Methylation determines the extracellular calcium sensitivity of the leak channel NALCN in hippocampal dentate granule cells

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. The addition of a methyl group to an arginine residue on the ion channel NALCN contributes to suppress the activity of this membrane protein and reduces neuronal excitability. Hana Cho, Jong-Sun Kang and colleagues at Sungkyunkwan University in South Korea found that neurons in the hippocampus of mice lacking an enzyme that mediates the transfer of methyl groups to proteins have increased NALCN activity and are more likely to fire an electrical signal. Furthermore, they showed that NALCN methylation facilitates the phosphorylation of an adjacent amino acid that prevents channel activation in response to extracellular calcium concentrations. These findings suggest that NALCN methylation has a key role in regulating the channel’s sensitivity to calcium. Moreover, they reveal a new mechanism for regulating neuronal excitability that could be targeted therapeutically to ameliorate diseases characterised by neuronal hyperexcitability.