scholarly journals The Role of S4 Charges in Voltage-dependent and Voltage-independent KCNQ1 Potassium Channel Complexes

2007 ◽  
Vol 129 (2) ◽  
pp. 121-133 ◽  
Author(s):  
Gianina Panaghie ◽  
Geoffrey W. Abbott
Keyword(s):  
Voltage Dependence ◽  
Single Amino Acid ◽  
Alanine Scanning Mutagenesis ◽  
Sequence Alignments ◽  
Voltage Gated ◽  
Constitutive Activation ◽  
Voltage Dependent ◽  
Α Subunits ◽  
Constitutively Active

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 α subunits to generate the voltage-dependent IKs channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium “leak” currents in gastrointestinal epithelia. Other Kv α subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2.

scholarly journals The Role of Na,k-Atpase α Subunit Serine 775 and Glutamate 779 in Determining the Extracellular K+And Membrane Potential–Dependent Properties of the Na,k -Pump

2000 ◽  
Vol 116 (1) ◽  
pp. 47-60 ◽  
Author(s):  
R. Daniel Peluffo ◽  
José M. Argüello ◽  
Joshua R. Berlin
Keyword(s):  
Membrane Potential ◽  
Voltage Dependence ◽  
Protein Structures ◽  
Pump Current ◽  
Transmembrane Segment ◽  
Α Subunit ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
Α1 Subunit

The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K -ATPase α subunit, in determining the voltage and extracellular K + (K +o) dependence of enzyme-mediated ion transport, were examined in this study. HeLa cells expressing the α1 subunit of sheep Na,K -ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+ (37°C). Na,K -pump current produced by the ouabain-resistant control enzyme (RD), containing amino acid substitutions Gln111Arg and Asn122Asp, displayed a membrane potential and K +o dependence similar to wild-type Na,K -ATPase during superfusion with 0 and 148 mM Na+-containing salt solutions. Additional substitution of alanine at Ser775 or Glu779 produced 155- and 15-fold increases, respectively, in the K +o concentration that half-maximally activated Na,K -pump current at 0 mV in extracellular Na+-free solutions. However, the voltage dependence of Na,K -pump current was unchanged in RD and alanine-substituted enzymes. Thus, large changes in apparent K +o affinity could be produced by mutations in the fifth transmembrane segment of the Na,K -ATPase with little effect on voltage-dependent properties of K + transport. One interpretation of these results is that protein structures responsible for the kinetics of K +o binding and/or occlusion may be distinct, at least in part, from those that are responsible for the voltage dependence of K +o binding to the Na,K -ATPase.

Identification of the target of gabapentinoid action in neuropathic pain

2018 ◽  
Author(s):  
Turo J. Nurmikko
Keyword(s):  
Neuropathic Pain ◽  
Scientific Community ◽  
Oral Absorption ◽  
Molecular Target ◽  
Wild Type ◽  
Seminal Paper ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels

The landmark paper discussed in this chapter is ‘Identification of the α‎2-δ‎-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin’, published by Field et al. in 2006. In this seminal paper, Field et al. demonstrated that the anti-allodynic effect of pregabalin is related to its binding to the α‎2δ‎-1 subunit of the voltage-gated calcium channel. In transgenic mice lacking this subunit, pregabalin had no effect on allodynia induced by sciatic nerve ligation, whereas, in wild-type mice, there was a substantial anti-allodynic response. This discovery was well received by the scientific community and was considered to conclusively establish the mechanism of action of pregabalin, which has remarkably similar properties to gabapentin but with increased potency and oral absorption. This exciting result acted as an impetus for further studies on the role of the subunit in the development and maintenance of neuropathic pain.

scholarly journals Neuropathic Pain Develops Normally in Mice Lacking both Nav1.7 and Nav1.8

