scholarly journals Syndromic deafness mutations at Asn 14 differentially alter the open stability of Cx26 hemichannels

2016 ◽  
Vol 148 (1) ◽  
pp. 25-42 ◽  
Author(s):  
Helmuth A. Sanchez ◽  
Nefeli Slavi ◽  
Miduturu Srinivas ◽  
Vytas K. Verselis
Keyword(s):  
Electrostatic Interactions ◽  
Ph Regulation ◽  
Transmembrane Protein ◽  
Cutaneous Manifestations ◽  
Gap Junction Channels ◽  
Substituted Cysteine Accessibility ◽  
Deactivation Kinetics ◽  
Cytoplasmic Extension ◽  
Voltage Dependent ◽  
Differential Properties

Connexin 26 (Cx26) is a transmembrane protein that forms hexameric hemichannels that can function when unopposed or dock to form intercellular gap junction channels. Aberrantly functioning unopposed hemichannels are a common feature of syndromic deafness associated with mutations in Cx26. In this study, we examine two different mutations at the same position in the N-terminal domain of Cx26, N14K and N14Y, which have been reported to produce different phenotypes in patients. We find that both N14K and N14Y, when expressed alone or together with wild-type (WT) Cx26, result in functional hemichannels with widely disparate functional properties. N14K currents are robust, whereas N14Y currents are small. The two mutants also exhibit opposite shifts in voltage-dependent loop gating, such that activation of N14K and N14Y is shifted in the hyperpolarizing and depolarizing directions, respectively. Deactivation kinetics suggests that N14K stabilizes and N14Y destabilizes the open state. Single N14K hemichannel recordings in low extracellular Ca2+ show no evidence of stable closing transitions associated with loop gating, and N14K hemichannels are insensitive to pH. Together, these properties cause N14K hemichannels to be particularly refractory to closing. Although we find that the unitary conductance of N14K is indistinguishable from WT Cx26, mutagenesis and substituted cysteine accessibility studies suggest that the N14 residue is exposed to the pore and that the differential properties of N14K and N14Y hemichannels likely result from altered electrostatic interactions between the N terminus and the cytoplasmic extension of TM2 in the adjacent subunit. The combined effects that we observe on loop gating and pH regulation may explain the unusual buccal cutaneous manifestations in patients carrying the N14K mutation. Our work also provides new considerations regarding the underlying molecular mechanism of loop gating, which controls hemichannel opening in the plasma membrane.

scholarly journals Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 475-488 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Katja Kohler ◽  
Andrea Bacconi ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Reaction Rates ◽  
Transport Pathway ◽  
Extracellular Medium ◽  
Voltage Range ◽  
Substituted Cysteine Accessibility ◽  
Voltage Dependent ◽  
Opposite Behavior ◽  
Rate Limiting ◽  
Kinetics Of

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

scholarly journals Differential voltage-dependent modulation of the ACh-gated K+ current by adenosine and acetylcholine

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261960
Author(s):  
Ana Laura López-Serrano ◽  
Rodrigo Zamora-Cárdenas ◽  
Iván A. Aréchiga-Figueroa ◽  
Pedro D. Salazar-Fajardo ◽  
Tania Ferrer ◽  
...  
Keyword(s):  
Voltage Dependence ◽  
Gradual Increase ◽  
Relaxation Property ◽  
Voltage Sensitivity ◽  
Response Curves ◽  
Deactivation Kinetics ◽  
Relative Affinity ◽  
Voltage Dependent ◽  
K Current ◽  
Inhibitory Regulation

Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the ‘relaxation’ property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.

scholarly journals Shisa7 is a GABAA receptor auxiliary subunit controlling benzodiazepine actions

