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The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four intramembrane fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145 and Y1436) that block these openings. Structural data suggest that occluded lateral fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of NaV, CaVand NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels, highlighting the existence of drug binding sites beyond the occluded fenestrations of NALCN. Further experiments using single alanine mutants revealed that phenytoin and 2-APB access their putative binding sites through distinct fenestrations, implying structural specificity to their modes of access. Intrigued by the activity of 2-APB and its analogues, we tested more compounds containing the diphenylmethane/amine moiety on WT channels. We identified novel compounds that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the resistance of NALCN to pharmacological targeting, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties.

Unlike classical voltage-gated sodium (Na

Depolarizing sodium (Na+) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity1–8. NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood3,7,9–12. NALCN, UNC79 and UNC80 are essential in rodents2,9,13, and mutations in human NALCN and UNC80 cause severe developmental and neurological disease14,15. Since fundamental aspects about the composition, assembly, and gating of the NALCN channelosome remain obscure, we determined the structure of this ~1 megadalton complex. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6-gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers are identified that lead to a model of channelosome gating. Our results provide a structural blueprint to understand NALCN channelosome physiology and a template for drug discovery to modulate the resting membrane potential.

Although several ionic mechanisms are known to control rate and regularity of the slow pacemaker in dopamine (DA) neurons, the core mechanism of pacing is controversial. Here we tested the hypothesis that pacemaking of SNc DA neurons is enabled by an unconventional conductance. We found that 1-(2,4-xylyl)guanidinium (XG), an established blocker of gating pore currents, selectively inhibits pacemaking of DA neurons. The compound inhibited all slow pacemaking DA neurons that were tested, both in the substantia nigra pars compacta, and in the ventral tegmental area. Interestingly, bursting behavior was not affected by XG. Furthermore, the drug did not affect fast pacemaking of GABAergic neurons from substantia nigra pars reticulata neurons or slow pacemaking of noradrenergic neurons. In DA neurons, current-clamp analysis revealed that XG did not appear to affect ion channels involved in the action potential. Its inhibitory effect persisted during blockade of all ion channels previously suggested to contribute to pacemaking. RNA sequencing and voltage-clamp recordings yielded no evidence for a gating pore current to underlie the conductance. However, we could isolate a small subthreshold XG-sensitive current, which was carried by both Na+ and Cl− ions. Although the molecular target of XG remains to be defined, these observations represent a step towards understanding pacemaking in DA neurons.

Although several ionic mechanisms are known to control rate and regularity of the pacemaker in dopamine (DA) neurons from the substantia nigra pars compacta (SNc), a conductance essential for pacing has yet to be defined. Here we provide pharmacological evidence that pacemaking of SNc DA neurons is enabled by an unconventional conductance. We found that 1-(2,4-xylyl)guanidine (XG), an established blocker of gating pore currents in mutant voltage gated sodium channels, selectively stops pacemaking of DA SNc neurons and is without effect on the main pore of their voltage-gated channels. We isolated a voltage-dependent, non-inactivating XG-sensitive current of 20-25 pA which operates in the relevant subthreshold range and is carried by both Na+ and Cl- ions. While the molecular identity of this conductance remains to be determined, we show that this XG-sensitive current is crucial to sustain pacemaking in these neurons.

Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, NaV1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods., Chemical modification of proteins can be used to decipher function or use that function for therapeutic purposes. Here, the authors insert synthetic peptides via tandem protein trans-splicing to add post-translational modifications or non-canonical amino acids.

Persistently depolarizing sodium (Na+) leak currents enhance electrical excitability1,2. The ion channel responsible for the major background Na+ conductance in neurons is the Na+ leak channel, non-selective (NALCN)3,4. NALCN-mediated currents regulate neuronal excitability linked to respiration, locomotion and circadian rhythm4–10. NALCN activity is under tight regulation11–14 and mutations in NALCN cause severe neurological disorders and early death15,16. NALCN is an orphan channel in humans, and fundamental aspects of channel assembly, gating, ion selectivity and pharmacology remain obscure. Here we investigate this essential leak channel and determined the structure of NALCN in complex with a distinct auxiliary subunit, family with sequence similarity 155 member A (FAM155A). FAM155A forms an extracellular dome that shields the ion-selectivity filter from neurotoxin attack. The pharmacology of NALCN is further delineated by a walled-off central cavity with occluded lateral pore fenestrations. Unusual voltage-sensor domains with asymmetric linkages to the pore suggest mechanisms by which NALCN activity is modulated. We found a tightly closed pore gate in NALCN where the majority of missense patient mutations cause gain-of-function phenotypes that cluster around the S6 gate and distinctive π-bulges. Our findings provide a framework to further study the physiology of NALCN and a foundation for discovery of treatments for NALCN channelopathies and other electrical disorders. Structural and functional studies of the sodium leak channel, non-selective (NALCN) in complex with a distinct auxiliary subunit reveal the structural basis of the channel function and pharmacology and the functional impact of mutations that cause NALCN channelopathies.

