nifedipine (10 microM), Zn2+ (500 microM) or Gd3+ (500 microM), were without effect. 4. Activation of ICCh was strongly attenuated by pretreatment with ryanodine (4 microM) plus caffeine (10 mM). Addition of neomycin (1 mM) into the bath or inclusion of heparin (3 mg ml-1) in the pipette abolished a substantial part of ICCh. These results suggest that ICCh is ICl(Ca), which is activated by inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release. 5. The nitric oxide donor S-nitroso-N-acetyl penicillamine (SNAP) reduced the amplitude of ICCh dose dependently (IC50, approximately 10 microM). Similar inhibition was also exerted by other types of NO donor such as glyceryl trinitrate (GTN) and (+/-)-E-methyl-2-(E-hydroxyimitol)-5-nitro-6-methoxy-3- hexeneamide (NO-R). 6. SNAP-induced ICCh inhibition was effectively antagonized by Methylene Blue (1-100 nM), and mimicked by dibutyryl cGMP (db-cGMP) (0.5-1 mM), whereas two structurally distinct types of cGMP-dependent (G)-kinase inhibitor, N-(2-aminoethyl)-5-isoquinilinesulphonamide (H-8, 2.5 microM) and KT5823 (1 microM), failed to counteract the inhibitory effects of SNAP or db-cGMP. Another G-kinase-specific inhibitor Rp-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS; 1 microM) itself caused a marked reduction in ICCh. 7. SNAP (100 microM) or db-cGMP (100 microM) exhibited no inhibitory actions, when caffeine (10 mM) or photolytically released IP3 were used instead of CCh to activate the inward current. 8. These results suggest that inhibition of ICCh by NO donors involves a cGMP-dependent but G-kinase-independent mechanism, which may operate at a site(s) between the muscarinic (M3) and IP3 receptors.

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.

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 in A7r5 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.

1. The excitatory actions of phenylephrine (Phe) and acetylcholine (ACh) on the smooth muscle of rabbit portal vein were investigated and compared by using whole-cell and single channel configurations of the patch clamp technique, in combination with a modified concentration jump method. 2. At negative holding potentials with KCl (0.1 mM EGTA) electrodes, rapid applications of Phe (> 1 microM) and ACh (> 10 microM) both resulted in biphasic responses consisting of fast outward or inward currents and a long-lasting inward current with an increased noise level. 3. The slow inward current was still recorded when the cell was dialysed with caesium aspartate solution complemented with 10 mM EGTA, in order to eliminate contributions of calcium-dependent conductances (K+ and Cl- currents). Phe was more potent at activating the current than ACh. Ion replacement experiments revealed that both the Phe- and ACh-induced slow inward currents are cation-selective conductances (CS currents). 4. The I-V relationships of the Phe- or ACh-induced CS currents were similar both for the instantaneous peak and the steady state. The tail current analysis over a wide range of membrane potentials (-150 to +100 mV) showed that depolarizations to very positive potentials (> +50 mV) from near the resting membrane potential (-40 mV) can produce a several-fold increase in the steady-state activation of the CS currents, but the maximal activations were in most cases not observed even at +100 mV. 5. Externally applied Cd2+ produced a quick and reversible inhibition of both the Phe- and ACh-induced CS currents. This inhibition seemed almost voltage independent and the concentrations of half-inhibition were 100 and 129 microM for Phe and ACh, respectively. 6. Single channel activities were recorded in the presence of Phe or ACh using the outside-out membrane patches. The unitary conductances and reversal potentials of the Phe- and ACh-activated channels were 23 pS, +5.2 mV and 25 pS, +4.1 mV, respectively, and the open lifetimes evaluated for 50 microM Phe and 500 microM ACh were of similar order (the longer open times at -60 mV for Phe and ACh were 4.3 and 4.4 ms, respectively). In addition, the relative open probability (Po,rel) was no more than 0.2 in the voltage range of -100 to -30 mV for both 50 microM Phe and 500 microM ACh, suggesting low open probabilities of the Phe- and ACh-activated CS channels near the resting membrane potential.(ABSTRACT TRUNCATED AT 400 WORDS)