2005 ◽  
Vol 1 ◽  
pp. 1744-8069-1-24 ◽  
Author(s):  
Mohammed A Nassar ◽  
Alessandra Levato ◽  
L Caroline Stirling ◽  
John N Wood
Keyword(s):  
Neuropathic Pain ◽  
Sodium Channels ◽  
Sensory Neurons ◽  
Inflammatory Pain ◽  
Pain Thresholds ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Pain Symptoms ◽  
Α Subunits

Two voltage gated sodium channel α-subunits, Nav1.7 and Nav1.8, are expressed at high levels in nociceptor terminals and have been implicated in the development of inflammatory pain. Mis-expression of voltage-gated sodium channels by damaged sensory neurons has also been implicated in the development of neuropathic pain, but the role of Nav1.7 and Nav1.8 is uncertain. Here we show that deleting Nav1.7 has no effect on the development of neuropathic pain. Double knockouts of both Nav1.7 and Nav1.8 also develop normal levels of neuropathic pain, despite a lack of inflammatory pain symptoms and altered mechanical and thermal acute pain thresholds. These studies demonstrate that, in contrast to the highly significant role for Nav1.7 in determining inflammatory pain thresholds, the development of neuropathic pain does not require the presence of either Nav1.7 or Nav1.8 alone or in combination.

scholarly journals The Role of a Conserved Lysine in Chloride- and Voltage-dependent ClC-0 Fast Gating

2007 ◽  
Vol 130 (4) ◽  
pp. 351-363 ◽  
Author(s):  
Anita M. Engh ◽  
José D. Faraldo-Gómez ◽  
Merritt Maduke
Keyword(s):  
Structural Changes ◽  
Computational Analysis ◽  
Voltage Dependence ◽  
Structural Information ◽  
Chloride Binding ◽  
Gate Opening ◽  
Voltage Dependent ◽  
Fast Gating ◽  
Homology Models

ClC-0 is a chloride channel whose gating is sensitive to voltage, chloride, and pH. In a previous publication, we showed that the K149C mutation causes a +70-mV shift in the voltage dependence of ClC-0 fast gating. In this paper we analyze the effects of a series of mutations at K149 on the voltage and chloride dependence of gating. By fitting our data to the previously proposed four-state model for ClC-0 fast gating, we show which steps in fast-gate opening are likely to be affected by these mutations. Computational analysis of mutant ClC-0 homology models show electrostatic contributions to chloride binding that may partially account for the effects of K149 on gating. The analysis of gating kinetics in combination with the available structural information suggests some of the structural changes likely to underpin fast-gate opening.

scholarly journals Irritable bowel syndrome patients have SCN5A channelopathies that lead to decreased NaV1.5 current and mechanosensitivity

2018 ◽  
Vol 314 (4) ◽  
pp. G494-G503 ◽  
Author(s):  
Peter R. Strege ◽  
Amelia Mazzone ◽  
Cheryl E. Bernard ◽  
Leila Neshatian ◽  
Simon J. Gibbons ◽  
...  
Keyword(s):  
Irritable Bowel Syndrome ◽  
Smooth Muscle ◽  
Voltage Dependence ◽  
Ethnically Diverse ◽  
Slow Waves ◽  
Missense Mutations ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Irritable Bowel ◽  
Negative Controls

