Dhananjay P. Thakur

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

Significance Transient Receptor Potential Canonical 4 (TRPC4) forms nonselective cation channels implicated in multiple functions in the brain, heart, vasculature, and gastrointestinal tract. However, mechanisms that govern TRPC4 channel activation remain mysterious, severely hampering their functional elucidation under physiological and pathological conditions. Uniquely, TRPC4 is activated following ligand stimulation of G protein-coupled receptors that function through either Gq/11 or Gi/o subgroups of G proteins. However, to what extent and how these two metabotropic pathways interact to regulate TRPC4 is unclear. Our study demonstrates the critical involvement of Gi/o signaling and phospholipase C activity in TRPC4 activation, providing detailed dissection of signaling steps of the phospholipase C pathway that contribute to channel gating. The mechanistic insights revealed should greatly facilitate evaluation and understanding of the physiological and pathological functions of these channels. 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 PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCβ 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 Ca2+ 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 PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of Gi/o-mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident Gi/o, Ca2+, 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.

Identification and optimization of 2‐aminobenzimidazole derivatives as novel inhibitors of TRPC4 and TRPC5 channels

Transient receptor potential canonical (TRPC) channels play important roles in a broad array of physiological functions and are involved in various diseases. However, due to a lack of potent subtype‐specific inhibitors the exact roles of TRPC channels in physiological and pathophysiological conditions have not been elucidated.

Overexpression of Smooth Muscle Myosin Heavy Chain Leads to Activation of the Unfolded Protein Response and Autophagic Turnover of Thick Filament-associated Proteins in Vascular Smooth Muscle Cells

Background: Genomic duplications involving the smooth muscle myosin heavy chain gene, MYH11, are associated with increased risk for acute aortic dissections. Results: MYH11 overexpression causes increased turnover of contractile proteins through increased autophagy. Conclusion: MYH11 duplications may predispose to aortic disease through increased turnover of contractile proteins and disruption of contractile signaling. Significance: Increased protein turnover may be an important mechanism by which genomic duplications cause human disease. Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications.

Dual depolarization responses generated within the same lateral septal neurons by TRPC4-containing channels

In the central nervous system, canonical transient receptor potential (TRPC) channels have been implicated in mediating neuronal excitation induced by stimulating metabotropic receptors, including group 1 metabotropic glutamate receptors (mGluRs). Lateral septal (LS) neurons express high levels of TRPC4 and group I mGluRs. However, to what extent native TRPC4-containing channels (TRPC4-cc) are activated as well as the impact of different levels of TRPC4-cc activation on neuronal excitability remain elusive. Here, we report that stimulating LS neurons with group I mGluR agonist, (S)-3,5-DHPG, causes either an immediate increase in firing rate or an initial burst followed by a pause of firing, which can be correlated with below-threshold-depolarization (BTD) or above-threshold-plateau-depolarization (ATPD), respectively, in whole-cell recordings. The early phase of BTD and the entire ATPD are completely absent in neurons from TRPC4−/− mice. Moreover, in the same LS neurons, BTD can be converted to ATPD at more depolarized potentials or with a brief current injection, suggesting that BTD and ATPD may represent partial and full activations of TRPC4-cc, respectively. We show that coincident mGluR stimulation and depolarization is required to evoke strong TRPC4-cc current, and Na+ and Ca2+ influx, together with dynamic changes of intracellular Ca2+, are essential for ATPD induction. Our results suggest that TRPC4-cc integrates metabotropic receptor stimulation with intracellular Ca2+ signals to generate two interconvertible depolarization responses to affect excitability of LS neurons in distinct fashions.