NALCN is the voltage-sensing, pore-forming subunit of a constitutively active, extracellular calcium-regulated complex., 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.

OBJECTIVE Unexplained cardiac arrest (UCA) is often the first manifestation of an inherited arrhythmogenic disease. Genetic testing in UCA is challenging due to the complexities of variant interpretation in the absence of supporting cardiac phenotype. We aimed to investigate if a KCNQ1 variant [p.(Pro64_Pro70del)], previously reported as pathogenic, contributes to the long-QT syndrome phenotype, co-segregates with disease or affects KCNQ1 function in vitro. METHODS DNA was extracted from peripheral blood of a 22-year-old male after resuscitation from UCA. Targeted exome sequencing was performed using the TruSight-One Sequencing Panel (Illumina). Variants in 190 clinically relevant cardiac genes with minor allele frequency A in PKP2 (p.Arg811Ser), classified as likely benign. Two asymptomatic family members with no evident phenotype hosted the KCNQ1 variant. Functional studies showed that the wild-type and mutant channels have no significant differences in current levels, conductance-voltage relationships, as well as activation and deactivation kinetics, in the absence and presence of the auxiliary subunit KCNE1. CONCLUSIONS Based on our data and previous reports, available evidence is insufficient to consider the variant KCNQ1:c.190_210del as pathogenic. Our findings call for cautious interpretation of genetic tests in UCA in the absence of a clinical phenotype.

SummaryThe 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 the robust function of NALCN in heterologous systems requires co-expression of UNC79, UNC80 and FAM155A. The resulting NALCN channel complex is constitutively active, 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 the putative voltage sensors of NALCN, the channel complex shows voltage-dependent modulation of the constitutive current, suggesting that voltage-sensing domains can give rise to a broader range of gating phenotypes than previously anticipated. Our work points towards formerly unknown contributions of NALCN to neuronal excitability and opens avenues for pharmacological targeting.HighlightsFunction of NALCN requires UNC79, UNC80 and FAM155AThe complex is permeable to monovalent cations, but is blocked by divalent cationsThe complex displays a constitutively active, voltage-modulated current phenotypePositively charged side chains in S4 of NALCN VSD I and II confer voltage sensitivity

Publisher's version (útgefin grein), γ-aminobutyric acid type A receptors (GABAARs), the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are arguably the most challenging member of the pentameric Cys-loop receptors to study due to their heteromeric structure. When two or more subunits are expressed together in heterologous systems, receptors of variable subunit type, ratio, and orientation can form, precluding accurate interpretation of data from functional studies. Subunit concatenation is a technique that involves the linking of individual subunits and in theory allows the precise control of the uniformity of expressed receptors. In reality, the resulting concatemers from widely used constructs are flexible in their orientation and may therefore assemble with themselves or free GABAAR subunits in unexpected ways. In this study, we examine functional responses of receptors from existing concatenated constructs and describe refinements necessary to allow expression of uniform receptor populations. We find that dimers from two commonly used concatenated constructs, β-23-α and α-10-β, assemble readily in both the clockwise and the counterclockwise orientations when coexpressed with free subunits. Furthermore, we show that concatemers formed from new tetrameric α-10-β-α-β and α-10-β-α-γ constructs also assemble in both orientations with free subunits to give canonical αβγ receptors. To restrict linker flexibility, we systematically shorten linker lengths of dimeric and pentameric constructs and find optimized constructs that direct the assembly of GABAARs only in one orientation, thus eliminating the ambiguity associated with previously described concatemers. Based on our data, we revisit some noncanonical GABAAR configurations proposed in recent years and explain how the use of some concatenated constructs may have led to wrong conclusions. Our results help clarify current contradictions in the literature regarding GABAAR subunit stoichiometry and arrangement. The lessons learned from this study may guide future efforts in understanding other related heteromeric receptors., This work was supported by the Australian Research Council (LP160100560) and the Australian National Health and Medical Research Council (APP1124567 and APP1081733 to M. Chebib, P.K. Ahring, and T. Balle). N.M. Kowal was supported by the Icelandic Research Fund (grant number 152604). The authors declare no competing financial interests. Author contributions: P.K. Ahring designed the research; V.W.Y. Liao, P.K. Ahring, H.C. Chua, N.M. Kowal, and T. Balle performed the research; P.K. Ahring analyzed the data and wrote the manuscript. P.K. Ahring, T. Balle, and M. Chebib acquired the necessary funding. All authors approved the final version of the manuscript. Richard W. Aldrich served as editor.

