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Single-molecule research techniques such as patch-clamp electrophysiology deliver unique biological insight by capturing the movement of individual proteins in real time, unobscured by whole-cell ensemble averaging. The critical first step in analysis is event detection, so called "idealisation", where noisy raw data are turned into discrete records of protein movement. To date there have been practical limitations in patch-clamp data idealisation; high quality idealisation is typically laborious and becomes infeasible and subjective with complex biological data containing many distinct native single-ion channel proteins gating simultaneously. Here, we show a deep learning model based on convolutional neural networks and long short-term memory architecture can automatically idealise complex single molecule activity more accurately and faster than traditional methods. There are no parameters to set; baseline, channel amplitude or numbers of channels for example. We believe this approach could revolutionise the unsupervised automatic detection of single-molecule transition events in the future.

Many types of ion channel localize to cholesterol and sphingolipid-enriched regions of the plasma membrane known as lipid microdomains or 'rafts'. The precise physiological role of these unique lipid microenvironments remains elusive due largely to difficulties associated with studying these potentially extremely small and dynamic domains. Nevertheless, increasing evidence suggests that membrane rafts regulate channel function in a number of different ways. Raft-enriched lipids such as cholesterol and sphingolipids exert effects on channel activity either through direct protein-lipid interactions or by influencing the physical properties of the bilayer. Rafts also appear to selectively recruit interacting signalling molecules to generate subcellular compartments that may be important for efficient and selective signal transduction. Direct interaction with raft-associated scaffold proteins such as caveolin can also influence channel function by altering gating kinetics or by affecting trafficking and surface expression. Selective association of ion channels with specific lipid microenvironments within the membrane is thus likely to be an important and fundamental regulatory aspect of channel physiology. This brief review highlights some of the existing evidence for raft modulation of channel function.

Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+)‐dependent signalling and modulates various cardiac ion channels. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS). LQTS patients display prolonged ventricular recovery times (QT interval), increasing their risk of incurring life‐threatening arrhythmic events. Loss‐of‐function mutations to Kv7.1 (which drives the slow delayed rectifier potassium current, IKs, a key ventricular repolarising current) are the largest contributor to congenital LQTS (>50% of cases). CaM modulates Kv7.1 to produce a Ca2+‐sensitive IKs, but little is known about the consequences of LQTS‐associated CaM mutations on Kv7.1 function. Here, we present novel data characterising the biophysical and modulatory properties of three LQTS‐associated CaM variants (D95V, N97I and D131H). We showed that mutations induced structural alterations in CaM and reduced affinity for Kv7.1, when compared with wild‐type (WT). Using HEK293T cells expressing Kv7.1 channel subunits (KCNQ1/KCNE1) and patch‐clamp electrophysiology, we demonstrated that LQTS‐associated CaM variants reduced current density at systolic Ca2+ concentrations (1 μm), revealing a direct QT‐prolonging modulatory effect. Our data highlight for the first time that LQTS‐associated perturbations to CaM's structure impede complex formation with Kv7.1 and subsequently result in reduced IKs. This provides a novel mechanistic insight into how the perturbed structure–function relationship of CaM variants contributes to the LQTS phenotype. Key points: Calmodulin (CaM) is a ubiquitous, highly conserved calcium (Ca2+) sensor playing a key role in cardiac muscle contraction.Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS), a life‐threatening cardiac arrhythmia syndrome.LQTS‐associated CaM variants (D95V, N97I and D131H) induced structural alterations, altered binding to Kv7.1 and reduced IKs.Our data provide a novel mechanistic insight into how the perturbed structure–function relationship of CaM variants contributes to the LQTS phenotype. [ABSTRACT FROM AUTHOR]

Potassium (K+) channels, with their diversity, often tissue-defined distribution, and critical role in controlling cellular excitability, have long held promise of being important drug targets for the treatment of dysrhythmias in the heart and abnormal neuronal activity within the brain. With the exception of drugs that target one particular class, ATP-sensitive K+ (KATP) channels, very few selective K+ channel activators or inhibitors are currently licensed for clinical use in cardiovascular and neurological disease. Here we review what a range of human genetic disorders have told us about the role of specific K+ channel subunits, explore the potential of activators and inhibitors of specific channel populations as a therapeutic strategy, and discuss possible reasons for the difficulty in designing clinically relevant K+ channel modulators. [ABSTRACT FROM AUTHOR]

Many cell types have significant negative resting membrane potentials (RMPs) resulting from the activity of potassium-selective and chloride-selective ion channels. In excitable cells, such as neurones, rapid changes in membrane permeability underlie the generation of action potentials. Chondrocytes have less negative RMPs and the role of the RMP is not clear. Here we examine the basis of the chondrocyte RMP and possible physiological benefits. We demonstrate that maintenance of the chondrocyte RMP involves gadolinium-sensitive cation channels. Pharmacological inhibition of these channels causes the RMP to become more negative (100 µM gadolinium: ΔVm = −30 ± 4 mV). Analysis of the gadolinium-sensitive conductance reveals a high permeability to calcium ions (PCa/PNa ≈80) with little selectivity between monovalent ions; similar to that reported elsewhere for TRPV5. Detection of TRPV5 by PCR and immunohistochemistry and the sensitivity of the RMP to the TRPV5 inhibitor econazole (ΔVm = −18 ± 3 mV) suggests that the RMP may be, in part, controlled by TRPV5. We investigated the physiological advantage of the relatively positive RMP using a mathematical model in which membrane stretch activates potassium channels allowing potassium efflux to oppose osmotic water uptake. At very negative RMP potassium efflux is negligible, but at more positive RMP it is sufficient to limit volume increase. In support of our model, cells clamped at −80 mV and challenged with a reduced osmotic potential swelled approximately twice as much as cells at +10 mV. The positive RMP may be a protective adaptation that allows chondrocytes to respond to the dramatic osmotic changes, with minimal changes in cell volume. J. Cell. Physiol. 226: 2979-2986, 2011. © 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Inwardly rectifying potassium (Kir) channels are prime determinants of resting membrane potential in neurons. Their subcellular distribution and surface density thus help shape neuronal excitability, yet mechanisms governing the membrane targeting and localization of Kir channels are poorly understood. Here we report a direct interaction between the strong inward rectifier, Kir2.1, and a recently identified splice variant of postsynaptic density-93 (PSD-93), a protein involved the subcellular targeting of ion channels and glutamate receptors at excitatory synapses. Yeast two-hybrid screening of a human brain cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding the first two PDZ domains of PSD-93, but with an extended N-terminal region that diverged from other PSD-93 isoforms. This clone represented the human homologue of the mouse PSD-93 splice variant, PSD-93δ. Reverse transcription-polymerase chain reaction analysis showed diffuse low level PSD-93δ expression throughout the brain, with significantly higher levels in spinal cord. In vitro binding studies revealed that a type I PDZ recognition motif at the extreme C terminus of the Kir2.1 mediates interaction with all three PDZ domains of PSD-93δ, and association between Kir2 channels and PSD-93δ was confirmed further by the ability of anti- Kir2.1 antibodies to coimmunoprecipitate PSD-93δ from rat spinal cord lysates. Functionally, coexpression of Kir2.1 and PSD-93δ had no discernible effect upon channel kinetics but resulted in cell surface Kir2.1 clustering and suppression of channel internalization. We conclude that PSD-93δ is potentially an important regulator of the spatial and temporal distribution of Kir2 channels within neuronal membranes of the central nervous system. [ABSTRACT FROM AUTHOR]