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Critical roles of Gi/o proteins and phospholipase C-?1 in the activation of receptor-operated TRPC4 channels.

ABSTRACT: Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G proteins and PLC?, with a preference for PLC?1 over PLC?3, but not necessarily the PLC? pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and Gi/o-coupled µ opioid receptor, µ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca(2+) and PIP2. Reducing PIP2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak Gi/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLC?1 levels and almost completely eliminated by a dominant-negative PLC?1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of Gi/o-mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLC? function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident Gi/o, Ca(2+), and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems.

SUBMITTER: Thakur DP

PROVIDER: S-EPMC4743816 | biostudies-other | 2016 Jan

REPOSITORIES: biostudies-other

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Critical roles of Gi/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels.

Thakur Dhananjay P DP Tian Jin-bin JB Jeon Jaepyo J Xiong Jian J Huang Yu Y Flockerzi Veit V Zhu Michael X MX

Proceedings of the National Academy of Sciences of the United States of America 20160111 4

Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G protei ...[more]

PMID: 26755577

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Project description:Background and purposeDespite the view that only ?2- as opposed to ?1-adrenoceptors (?ARs) couple to G(i), some data indicate that the ?1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Experimental approachWe used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate ?2ARs (?1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.Key resultsPTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.Conclusions and implicationsTogether, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

Project description:Kisspeptin signaling via its G?q-coupled receptor GPR54 plays a crucial role in modulating GnRH neuronal excitability, which controls pituitary gonadotropins secretion and ultimately reproduction. Kisspeptin potently depolarizes GnRH neurons primarily through the activation of canonical transient receptor potential (TRPC) channels, but the intracellular signaling cascade has not been elucidated. Presently, we have established that kisspeptin activation of TRPC channels requires multiple membrane and intracellular signaling molecules. First, phosphatidylinositol-4,5-bisphosphate (PIP(2)) hydrolysis by phospholipase C? is required because whole-cell dialysis of Dioctanoylglycerol-PIP(2) (DiC8-PIP(2)) inhibited the kisspeptin activation of TRPC channels, and the phosphatidylinositol 4-kinase inhibitor wortmannin, which attenuates PIP(2) synthesis, prolonged TRPC channel activation. Using single cell RT-PCR, we identified that the mRNA for the PIP(2)-interacting TRPC channel subunit, TRPC4?, is expressed in GnRH neurons. Depletion of intracellular Ca(2+) stores by thapsigargin and inositol 1,4,5-trisphosphate had no effect, indicating that the TRPC channels are not store-operated. Neither removing extracellular Ca(2+) nor buffering intracellular Ca(2+) with EGTA or BAPTA had any effect on the kisspeptin activation of the TRPC channels. However, the Ca(2+) channel blocker Ni(2+) inhibited the kisspeptin-induced inward current. Moreover, inhibition of protein kinase C by bisindolylmaleimide-I or calphostin C had no effect, but activation of protein kinase C by phorbol 12,13-dibutyrate occluded the kisspeptin-activated current. Finally, inhibition of the cytoplasmic tyrosine kinase cSrc by genistein or the pyrazolo-pyrimidine PP2 blocked the activation of TRPC channels by kisspeptin. Therefore, TRPC channels in GnRH neurons are receptor-operated, and kisspeptin activates TRPC channels through PIP(2) depletion and cSrc tyrosine kinase activation, which is a novel signaling pathway for peptidergic excitation of GnRH neurons.

Dataset Information

Critical roles of Gi/o proteins and phospholipase C-?1 in the activation of receptor-operated TRPC4 channels.

Publications

Critical roles of Gi/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels.

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