Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.
Our study has demonstrated for the first time that the infant calming response to maternal carrying is a coordinated set of central, motor, and cardiac regulations and is a conserved component of mammalian mother-infant interactions. Our findings provide evidence for and have the potential to impact current parenting theory and practice, since unsoothable crying is the major risk factor for child abuse.
Mutations, exclusively missense, of voltage-gated sodium channel ␣ subunit type 1 (SCN1A) and type 2 (SCN2A) genes were reported in patients with idiopathic epilepsy: generalized epilepsy with febrile seizures plus. Nonsense and frameshift mutations of SCN1A, by contrast, were identified in intractable epilepsy: severe myoclonic epilepsy in infancy (SMEI). Here we describe a first nonsense mutation of SCN2A in a patient with intractable epilepsy and severe mental decline. The phenotype is similar to SMEI but distinct because of partial epilepsy, delayed onset (1 year 7 months), and absence of temperature sensitivity. A mutational analysis revealed that the patient had a heterozygous de novo nonsense mutation R102X of SCN2A. Patch-clamp analysis of Na v 1.2 wild-type channels and the R102X mutant protein coexpressed in human embryonic kidney 293 cells showed that the truncated mutant protein shifted the voltage dependence of inactivation of wild-type channels in the hyperpolarizing direction. Analysis of the subcellular localization of R102X truncated protein suggested that its dominant negative effect could arise from direct or indirect cytoskeletal interactions of the mutant protein. Haploinsufficiency of Na v 1.2 protein is one plausible explanation for the pathology of this patient; however, our biophysical findings suggest that the R102X truncated protein exerts a dominant negative effect leading to the patient's intractable epilepsy.
Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.
Kawano and colleagues show two distinct ion conductance states by embedding a single metal-organic porous molecule with Archimedean cuboctahedron geometry into a planar lipid bilayer. The triangular and square apertures in the cuboctahedron work independently as ion-transporting pathways. By changing the aliphatic chain length introduced on the periphery of the cuboctahedron, the authors identified that the rotational dynamics of the cuboctahedron regulate the open pore time of each conductance state through distinct apertures and the switching between them.
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