Channels (Austin, Tex.)最新文献

Voltage-gated calcium channels (VGCCs) are critical for Ca²⁺ influx into all types of excitable cells, but their exact function is still poorly understood. Recent reconstruction of homology models for all human VGCCs at atomic resolution provides the opportunity for a structure-based discussion of VGCC function and novel insights into the mechanisms underlying Ca²⁺ selective flux through these channels. In the present review, we use these data as a basis to examine the structure, function, and Zn²⁺-induced modulation of Ca_v2.3 VGCCs, which mediate native R-type currents and belong to the most enigmatic members of the family. Their unique sensitivity to Zn²⁺ and the existence of multiple mechanisms of Zn²⁺ action strongly argue for a role of these channels in the modulatory action of endogenous loosely bound Zn²⁺, pools of which have been detected in a number of neuronal, endocrine, and reproductive tissues. Following a description of the different mechanisms by which Zn²⁺ has been shown or is thought to alter the function of these channels, we discuss their potential (patho)physiological relevance, taking into account what is known about the magnitude and function of extracellular Zn²⁺ signals in different tissues. While still far from complete, the picture that emerges is one where Ca_v2.3 channel expression parallels the occurrence of loosely bound Zn²⁺ pools in different tissues and where these channels may serve to translate physiological Zn²⁺ signals into changes of electrical activity and/or intracellular Ca²⁺ levels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed throughout the mammalian central nervous system (CNS). These channels have been implicated in a wide range of diseases, including Major Depressive Disorder and multiple subtypes of epilepsy. The diversity of functions that HCN channels perform is in part attributable to differences in their subcellular localization. To facilitate a broad range of subcellular distributions, HCN channels are bound by auxiliary subunits that regulate surface trafficking and channel function. One of the best studied auxiliary subunits is tetratricopeptide-repeat containing, Rab8b-interacting protein (TRIP8b). TRIP8b is an extensively alternatively spliced protein whose only known function is to regulate HCN channels. TRIP8b binds to HCN pore-forming subunits at multiple interaction sites that differentially regulate HCN channel function and subcellular distribution. In this review, we summarize what is currently known about the structure and function of TRIP8b isoforms with an emphasis on the role of this auxiliary subunit in health and disease.

The voltage-gated calcium channel Ca_V1.1a primarily functions as voltage-sensor in skeletal muscle excitation-contraction (EC) coupling. In embryonic muscle the splice variant Ca_V1.1e, which lacks exon 29, additionally function as a genuine L-type calcium channel. Because previous work in most laboratories used a Ca_V1.1 expression plasmid containing a single amino acid substitution (R165K) of a critical gating charge in the first voltage-sensing domain (VSD), we corrected this substitution and analyzed its effects on the gating properties of the L-type calcium currents in dysgenic myotubes. Reverting K165 to R right-shifted the voltage-dependence of activation by ~12 mV in both Ca_V1.1 splice variants without changing their current amplitudes or kinetics. This demonstrates the exquisite sensitivity of the voltage-sensor function to changes in the specific amino acid side chains independent of their charge. Our results further indicate the cooperativity of VSDs I and IV in determining the voltage-sensitivity of Ca_V1.1 channel gating.

Voltage-gated Kv1.1 potassium channel α-subunits are broadly expressed in the nervous system where they act as critical regulators of neuronal excitability. Mutations in the KCNA1 gene, which encodes Kv1.1, are associated with the neurological diseases episodic ataxia and epilepsy. Studies in mouse models have shown that Kv1.1 is important for neural control of the heart and that Kcna1 deletion leads to cardiac dysfunction that appears to be brain-driven. Traditionally, KCNA1 was not believed to be expressed in the heart. However, recent studies have revealed that Kv1.1 subunits are not only present in cardiomyocytes, but that they also make an important heart-intrinsic functional contribution to outward K⁺ currents and action potential repolarization. This review recounts the winding history of discovery of KCNA1 gene expression and neurocardiac function from fruit flies to mammals and from brain to heart and looks at some of the salient questions that remain to be answered regarding emerging cardiac roles of Kv1.1.

