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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 (PIP₂) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C-terminal PDZ-binding motif allowing for binding of the scaffolding proteins Na⁺/H⁺ 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. PIP₂ 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.

SUBMITTER: Storch U

PROVIDER: S-EPMC5224389 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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

Storch Ursula U Forst Anna-Lena AL Pardatscher Franziska F Erdogmus Serap S Philipp Maximilian M Gregoritza Manuel M Mederos Y Schnitzler Michael M Gudermann Thomas T

Proceedings of the National Academy of Sciences of the United States of America 20161219 1

The activation mechanism of the classical transient receptor potential channels TRPC4 and -5 via the G<sub>q/11</sub> 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<sub>2</sub>) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C ...[more]

PMID: 27994151

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Project description:Human ether-á-go-go (eag)-related gene (hERG) potassium channel kinetics are characterized by rapid inactivation upon depolarization, along with rapid recovery from inactivation and very slow closing (deactivation) upon repolarization. These factors combine to create a resurgent hERG current, where the current amplitude is paradoxically larger with repolarization than with depolarization. Previous data showed that the hERG N-terminal eag domain regulated deactivation kinetics by making a direct interaction with the C-terminal region of the channel. A primary mechanism for fast inactivation depends on residues in the channel pore; however, inactivation was also shown to be slower after deletion of a large N-terminal region. The mechanism for N-terminal region regulation of inactivation is unclear. Here, we investigated the contributions of the large N-terminal domains (amino acids 1-354), including the eag domain (amino acids 1-135), to hERG channel inactivation kinetics and steady-state inactivation properties. We found that N-deleted channels lacking just the eag domain (Δ2-135) or both the eag domain and the adjacent proximal domain (Δ2-354) had less rectifying current-voltage (I-V) relationships, slower inactivation, faster recovery from inactivation, and lessened steady-state inactivation. We coexpressed genetically encoded N-terminal fragments for the eag domain (N1-135) or the eag domain plus the proximal domain (N1-354) with N-deleted hERG Δ2-135 or hERG Δ2-354 channels and found that the resulting channels had more rectifying I-V relationships, faster inactivation, slower recovery from inactivation, and increased steady-state inactivation, similar to those properties measured for wild-type (WT) hERG. We also found that the eag domain-containing fragments regulated the time to peak and the voltage at the peak of a resurgent current elicited with a ramp voltage protocol. The eag domain-containing fragments effectively converted N-deleted channels into WT-like channels. Neither the addition of the proximal domain to the eag domain in N1-354 fragments nor the presence of the proximal domain in hERG Δ2-135 channels measurably affected inactivation properties; in contrast, the proximal region regulated steady-state activation in hERG Δ2-135 channels. The results show that N-terminal region-dependent regulation of channel inactivation and resurgent current properties are caused by a direct interaction of the eag domain with the rest of the hERG channel.

Dataset Information

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

Publications

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

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