The SCN5A-encoded voltage-gated mechanosensitive Na+ channel NaV1.5 is expressed in human gastrointestinal smooth muscle cells and interstitial cells of Cajal. NaV1.5 contributes to smooth muscle electrical slow waves and mechanical sensitivity. In predominantly Caucasian irritable bowel syndrome (IBS) patient cohorts, 2–3% of patients have SCN5A missense mutations that alter NaV1.5 function and may contribute to IBS pathophysiology. In this study we examined a racially and ethnically diverse cohort of IBS patients for SCN5A missense mutations, compared them with IBS-negative controls, and determined the resulting NaV1.5 voltage-dependent and mechanosensitive properties. All SCN5A exons were sequenced from somatic DNA of 252 Rome III IBS patients with diverse ethnic and racial backgrounds. Missense mutations were introduced into wild-type SCN5A by site-directed mutagenesis and cotransfected with green fluorescent protein into HEK-293 cells. NaV1.5 voltage-dependent and mechanosensitive functions were studied by whole cell electrophysiology with and without shear force. Five of 252 (2.0%) IBS patients had six rare SCN5A mutations that were absent in 377 IBS-negative controls. Six of six (100%) IBS-associated NaV1.5 mutations had voltage-dependent gating abnormalities [current density reduction (R225W, R433C, R986Q, and F1293S) and altered voltage dependence (R225W, R433C, R986Q, G1037V, and F1293S)], and at least one kinetic parameter was altered in all mutations. Four of six (67%) IBS-associated SCN5A mutations (R225W, R433C, R986Q, and F1293S) resulted in altered NaV1.5 mechanosensitivity. In this racially and ethnically diverse cohort of IBS patients, we show that 2% of IBS patients harbor SCN5A mutations that are absent in IBS-negative controls and result in NaV1.5 channels with abnormal voltage-dependent and mechanosensitive function. NEW & NOTEWORTHY The voltage-gated Na+ channel NaV1.5 contributes to smooth muscle physiology and electrical slow waves. In a racially and ethnically mixed irritable bowel syndrome cohort, 2% had mutations in the NaV1.5 gene SCN5A. These mutations were absent in irritable bowel syndrome-negative controls. Most mutant NaV1.5 channels were loss of function in voltage dependence or mechanosensitivity.

PTEN Regulates SYK-Directed AKT Activation in MCL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2941-2941
Author(s):  
Martina Rudelius ◽  
Stefania Pittaluga ◽  
Denise Sebasigari ◽  
Theresa Davies-Hill ◽  
Margit Klier ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Akt Pathway ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
Constitutive Activation ◽  
Survival Pathways ◽  
Mantle Cell ◽  
Constitutively Active

Abstract Abstract 2941 Poster Board II-917 Mantle cell lymphoma (MCL) can be divided into two clinicopathologic subtypes, a common or “typical” form, and an aggressive “blastoid” variant. A subset of MCL, including all of the aggressive blastoid variants, shows constitutive activation of the PI3K/AKT pathway. Since the BCR-associated SYK tyrosine kinase can activate the PI3K/AKT pathway during normal BCR signaling, we wished to assess its role in MCL. Five MCL cell lines (Granta 519, Rec-1, Jeko, Z138C and JVM-2) and 32 primary cases were studied for SYK activation and phosphorylation of several SYK targets, including blnk and PLC-gamma. The functional role of SYK was assessed by pharmacologic inhibition with R406, Piceatannol and SYK II inhibitor (2-(2-Aminoethylamino)-4-(3-trifluoromethyl-anilino)-pyrimidine-5-carboxamide, Dihydrochloride, Dihydrate). To rule out off target effects functional data was validated by siRNA experiments. Subsequently we analysed the effects of PTEN and constitutively active (S380A) PTEN in 2 cell lines (Granta 519 and Z138C). All cell lines were found to have constitutively activated SYK accompanied by phosphorylation of its downstream targets blnk and PLC-gamma. Inhibition of SYK with pharmacologic inhibitors or by specific siRNA resulted in AKT inactivation, suggesting a causal relationship between SYK activation and AKT activation in the cell lines. However, all primary MCL cases demonstrated constitutive activation of SYK, regardless of the activation state of AKT. Interestingly, p-AKT expression correlated with PTEN inactivation, as assessed by p-PTEN expression, in both primary cases and MCL cell lines. Increasing PTEN phosphatase activity, by introducing a constitutively active PTEN, abrogated AKT phosphorylation, without affecting SYK activity. These data suggest that SYK activity in MCL is necessary, but not sufficient to activate the PI3K/AKT pathway, and that an additional critical step is the inactivation of PTEN. This study reveals new complexity in the dysregulation of survival pathways in MCL that may be relevant to pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Reduced voltage dependence of inactivation in the SCN5A sodium channel mutation delF1617