Ion channels display conformational changes in response to binding of their agonists and antagonists. The study of the relationships between the structure and the function of these proteins has witnessed considerable advances in the last two decades using a combination of techniques, which include electrophysiology, optical approaches (i.e. patch clamp fluorometry, incorporation of non-canonic amino acids, etc.), molecular biology (mutations in different regions of ion channels to determine their role in function) and those that have permitted the resolution of their structures in detail (X-ray crystallography and cryo-electron microscopy). The possibility of making correlations among structural components and functional traits in ion channels has allowed for more refined conclusions on how these proteins work at the molecular level. With the cloning and description of the family of Transient Receptor Potential (TRP) channels, our understanding of several sensory-related processes has also greatly moved forward. The response of these proteins to several agonists, their regulation by signaling pathways as well as by protein-protein and lipid-protein interactions and, in some cases, their biophysical characteristics have been studied thoroughly and, recently, with the resolution of their structures, the field has experienced a new boom. This review article focuses on the conformational changes in the pores, concentrating on some members of the TRP family of ion channels (TRPV and TRPA subfamilies) that result in changes in their single-channel conductances, a phenomenon that may lead to fine-tuning the electrical response to a given agonist in a cell.

Objective: To verify the diagnosis of channelopathies in two families and explore the mechanism of the overlap between periodic paralysis (PP) and paramyotonia congenita (PMC).

Methods: We have studied two cases with overlapping symptoms of episodic weakness and stiffness in our clinical center using a series of assessment including detailed medical history, careful physical examination, laboratory analyses, muscle biopsy, electrophysiological evaluation, and genetic analysis.

Results: The first proband and part of his family with the overlap of PMC and hyperkalemic periodic paralysis (HyperPP) has been identified as c.2111C > T (T704M) substitution of the gene SCN4A. The second proband and part of his family with the overlap of PMC and hypokalemic periodic paralysis type 2 (HypoPP2) has been identified as c.4343G > A (R1448H) substitution of the gene SCN4A. In addition, one member of the second family with overlapping symptoms has been identified as a novel mutation c.2111C > T without the mutation c.4343G > A.

Conclusions: SCN4A gene mutations can cause the overlap of PMC and PP (especially the HypoPP2). The clinical symptoms of episodic weakness and stiffness could happen at a different time or temperature. Based on diagnosis assessments such as medical history and muscle biopsy, further evaluations on long-time exercise test, genetic analysis, and patch clamp electrophysiology test need to be done in order to verify the specific subtype of channelopathies. Furthermore, the improvement of one member in the pregnancy period can be used as a reference for the other female in the child-bearing period with T704M.

SCN5A is expressed in cardiomyocytes and gastrointestinal (GI) smooth muscle cells (SMCs) as the voltage-gated mechanosensitive sodium channel Na_V1.5. The influx of Na⁺ through Na_V1.5 produces a fast depolarization in membrane potential, indispensable for electrical excitability in cardiomyocytes and important for electrical slow waves in GI smooth muscle. As such, abnormal Na_V1.5 voltage gating or mechanosensitivity may result in channelopathies. SCN5A mutation G615E - found separately in cases of acquired long-QT syndrome, sudden cardiac death, and irritable bowel syndrome - has a relatively minor effect on Na_V1.5 voltage gating. The aim of this study was to test whether G615E impacts mechanosensitivity. Mechanosensitivity of wild-type (WT) or G615E-Na_V1.5 in HEK-293 cells was examined by shear stress on voltage- or current-clamped whole cells or pressure on macroscopic patches. Unlike WT, voltage-clamped G615E-Na_V1.5 showed a loss in shear- and pressure-sensitivity of peak current yet a normal leftward shift in the voltage-dependence of activation. In current-clamp, shear stress led to a significant increase in firing spike frequency with a decrease in firing threshold for WT but not G615E-Na_V1.5. Our results show that the G615E mutation leads to functionally abnormal Na_V1.5 channels, which cause disruptions in mechanosensitivity and mechano-electrical feedback and suggest a potential contribution to smooth muscle pathophysiology.

Mechanosensitive ion channels are membrane gated pores which are activated by mechanical stimuli. The focus of this study is on Piezo1, a newly discovered, large, mammalian, mechanosensitive ion channel, which has been linked to diseases such as dehydrated hereditary stomatocytosis (Xerocytosis) and lymphatic dysplasia. Here we utilize an established in-vitro artificial bilayer system to interrogate single Piezo1 channel activity. The droplet-hydrogel bilayer (DHB) system uniquely allows the simultaneous recording of electrical activity and fluorescence imaging of labelled protein. We successfully reconstituted fluorescently labelled Piezo1 ion channels in DHBs and verified activity using electrophysiology in the same system. We demonstrate successful insertion and activation of hPiezo1-GFP in bilayers of varying composition. Furthermore, we compare the Piezo1 bilayer reconstitution with measurements of insertion and activation of KcsA channels to reproduce the channel conductances reported in the literature. Together, our results showcase the use of DHBs for future experiments allowing simultaneous measurements of ion channel gating while visualising the channel proteins using fluorescence.