Science ◽  
2019 ◽  
Vol 366 (6462) ◽  
pp. 246-250 ◽  
Author(s):  
Wenyan Han ◽  
Jun Li ◽  
Kenneth A. Pelkey ◽  
Saurabh Pandey ◽  
Xiumin Chen ◽  
...  
Keyword(s):  
Transmembrane Protein ◽  
Clinical Importance ◽  
Aminobutyric Acid ◽  
Inhibitory Synapses ◽  
Channel Pore ◽  
Auxiliary Subunit ◽  
Deactivation Kinetics ◽  
Acid Type ◽  
Genetic Deletion ◽  
The Brain

The function and pharmacology of γ-aminobutyric acid type A receptors (GABAARs) are of great physiological and clinical importance and have long been thought to be determined by the channel pore–forming subunits. We discovered that Shisa7, a single-passing transmembrane protein, localizes at GABAergic inhibitory synapses and interacts with GABAARs. Shisa7 controls receptor abundance at synapses and speeds up the channel deactivation kinetics. Shisa7 also potently enhances the action of diazepam, a classic benzodiazepine, on GABAARs. Genetic deletion of Shisa7 selectively impairs GABAergic transmission and diminishes the effects of diazepam in mice. Our data indicate that Shisa7 regulates GABAAR trafficking, function, and pharmacology and reveal a previously unknown molecular interaction that modulates benzodiazepine action in the brain.

scholarly journals Properties of a nonjunctional current expressed from a rat connexin46 cDNA in Xenopus oocytes.

1993 ◽  
Vol 102 (1) ◽  
pp. 59-74 ◽  
Author(s):  
L Ebihara ◽  
E Steiner
Keyword(s):  
Xenopus Oocytes ◽  
Inactivation Kinetics ◽  
Induced Current ◽  
Strongly Coupled ◽  
Deactivation Kinetics ◽  
Voltage Dependent ◽  
Junctional Protein ◽  
Gap Junctional ◽  
Kinetics Of ◽  
External Calcium

Connexin46 (cxn46) is a gap junctional protein that was cloned from a rat lens cDNA library. Expression of cxn46 in solitary Xenopus oocytes resulted in the development of a large time- and voltage-dependent current that was not observed in noninjected control oocytes or in oocytes injected with mRNA for cxn43 or cxn32. The cxn46-induced current activated at potentials positive to -20 mV. On repolarization to -40 mV, the current deactivated over a period of several seconds. Removal of external calcium caused a marked increase in the amplitude of the cxn46-induced current, shifted the steady-state activation curve to more negative potentials, and altered the kinetics of activation and deactivation. Increasing external calcium had the opposite effect. The ability of cxn46 to induce the formation of cell-to-cell channels was tested in the oocyte pair system. Oocyte pairs injected with cxn46 mRNA + antisense oligonucleotides for Xenopus cxn38 were strongly coupled. In contrast, oocyte pairs injected with antisense alone showed no coupling. The inactivation kinetics of the gap junctional channels resembled the deactivation kinetics of the cxn46-induced current in solitary oocytes.

scholarly journals Discovery and characterization of Hv1-type proton channels in reef-building corals.

2021 ◽  
Author(s):  
Gisela Rangel-Tescas ◽  
Cecilia Cervantes ◽  
Miguel A Cervantes-Rocha ◽  
Esteban Suarez-Delgado ◽  
Anastazia T Banaszak ◽  
...  
Keyword(s):  
Voltage Dependence ◽  
Ph Regulation ◽  
Functional Characterization ◽  
Gene Encoding ◽  
Proton Channels ◽  
Gating Model ◽  
Mechanistic Insight ◽  
Voltage Dependent ◽  
Insight Into

Voltage-dependent proton-permeable channels are membrane proteins mediating a number of important physiological functions. Here we report the presence of a gene encoding for Hv1 voltage-dependent, proton-permeable channels in two species of reef-building corals. We performed a characterization of their biophysical properties and found that these channels are fast-activating and modulated by the pH gradient in a manner that makes them interesting models for studying these processes more easily. We have also developed an allosteric gating model that provides mechanistic insight into the modulation of voltage-dependence by protons. This work also represents the first functional characterization of any ion channel in scleractinian corals. We discuss the implications of the presence of these channels in the membranes of coral cells in the calcification and pH regulation processes and possible consequences of ocean acidification related to the function of these channels.