2005 ◽  
Vol 288 (6) ◽  
pp. H2666-H2676 ◽  
Author(s):  
Tiehua Chen ◽  
Masashi Inoue ◽  
Michael F. Sheets
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Expression System ◽  
Single Amino Acid ◽  
Wild Type ◽  
Gating Charge ◽  
Voltage Curve ◽  
Voltage Dependent ◽  
Single Channel Currents

Deletion of a phenylalanine at position 1617 (delF1617) in the extracellular linker between segments S3 and S4 in domain IV of the human heart Na+ channel (hH1a) has been tentatively associated with long QT syndrome type 3 (LQT3). In a mammalian cell expression system, we compared whole cell, gating, and single-channel currents of delF1617 with those of wild-type hH1a. The half points of the peak activation-voltage curve for the two channels were similar, as were the deactivation time constants at hyperpolarized test potentials. However, delF1617 demonstrated a significant negative shift of −7 mV in the half point of the voltage-dependent Na+ channel availability curve compared with wild type. In addition, both the time course of decay of Na+ current ( INa) and two-pulse development of inactivation of delF1617 were faster at negative test potentials, whereas they tended to be slower at positive potentials compared with wild type. Mean channel open times for delF1617 were shorter at potentials <0 mV, whereas they were longer at potentials >0 mV compared with wild type. Using anthopleurin-A, a site-3 toxin that inhibits movement of segment S4 in domain IV (S4-DIV), we found that gating charge contributed by the S4-DIV in delF1617 was reduced 37% compared with wild type. We conclude that deletion of a single amino acid in the S3-S4 linker of domain IV alters the voltage dependence of fast inactivation via a reduction in the gating charge contributed by S4-DIV and can cause either a gain or loss of INa, depending on membrane potential.

scholarly journals Coupling of Voltage Sensing to Channel Opening Reflects Intrasubunit Interactions in Kv Channels

2004 ◽  
Vol 125 (1) ◽  
pp. 71-80 ◽  
Author(s):  
Alain J. Labro ◽  
Adam L. Raes ◽  
Dirk J. Snyders
Keyword(s):  
Voltage Dependence ◽  
Voltage Sensing ◽  
Α Subunit ◽  
K Channels ◽  
Voltage Gated ◽  
Kv Channels ◽  
Channel Opening ◽  
Subunit Stoichiometry ◽  
Voltage Dependent ◽  
Tetrameric Structure

Voltage-gated K+ channels play a central role in the modulation of excitability. In these channels, the voltage-dependent movement of the voltage sensor (primarily S4) is coupled to the (S6) gate that opens the permeation pathway. Because of the tetrameric structure, such coupling could occur within each subunit or between adjacent subunits. To discriminate between these possibilities, we analyzed various combinations of a S4 mutation (R401N) and a S6 mutation (P511G) in hKv1.5, incorporated into tandem constructs to constrain subunit stoichiometry. R401N shifted the voltage dependence of activation to negative potentials while P511G did the opposite. When both mutations were introduced in the same α-subunit of the tandem, the positive shift of P511G was compensated by the negative shift of R401N. With each mutation in a separate subunit of a tandem, this compensation did not occur. This suggests that for Kv channels, the coupling between voltage sensing and gating reflects primarily an intrasubunit interaction.

scholarly journals The NH2 terminus regulates voltage-dependent gating of CALHM ion channels

2017 ◽  
Vol 313 (2) ◽  
pp. C173-C186 ◽  
Author(s):  
Jessica E. Tanis ◽  
Zhongming Ma ◽  
J. Kevin Foskett
Keyword(s):  
Amino Acids ◽  
Ion Channels ◽  
Voltage Dependence ◽  
New Family ◽  
Voltage Dependent ◽  
Voltage Sensing Domain ◽  
Hyperpolarizing Shift ◽  
Voltage Relationship ◽  
Residual Conductance

