The sodium leak channel (NALCN) is essential for survival in mammals: NALCN mutations are life-threatening in humans and knockout is lethal in mice. However, the basic functional and pharmacological properties of NALCN have remained elusive. Here, we found that robust function of NALCN in heterologous systems requires co-expression of UNC79, UNC80, and FAM155A. The resulting NALCN channel complex is constitutively active and conducts monovalent cations but is blocked by physiological concentrations of extracellular divalent cations. Our data support the notion that NALCN is directly responsible for the increased excitability observed in a variety of neurons in reduced extracellular Ca2+. Despite the smaller number of voltage-sensing residues in NALCN, the constitutive activity is modulated by voltage, suggesting that voltage-sensing domains can give rise to a broader range of gating phenotypes than previously anticipated. Our work points toward formerly unknown contributions of NALCN to neuronal excitability and opens avenues for pharmacological targeting.

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Highlights •Function of NALCN requires UNC79, UNC80 and FAM155A• The complex is permeable to monovalent cations, but is blocked by divalent cations •The complex displays a constitutively active, voltage-modulated current phenotype• Positively charged side chains in S4 of NALCN VSD I and II confer voltage sensitivity

The ability of proteins to assemble at sites of high membrane curvature is essential for various membrane remodeling processes, including clathrinmediated endocytosis. This endocytic process is facilitated by a network of multivalent adaptor proteins that bind to the plasma membrane surface. Many of these adaptor proteins have been shown to sense regions of high membrane curvature, leading to local recruitment of the clathrin coat. Because clathrin does not bind to the membrane directly, it has remained unclear whether the clathrin coat plays an active role in sensing membrane curvature or is passively recruited by adaptor proteins. Using a synthetic tag to assemble clathrin directly on membrane surfaces, we examined protein binding equilibria to show that clathrin is a strong sensor of membrane curvature, comparable to previously studied adaptor proteins. interestingly, this sensitivity arises from clathrin assembly, rather than from the properties of unassembled clathrin triskelia. When clathrin lattice assembly was inhibited, curvature sensitivity was reduced substantially, suggesting that triskelia have preferred angles of interaction. Further, when clathrin was recruited by endocytic adaptor proteins, its curvature sensitivity was amplified by two to tenfold , such that the resulting protein complex was up to 100 times more likely to assemble on a highly curved surface, compared to a flatter one. This exquisite sensitivity points to a synergistic relationship between the coat and its adaptor proteins, which enables clathrin to pinpoint sites of high membrane curvature, an essential step in ensuring robust membrane traffic. More broadly, these findings suggest that supramolecular protein networks, rather than individual protein domains, are likely the critical drivers of membrane curvature sensing.