GABA

GABAA receptors (GABAARs) are a class of ligand-gated ion channels with high physiological and therapeutic significance. In the brain, these pentameric receptors occur with diverse subunit composition, which confers highly complex pharmacology to this receptor class. An impressive range of clinically used therapeutics are known to bind to distinct sites found on GABAARs to modulate receptor function. Numerous experimental approaches have been used over the years to elucidate the binding sites of these drugs, but unequivocal identification is challenging due to subtype- and ligand-dependent pharmacology. Here, we review the current structural and pharmacological understanding of GABAARs, besides highlighting recent evidence which has revealed greater complexity than previously anticipated.

Extracts of the pepper plant kava (Piper methysticum) are effective in alleviating anxiety in clinical trials. Despite the long-standing therapeutic interest in kava, the molecular target(s) of the pharmacologically active constituents, kavalactones have not been established. γ-Aminobutyric acid type A receptors (GABAARs) are assumed to be the in vivo molecular target of kavalactones based on data from binding assays, but evidence in support of a direct interaction between kavalactones and GABAARs is scarce and equivocal. In this study, we characterised the functional properties of the major anxiolytic kavalactone, kavain at human recombinant α1β2, β2γ2L, αxβ2γ2L (x = 1, 2, 3 and 5), α1βxγ2L (x = 1, 2 and 3) and α4β2δ GABAARs expressed in Xenopus oocytes using the two-electrode voltage clamp technique. We found that kavain positively modulated all receptors regardless of the subunit composition, but the degree of enhancement was greater at α4β2δ than at α1β2γ2L GABAARs. The modulatory effect of kavain was unaffected by flumazenil, indicating that kavain did not enhance GABAARs via the classical benzodiazepine binding site. The β3N265M point mutation which has been previously shown to profoundly decrease anaesthetic sensitivity, also diminished kavain-mediated potentiation. To our knowledge, this study is the first report of the functional characteristics of a single kavalactone at distinct GABAAR subtypes, and presents the first experimental evidence in support of a direct interaction between a kavalactone and GABAARs.

2'-Methoxy-6-methylflavone (2'MeO6MF) is an anxiolytic flavonoid which has been shown to display GABAA receptor (GABAAR) β2/3-subunit selectivity, a pharmacological profile similar to that of the general anaesthetic etomidate. Electrophysiological studies suggest that the full agonist action of 2'MeO6MF at α2β3γ2L GABAARs may mediate the flavonoid's in vivo effects. However, we found variations in the relative efficacy of 2'MeO6MF (2'MeO6MF-elicited current responses normalised to the maximal GABA response) at α2β3γ2L GABAARs due to the presence of mixed receptor populations. To understand which receptor subpopulation(s) underlie the variations observed, we conducted a systematic investigation of 2'MeO6MF activity at all receptor combinations that could theoretically form (α2, β3, γ2L, α2β3, α2γ2L, β3γ2L and α2β3γ2L) in Xenopus oocytes using the two-electrode voltage clamp technique. We found that 2'MeO6MF activated non-α-containing β3γ2L receptors. In an attempt to establish the optimal conditions to express a uniform population of these receptors, we found that varying the relative amounts of β3:γ2L subunit mRNAs resulted in differences in the level of constitutive activity, the GABA concentration-response relationships, and the relative efficacy of 2'MeO6MF activation. Like 2'MeO6MF, general anaesthetics such as etomidate and propofol also showed distinct levels of relative efficacy across different injection ratios. Based on these results, we infer that β3γ2L receptors may form with different subunit stoichiometries, resulting in the complex pharmacology observed across different injection ratios. Moreover, the discovery that GABA and etomidate have direct actions at the α-lacking β3γ2L receptors raises questions about the structural requirements for their respective binding sites at GABAARs.