Calcium homeostasis modulator protein-1 (CALHM1) and its Caenorhabditis elegans (ce) homolog, CLHM-1, belong to a new family of physiologically important ion channels that are regulated by voltage and extracellular Ca2+ (Ca2+o) but lack a canonical voltage-sensing domain. Consequently, the intrinsic voltage-dependent gating mechanisms for CALHM channels are unknown. Here, we performed voltage-clamp experiments on ceCLHM-1 chimeric, deletion, insertion, and point mutants to assess the role of the NH2 terminus (NT) in CALHM channel gating. Analyses of chimeric channels in which the ceCLHM-1 and human (h)CALHM1 NH2 termini were interchanged showed that the hCALHM1 NT destabilized channel-closed states, whereas the ceCLHM-1 NT had a stabilizing effect. In the absence of Ca2+o, deletion of up to eight amino acids from the ceCLHM-1 NT caused a hyperpolarizing shift in the conductance-voltage relationship with little effect on voltage-dependent slope. However, deletion of nine or more amino acids decreased voltage dependence and induced a residual conductance at hyperpolarized voltages. Insertion of amino acids into the NH2-terminal helix also decreased voltage dependence but did not prevent channel closure. Mutation of ceCLHM-1 valine 9 and glutamine 13 altered half-maximal activation and voltage dependence, respectively, in 0 Ca2+. In 2 mM Ca2+o, ceCLHM-1 NH2-terminal deletion and point mutant channels closed completely at hyperpolarized voltages with apparent affinity for Ca2+o indistinguishable from wild-type ceCLHM-1, although the ceCLHM-1 valine 9 mutant exhibited an altered conductance-voltage relationship and kinetics. We conclude that the NT plays critical roles modulating voltage dependence and stabilizing the closed states of CALHM channels.

The role of transmembrane domain 9 in substrate recognition by the fungal high-affinity glutathione transporters

2010 ◽  
Vol 429 (3) ◽  
pp. 593-602 ◽  
Author(s):  
Anil Thakur ◽  
Anand K. Bachhawat
Keyword(s):  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Substrate Recognition ◽  
Fold Increase ◽  
Alanine Scanning Mutagenesis ◽  
Sequence Alignments ◽  
Substrate Specificities ◽  
High Affinity ◽  
Key Features

Hgt1p, a high-affinity glutathione transporter from Saccharomyces cerevisiae belongs to the recently described family of OPTs (oligopeptide transporters), the majority of whose members still have unknown substrate specificity. To obtain insights into substrate recognition and translocation, we have subjected all 21 residues of TMD9 (transmembrane domain 9) to alanine-scanning mutagenesis. Phe523 was found to be critical for glutathione recognition, since F523A mutants showed a 4-fold increase in Km without affecting expression or localization. Phe523 and the previously identified polar residue Gln526 were on the same face of the helix suggesting a joint participation in glutathione recognition, whereas two other polar residues, Ser519 and Asn522, of TMD9, although also orientated on the same face, did not appear to be involved. The size and hydrophobicity of Phe523 were both key features of its functionality, as seen from mutational analysis. Sequence alignments revealed that Phe523 and Gln526 were conserved in a cluster of OPT homologues from different fungi. A second cluster contained isoleucine and glutamate residues in place of phenylalanine and glutamine residues, residues that are best tolerated in Hgt1p for glutathione transporter activity, when introduced together. The critical nature of the residues at these positions in TMD9 for substrate recognition was exploited to assign substrate specificities of several putative fungal orthologues present in these and other clusters. The presence of either phenylalanine and glutamine or isoleucine and glutamate residues at these positions correlated with their function as high-affinity glutathione transporters based on genetic assays and the Km of these transporters towards glutathione.

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