Functional diversity of potassium channel voltage-sensing domains

Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology.     

Modulation of Cav3.2 T-type calcium channel permeability by asparagine-linked glycosylation

Low-voltage-gated T-type calcium channels are expressed throughout the nervous system where they play an essential role in shaping neuronal excitability. Defects in T-type channel expression have been linked to various neuronal disorders including neuropathic pain and epilepsy. Currently, little is known about the cellular mechanisms controlling the expression and function of T-type channels. Asparagine-linked glycosylation has recently emerged as an essential signaling pathway by which the cellular environment can control expression of T-type channels. However, the role of N-glycans in the conducting function of T-type channels remains elusive. In the present study, we used human Ca_v3.2 glycosylation-deficient channels to assess the role of N-glycosylation on the gating of the channel. Patch-clamp recordings of gating currents revealed that N-glycans attached to hCa_v3.2 channels have a minimal effect on the functioning of the channel voltage-sensor. In contrast, N-glycosylation on specific asparagine residues may have an essential role in the conducting function of the channel by enhancing the channel permeability and / or the pore opening of the channel. Our data suggest that modulation of N-linked glycosylation of hCa_v3.2 channels may play an important physiological role, and could also support the alteration of T-type currents observed in disease states.     

Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels

Voltage-gated potassium channels are important regulators of electrical excitation in many tissues, with Kv1.2 standing out as an essential contributor in the CNS. Genetic deletion of Kv1.2 invariably leads to early lethality in mice. In humans, mutations affecting Kv1.2 function are linked to epileptic encephalopathy and movement disorders. We have demonstrated that Kv1.2 is subject to a unique regulatory mechanism in which repetitive stimulation leads to dramatic potentiation of current. In this study, we explore the properties and molecular determinants of this use-dependent potentiation/activation. First, we examine how alterations in duty cycle (depolarization and repolarization/recovery times) affect the onset and extent of use-dependent activation. Also, we use trains of repetitive depolarizations to test the effects of a variety of Thr252 (S2-S3 linker) mutations on use-dependent activation. Substitutions of Thr with some sterically similar amino acids (Ser, Val, and Met, but not Cys) retain use-dependent activation, while bulky or charged amino acid substitutions eliminate use-dependence. Introduction of Thr at the equivalent position in other Kv1 channels (1.1, 1.3, 1.4), was not sufficient to transfer the phenotype. We hypothesize that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulator that binds preferentially to the channel closed state, with Thr252 being necessary but not sufficient for this interaction to alter channel function. These findings extend the conclusions of our recent demonstration of use-dependent activation of Kv1.2-containing channels in hippocampal neurons, by adding new details about the molecular mechanism underlying this effect.     

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.     

Three pairs of weak interactions precisely regulate the G‐loop gate of Kir2.1 channel

Kir2.1 (also known as IRK1) plays key roles in regulation of resting membrane potential and cell excitability. To achieve its physiological roles, Kir2.1 performs a series of conformational transition, named as gating. However, the structural basis of gating is still obscure. Here, we combined site‐directed mutation, two‐electrode voltage clamp with molecular dynamics simulations and determined that H221 regulates the gating process of Kir2.1 by involving a weak interaction network. Our data show that the H221R mutant accelerates the rundown kinetics and decelerates the reactivation kinetics of Kir2.1. Compared with the WT channel, the H221R mutation strengthens the interaction between the CD‐ and G‐loops (E303‐R221) which stabilizes the close state of the G‐loop gate and weakens the interactions between C‐linker and CD‐loop (R221‐R189) and the adjacent G‐loops (E303‐R312) which destabilizes the open state of G‐loop gate. Our data indicate that the three pairs of interactions (E303‐H221, H221‐R189 and E303‐R312) precisely regulate the G‐loop gate by controlling the conformation of G‐loop. Proteins 2016; 84:1929–1937. © 2016 Wiley Periodicals, Inc.     

L‐type voltage‐gated calcium channels in stem cells and tissue engineering

L‐type voltage‐gated calcium ion channels (L‐VGCCs) have been demonstrated to be the mediator of several significant intracellular activities in excitable cells, such as neurons, chromaffin cells and myocytes. Recently, an increasing number of studies have investigated the function of L‐VGCCs in non‐excitable cells, particularly stem cells. However, there appear to be no systematic reviews of the relationship between L‐VGCCs and stem cells, and filling this gap is prescient considering the contribution of L‐VGCCs to the proliferation and differentiation of several types of stem cells. This review will discuss the possible involvement of L‐VGCCs in stem cells, mainly focusing on osteogenesis mediated by mesenchymal stem cells (MSCs) from different tissues and neurogenesis mediated by neural stem/progenitor cells (NSCs). Additionally, advanced applications that use these channels as the target for tissue engineering, which may offer the hope of tissue regeneration in the future, will also be explored.     

Efficient Pre-mRNA Cleavage Prevents Replication-Stress-Associated Genome Instability

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Mutations in DIIS5 and the DIIS4-S5 linker of Drosophila melanogaster sodium channel define binding domains for pyrethroids and DDT

Mutations in the DIIS4-S5 linker and DIIS5 have identified hotspots of pyrethroid and DDT interaction with the Drosophila para sodium channel. Wild-type and mutant channels were expressed in Xenopus oocytes and subjected to voltage-clamp analysis. Substitutions L914I, M918T, L925I, T929I and C933A decreased deltamethrin potency, M918T, L925I and T929I decreased permethrin potency and T929I, L925I and I936V decreased fenfluthrin potency. DDT potency was unaffected by M918T, but abolished by T929I and reduced by L925I, L932F and I936V, suggesting that DIIS5 contains at least part of the DDT binding domain. The data support a computer model of pyrethroid and DDT binding.