Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol

Abstract

The activation mechanism of the classical transient receptor potential channels TRPC4 and -5 via the G q/11 protein-phospholipase C (PLC) signaling pathway has remained elusive so far. In contrast to all other TRPC channels, the PLC product diacylglycerol (DAG) is not sufficient for channel activation, whereas TRPC4/5 channel activity is potentiated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C-terminal PDZ-binding motif allowing for binding of the scaffolding proteins Na+ exchanger regulatory factor (NHERF) 1 and 2. PKC inhibition or the exchange of threonine for alanine in the C-terminal PDZ-binding motif conferred DAG sensitivity to the channel. Altogether, we present a DAG-mediated activation mechanism for TRPC4/5 channels tightly regulated by NHERF1/2 interaction. PIP 2 depletion evokes a C-terminal conformational change of TRPC5 proteins leading to dynamic dissociation of NHERF1/2 from the C terminus of TRPC5 as a prerequisite for DAG sensitivity. We show that NHERF proteins are direct regulators of ion channel activity and that DAG sensitivity is a distinctive hallmark of TRPC channels.and -5 channels are members of the classical transient receptor potential cation (TRPC) family of nonselective, calcium permeable receptor-operated cation channels. They are widely expressed in many tissues, including brain, kidney, and the vascular system. High expression levels are found in the central nervous system where TRPC4 and -5 are involved in amygdala function and fear-related behavior (1, 2), seizure, and excitotoxicity (3). Furthermore, TRPC5 channels are implicated in neuronal depolarization and bursting during epiletiform seizures (4) and regulate hippocampal neurite length and growth cone morphology (5). In the kidney, TRPC5 channels are proposed to be protective against renal failure (6). TRPC channels are usually activated by G q/11 proteincoupled receptors via phospholipase C (PLC) activation resulting in cleavage of phosphatidylinositol-3,4-bisphosphate (PIP 2 ) into the second messengers inositol-1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). DAG is known to activate TRPC2, -3, -6, and -7 (7-9) channels, whereas TRPC4 and -5 are supposed to be insensitive to the PLC product DAG (8) and are even inhibited by DAG or its membrane-permeable analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) (10). DAG-mediated TRPC5 channel inhibition was shown to be PKC dependent (10). Furthermore, TRPC4 and -5 channels can be activated by depleting PIP 2 (11, 12), contrary to TRPC6 and -7 channels, which are inhibited by PIP 2 depletion (13). However, there are first hints to show that endogenously expressed TRPC5 channels might be DAG sensitive (14) but mechanistic insight is lacking so far.A noteworthy structural difference between TRPC4 and -5 and the established DAG-sensitive TRPC3, -6, and -7 channels is the PDZbinding motif VTTLR in the C termini of TRPC4 and -5 channels (15-17) as a structural basis of the interaction with Na + /H + e...

Potent, selective, and subunit‐dependent activation of TRPC5 channels by a xanthine derivative

“…The TRPC proteins are widely expressed in excitable and non-excitable cells, and TRPC channels are modulated by many physiological mechanisms and chemical and physical factors, including G-protein signalling, receptor tyrosine kinase signalling, temperature, heavy metal ions, lipids, and dietary compounds. This has led to the idea that the channels are complex integrators of multiple physiological and environmental stimuli (Bon & Beech, 2013;Clapham, 2003;Mederos y Schnitzler, Gudermann, & Storch, 2018;Naylor et al, 2016;Storch et al, 2017).…”

Treasure troves of pharmacological tools to study transient receptor potential canonical 1/4/5 channels

“…In addition to PIP 2 , several other lipid factors, such as oxidized phospholipids, lysophosphatidic acid, some steroidal derivatives, and PAF, also regulate the activity of the TRPC1/4/5 channels (Beech, ). The depletion of PIP 2 which occurs after GPCR‐mediated activation of the Gq‐PLCβ and/or receptor TK‐mediated activation of the PLCγ signaling, stimulates the TRPC1/4/5 channels (Beech, ; Clapham, ; Kim et al, ; Storch et al, ). Notably, in contrast to other TRPC channel members (TRPC2, 3, 6, and 7), TRPC1/4/5 are not activated by DAG.…”

Store-operated Ca2+ entry in primary murine lung fibroblasts is independent of classical transient receptor potential (TRPC) channels and contributes to cell migration

“…These so called DAG-sensitive channels are mainly expressed in brain, endothelial and smooth muscle cells of the vasculature and mediate ROCE (reviewed in 34 ). Recent findings indicate that Na + /H + exchanger regulatory factors dynamically determine the DAG sensitivity of TRPC4 and TRPC5 channels 35 and that TRPC1 proteins work as channel regulators in heteromeric complexes with all other six channels of the TRPC family 25 . Therefore, all TRPC channels are responsible for ROCE and global ablation of TRPC1 and TRPC6 proteins is sufficient to reduce intracellular Ca 2+ levels during ROCE in pmLF from TRPC1/6-deficient mice (Fig.…”