We investigated the inhibitory role of the nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway on receptor-activated TRPC6 channels in both a heterologous expression system (HEK293 cells) and A7r5 vascular myocytes. Cationic currents due to TRPC6 expression were strongly suppressed (by approximately 70%) by a NO donor SNAP (100 microm) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 microm) or after G-protein activation with intracellular perfusion of GTPgammaS (100 microm). A similar extent of suppression was also observed with a membrane-permeable analogue of cGMP, 8Br-cGMP (100 microm). The inhibitory effects of SNAP and 8Br-cGMP on TRPC6 channel currents were strongly attenuated by the presence of inhibitors for guanylyl cyclase and PKG such as ODQ, KT5823 and DT3. Alanine substitution for the PKG phosphorylation candidate site at T69 but not at other sites (T14A, S28A, T193A, S321A) of TRPC6 similarly attenuated the inhibitory effects of SNAP and 8Br-cGMP. SNAP also significantly reduced single TRPC6 channel activity recorded in the inside-out configuration in a PKG-dependent manner. SNAP-induced PKG activation stimulated the incorporation of (32)P into wild-type and S321A-mutant TRPC6 proteins immunoprecipitated by TRPC6-specific antibody, but this was greatly attenuated in the T69A mutant. SNAP or 8Br-cGMP strongly suppressed TRPC6-like cation currents and membrane depolarization evoked by Arg(8)-vasopressin in A7r5 myocytes. These results strongly suggest that TRPC6 channels can be negatively regulated by the NO-cGMP-PKG pathway, probably via T69 phosphorylation of the N-terminal. This mechanism may be physiologically important in vascular tissues where NO is constantly released from vascular endothelial cells or nitrergic nerves.

1. To investigate the distribution profile of functional inhibitory non-adrenergic non-cholinergic (i-NANC) nerves and the contribution of NO to the NANC relaxation in the cat, we studied the effects of N omega-nitro-L-arginine methyl ester (L-NAME) on NANC relaxation elicited by electrical field stimulation (EFS) in the trachea, bronchus and bronchiole. 2. EFS applied to the tracheal smooth muscle during contraction induced by 5-HT (10(-5) M) in the presence of atropine (10(-6) M) and guanethidine (10(-6) M) elicited a monophasic NANC relaxation. By contrast, NANC relaxation elicited in the peripheral airway was biphasic, comprising an initial fast followed by a second slow component and L-NAME (10(-5) M) selectively abolished the first component without affecting the second one. In the trachea, L-NAME (10(-5) M) completely suppressed the monophasic NANC relaxation when single or short repetitive stimuli (< 5) with 1 ms pulse duration were applied. However, at higher repetitive stimuli (> 10) with 1 or 4 ms pulse duration, suppression of NANC relaxation was incomplete. 3. In the small bronchi obtained from L-NAME-pretreated cats, EFS applied during contraction induced by 5-HT (10(-5) M) elicited only the slow component of NANC relaxation which is sensitive to tetrodotoxin. 4. In the peripheral airway, a newly synthesized VIP antagonist (10(-6) M) or alpha-chymotrypsin (1 U ml-1) considerably attenuated the amplitude of L-NAME-insensitive relaxation. 5. Single or repetitive EFS consistently evoked excitatory junction potentials (EJPs) in the central and peripheral airways. When tissues were exposed to atropine (10(-6) M) and guanethidine (10(-6) M), single or repetitive EFS did not alter the resting membrane potential. 6. These results indicate that at least two neurotransmitters, possibly NO or NO-containing compounds and VIP, are involved in i-NANC neurotransmission and the distribution profile of the two components differs in the central and peripheral airway of the cat.

1. The effects of nitrosocysteine (cys-NO), L-N omega-nitroarginine (L-NNA) and L-N omega-nitro-L-arginine methylester (L-NAME), oxyhaemoglobin and Methylene Blue were observed on the resting membrane potential, muscle tone and excitatory junction potentials (EJPs) of cat tracheal smooth muscle tissue. 2. Cys-NO (10(-9) to 10(-6) M) showed no effect on the resting membrane potential of smooth muscle cells of the cat trachea but it dose-dependently relaxed the tracheal tissue in the presence of 5-HT, atropine and guanethidine. 3. Electrical field stimulation (EFS) applied during contraction evoked by 5-HT in the presence of atropine and guanethidine evoked non-adrenergic, non-cholinergic (NANC) muscle relaxation. L-NNA (10(-4) M) and L-NAME (10(-4) M) completely suppressed the relaxation when single or short repetitive stimuli were applied, but suppression was incomplete with repetitive stimuli of 4 ms pulse duration applied at 20 Hz. A substantial part of the L-NNA- or L-NAME-insensitive relaxation was abolished by tetrodotoxin. 4. Cys-NO dose-dependently suppressed the EJPs without changing the resting membrane potential, and L-NNA, L-NAME, Methylene Blue and oxyhaemoglobin enhanced the amplitude of the EJP to 1.2-1.5 times the control value. 5. EJPs showed some summation when repetitive field stimulation was applied at 20 Hz. L-NNA or L-NAME enhanced the summation, and the mean slopes were increased from 0.61 +/- 0.22 to 2.0 +/- 0.3, or 1.9 +/- 0.2 mV per stimulus. Vasoactive intestinal polypeptide (VIP) antiserum and VIP antagonists further enhanced the summation in the presence of L-NNA. 6. These results indicate that NANC relaxation can be classified into two different components according to the threshold for activation, and nitric oxide is involved in one. The present results also suggest that endogenous or exogenous nitric oxide has a prejunctional action in inhibiting excitatory neuroeffector transmission in addition to a direct action on the smooth muscle cells, presumably by suppressing transmitter release from the vagus nerve.