Phosphorylation of TRPC6 Channels at Thr69 Is Required for Anti-hypertrophic Effects of Phosphodiesterase 5 Inhibition

Abstract

Activation of Ca2؉ signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca 2؉ signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-, diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca 2؉ influx in rat neonatal cardiomyocytes. influx. Substitution of Ala for Thr 69 in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce G␣ q -mediated signaling, did not affect the suppression of receptor-activated Ca 2؉ influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition.Pathological hypertrophy of the heart, induced by pressure overload, such as chronic hypertension and aortic stenosis, is a major risk factor for heart failure and cardiovascular mortality (1). Neurohumoral factors, such as norepinephrine, angiotensin II (Ang II), 2 and endothelin-1 (ET-1), and mechanical stress are believed to be prominent contributors for pressure overloadinduced cardiac hypertrophy (2, (9). A partial depolarization of plasma membrane by receptor stimulation is reported to increase the frequency of Ca 2ϩ oscillations, leading to activation of nuclear factor of activated T cells (NFAT), a transcription factor that is predominantly regulated by calcineurin (10). Recent reports have indicated that transient receptor potential canonical (TRPC) subfamily proteins play an essential role in agonistinduced membrane depolarization (11, 12). The relevance of TRPC channels to pathological hypertrophy is underscored by the observations that heart-targeted transgenic mice expressing TRPC channels caused hypertrophy (13,14) and that TRPC proteins were up-regulated in hypertrophied and failing hearts (14 -17). Among seven TRPC subfamilies, increased channel activities of TRPC1, TRPC3, and TRPC6 have been implicated in cardiac hypertrophy in vivo. TRPC1 is known to function not * This study was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M. Nishida, M. Nakaya, and H. Kurose), a grant-in-aid for scientific research on Innovative Areas (to M. Nishida), a grant-in-aid for scientific research on Priority Areas (H. Kurose), and grants from the Naito Foundation, the Nakatomi Foundation, the Sapporo ...

Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2)

“…In contrast, in pituitary cells, nonspecific cation channels, probably including TRPC6, are involved in PKA-stimulated calcium influx (106). PKA has been reported to phosphorylate mouse TRPC6 at two sites, Ser-28 and Thr-69 (corresponding to Thr-70 in humans), leading to channel inhibition (86,100). However, in one study, Ser-28 was felt to be the critical inhibitory site (100), whereas the other study noted that phosphorylation of Thr-69 was key (98).…”

“…9, A and C). In addition, abolishing the PKA/PKG phosphorylation site on Thr-70 (86,100) or the dual tyrosine phosphorylation sites at Tyr-31 and Tyr-285 (8) failed to significantly alter ERK activation by TRPC6 R895C (Fig. 9, A and C).…”

“…3C), further arguing against a role for CaMKII in mediating ERK activation by mutant TRPC6 and suggesting that the effect of KN-93 on this process is mediated, at least in part, independently of its effect on CaMKII activity. Previous studies have suggested that PKA and cGMP-dependent protein kinase (PKG) might play a role in limiting the activity of the TRPC6 channel (82)(83)(84)(85)(86)(87). We therefore examined whether inhibition of PKA through H89 might further enhance ERK phosphorylation in cells expressing TRPC6 R895C.…”

“…TRPC6 contains several potential consensus PKA phosphorylation sites, including Ser-13, Thr-70, and Ser-322 (84). Phosphorylation of threonine 70 in TRPC6 and of the corresponding threonine in TRPC3 by both PKA and PKG has been reported (85,86,98). The role of threonine 70 as a site for PKA phosphorylation was therefore investigated (Fig.…”

TRP Channels: Their Function and Potentiality as Drug Targets

“…For example, α1 adrenergic receptorstimulated hypertrophic responses were blocked by 2-aminoethoxydiphenylborane (2-APB) and N-{4-[3,5-bis(trifl uoromethyl)-1H-pyrazol-1yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2; also called Pyr2), but not by verapamil, a voltage-dependent L-type Ca 2+ channel blocker [ 142 ]. Indirect inhibition of TRPC3/6 channel activities by PDE-5 inhibitors [ 159 ] and ANP [ 158 ] can also suppresses pathological hypertrophy through phosphorylation of TRPC6 at Thr69. Mori developed a pyrazole compound, Pyr3, which selectively inhibits TRPC3 channel activity with an IC 50 value of 0.7 μM [ 36 ].…”