Abstract
The sodium leak channel NALCN is a key player in establishing the resting membrane potential (RMP) in neurons and transduces changes in extracellular Ca2+ concentration ([Ca2+]e) into increased neuronal excitability as the downstream effector of calcium-sensing receptor (CaSR). Gain-of-function mutations in the human NALCN gene cause encephalopathy and severe intellectual disability. Thus, understanding the regulatory mechanisms of NALCN is important for both basic and translational research. This study reveals a novel mechanism for NALCN regulation by arginine methylation. Hippocampal dentate granule cells in protein arginine methyltransferase 7 (PRMT7)-deficient mice display a depolarization of the RMP, decreased threshold currents, and increased excitability compared to wild-type neurons. Electrophysiological studies combined with molecular analysis indicate that enhanced NALCN activities contribute to hyperexcitability in PRMT7−/− neurons. PRMT7 depletion in HEK293T cells increases NALCN activity by shifting the dose-response curve of NALCN inhibition by [Ca2+]e without affecting NALCN protein levels. In vitro methylation studies show that PRMT7 methylates a highly conserved Arg1653 of the NALCN gene located in the carboxy-terminal region that is implicated in CaSR-mediated regulation. A kinase-specific phosphorylation site prediction program shows that the adjacent Ser1652 is a potential phosphorylation site. Consistently, our data from site-specific mutants and PKC inhibitors suggest that Arg1653 methylation might modulate Ser1652 phosphorylation mediated by CaSR/PKC-delta, leading to [Ca2+]e-mediated NALCN suppression. Collectively, these data suggest that PRMT7 deficiency decreases NALCN methylation at Arg1653, which, in turn, decreases CaSR/PKC-mediated Ser1652 phosphorylation, lifting NALCN inhibition, thereby enhancing neuronal excitability. Thus, PRMT7-mediated NALCN inhibition provides a potential target for the development of therapeutic tools for neurological diseases.
Highlights
NALCN is a nonselective sodium leak channel that is highly conserved evolutionarily and is expressed widely in neurons throughout the brain[1]
Our results demonstrate that protein arginine methyltransferase 7 (PRMT7)−/− (KO) dentate gyrus (DG) neurons exhibit increased intrinsic excitability compared to wild type (WT) DG neurons
Immunoblot analysis demonstrated that PRMT7 proteins were expressed in all examined brain areas; PRMT7 is highly expressed in the hippocampus and cortex. (Fig. 1a)
Summary
NALCN is a nonselective sodium leak channel that is highly conserved evolutionarily and is expressed widely in neurons throughout the brain[1]. Diverse mechanisms have been linked to the control of ion channel activity. One mechanism is related to the control of expression and membrane targeting, leading to alterations in ion-channel density at the membrane[7]. The posttranslational modification of ion channels has been shown to play important roles in the control of a channel’s functional properties[8,9,10]. Arginine methylation has newly emerged as a key posttranslational modification that can regulate ion channel activity. Protein arginine methylransferases (PRMTs) are enzymes that catalyze the mono- and di-methylation of arginine residues of histone or nonhistone substrates[13]. The type III PRMT, including PRMT7, catalyzes the mono- and symmetrically di-methylated arginine residues. PRMTs are potentially intriguing targets for the modulation of channel activity and neural function
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