scholarly journals PtdIns4P-mediated electrostatic forces influence S-acylation of peripheral proteins at the Golgi complex

2020 ◽  
Vol 40 (1) ◽  
Author(s):  
Sabrina Chumpen Ramirez ◽  
Micaela R. Astrada ◽  
Jose L. Daniotti
Keyword(s):  
Amino Acids ◽  
Golgi Complex ◽  
Electrostatic Interactions ◽  
Transmembrane Protein ◽  
Basic Amino Acid ◽  
Soluble Proteins ◽  
Post Translational Modification ◽  
Hydrophobic Region ◽  
Membrane Interaction ◽  
Basic Amino Acids

Abstract Protein S-acylation is a reversible post-translational modification involving the addition of fatty acids to cysteines and is catalyzed by transmembrane protein acyltransferases (PATs) mainly expressed at the Golgi complex. In case of soluble proteins, S-acylation confers stable membrane attachment. Myristoylation or farnesylation of many soluble proteins constitutes the initial transient membrane adsorption step prior to S-acylation. However, some S-acylated soluble proteins, such as the neuronal growth-associated protein Growth-associated protein-43 (GAP-43), lack the hydrophobic modifications required for this initial membrane interaction. The signals for GAP-43 S-acylation are confined to the first 13 amino acids, including the S-acylatable cysteines 3 and 4 embedded in a hydrophobic region, followed by a cluster of basic amino acids. We found that mutation of critical basic amino acids drastically reduced membrane interaction and hence S-acylation of GAP-43. Interestingly, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) at the Golgi complex reduced GAP-43 membrane binding, highlighting a new, pivotal role for this anionic lipid and supporting the idea that basic amino acid residues are involved in the electrostatic interactions between GAP-43 and membranes of the Golgi complex where they are S-acylated.

Properties of gap junction channels formed of connexin 45 endogenously expressed in human hepatoma (SKHep1) cells

1995 ◽  
Vol 268 (2) ◽  
pp. C356-C365 ◽  
Author(s):  
A. P. Moreno ◽  
J. G. Laing ◽  
E. C. Beyer ◽  
D. C. Spray
Keyword(s):  
Gap Junction ◽  
Voltage Dependence ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Voltage Sensitivity ◽  
Gap Junction Channels ◽  
Unitary Conductance ◽  
Junction Protein ◽  
Voltage Dependent ◽  
Gap Junctional

We have evaluated the voltage dependence and unitary conductance of gap junctional channels that were recorded in a clone isolated from the hepatoma cell line SKHep1. In this clonal population (designated SKHep1A), Northern blots, immunoprecipitation, and immunohistochemical staining demonstrated the expression of connexin (Cx) 45; no other gap junction protein was identified by these techniques, although weak hybridization with Cx40 was detected. Macroscopic junctional conductance (gj) in these cells was low, averaging 1.3 nS, and was steeply voltage dependent. Parameters of voltage sensitivity were as follows: voltage at which voltage-sensitive conductance is reduced by 50%, 13.4 mV; steepness of relation, 0.115 (corresponding to 2.7 gating charges), and voltage-insensitive fraction of residual to total conductance approximately 0.06. Unitary conductance (gamma j) of these junctional channels averaged 32 +/- 8 pS; although gamma j was independent of transjunctional voltage (Vj), at high Vj values (> 50 mV), smaller conductance values were also detected. Open probabilities of the 30-pS channels at various Vj values closely matched the predicted voltage-dependent component of macroscopic gj, the residual conductance at high Vj might be attributable to the smaller conductance events. The voltage dependence of human Cx45 gap junction channels is as steep as that seen for channels formed by Xenopus Cx38 and is much steeper than that previously reported for channels formed of the highly homologous chick Cx45 and for other mammalian connexins expressed either endogenously or exogenously.

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