scholarly journals Single-Channel Characteristics of Wild-Type IKs Channels and Channels formed with Two MinK Mutants that Cause Long QT Syndrome

1998 ◽  
Vol 112 (6) ◽  
pp. 651-663 ◽  
Author(s):  
Federico Sesti ◽  
Steve A.N. Goldstein
Keyword(s):  
Long Qt Syndrome ◽  
Single Channel ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Long Qt ◽  
Conduction Pathway ◽  
Qt Syndrome ◽  
Single Channel Currents

IKs channels are voltage dependent and K+ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, IKs channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human IKs channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel “flicker.” At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of IKs channels was ∼16 pS (corresponding to ∼0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant IKs channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K+ flux through IKs channels due to lowered single-channel conductance and altered gating.

A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating

1997 ◽  
Vol 273 (4) ◽  
pp. F516-F529 ◽  
Author(s):  
Han Choe ◽  
Hao Zhou ◽  
Lawrence G. Palmer ◽  
Henry Sackin
Keyword(s):  
Single Channel ◽  
Ph Sensitivity ◽  
Channel Noise ◽  
Wild Type ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Hydrophobic Region ◽  
K Secretion ◽  
Single Channel Currents

ROMK channels play a key role in overall K balance by controlling K secretion across the apical membrane of mammalian cortical collecting tubule. In contrast to the family of strong inward rectifiers (IRKs), ROMK channels are markedly sensitive to intracellular pH. Using Xenopus oocytes, we have confirmed this pH sensitivity at both the single-channel and whole cell level. Reduction of oocyte pH from 6.8 to 6.4 (using a permeant acetate buffer) reduced channel open probability from 0.76 ± 0.02 to near zero ( n = 8), without altering single-channel conductance. This was due to the appearance of a long-lived closed state at low internal pH. We have confirmed that a lysine residue (K61 on ROMK2; K80 on ROMK1), NH2 terminal to the first putative transmembrane segment (M1), is primarily responsible for conferring a steep pH sensitivity to ROMK (B. Fakler, J. Schultz, J. Yang, U. Schulte, U. Bråandle, H. P. Zenner, L. Y. Jan, and J. P. Ruppersberg. EMBO J. 15: 4093–4099, 1996). However, the apparent p K a of ROMK also depends on another residue in a highly conserved, mildly hydrophobic area: T51 on ROMK2 (T70 on ROMK1). Replacing this neutral threonine (T51) with a negatively charged glutamate shifted the apparent p K a for inward conductance from 6.5 ± 0.01 ( n = 8, wild type) to 7.0 ± 0.02 ( n = 5, T51E). On the other hand, replacing T51 with a positively charged lysine shifted the apparent p K a in the opposite direction, from 6.5 ± 0.01 ( n = 8, wild type) to 6.0 ± 0.02 ( n = 9, T51K). The opposite effects of the glutamate and lysine substitutions at position 51 (ROMK2) are consistent with a model in which T51 is physically close to K61 and alters either the local pH or the apparent p K a via an electrostatic mechanism. In addition to its effects on pH sensitivity, the mutation T51E also decreased single-channel conductance from 34.0 ± 1.0 pS ( n = 8, wild type) to 17.4 ± 1 pS ( n = 9, T51E), reversed the voltage gating of the channel, and significantly increased open-channel noise. These effects on single-channel currents suggest that the T51 residue, located in a mildly hydrophobic area of ROMK2, also interacts with the hydrophobic region of the permeation pathway.

scholarly journals Anion permeation in an apical membrane chloride channel of a secretory epithelial cell.

1992 ◽  
Vol 99 (3) ◽  
pp. 339-366 ◽  
Author(s):  
D R Halm ◽  
R A Frizzell
Keyword(s):  
Epithelial Cell ◽  
Single Channel ◽  
Apical Membrane ◽  
Epithelial Cell Line ◽  
Low Permeability ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Conduction Pathway ◽  
Single Channel Currents ◽  
Secretory Epithelial Cell

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.

scholarly journals Intrinsic Electrostatic Potential in the BK Channel Pore: Role in Determining Single Channel Conductance and Block

2008 ◽  
Vol 131 (2) ◽  
pp. 147-161 ◽  
Author(s):  
Ingrid Carvacho ◽  
Wendy Gonzalez ◽  
Yolima P. Torres ◽  
Sebastian Brauchi ◽  
Osvaldo Alvarez ◽  
...  
Keyword(s):  
Electrostatic Potential ◽  
Single Channel ◽  
Molecular Model ◽  
Bk Channels ◽  
Bk Channel ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Electrostatic Effect

The internal vestibule of large-conductance Ca2+ voltage-activated K+ (BK) channels contains a ring of eight negative charges not present in K+ channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017–9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K+ was 18% lower than the control. Current–voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K+ (30 mM) and vanished at high K+ (1 M K+). We determine the electrostatic potential change, Δφ, caused by the charge neutralization using TEA+ block for the extracellular charges and Ba2+ for intracellular charges. We measured 13 ± 2 mV for Δφ at the TEA+ site when turning off the extracellular charges, and 17 ± 2 mV for the Δφ at the Ba2+ site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Δφ using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.

scholarly journals How does ryanodine modify ion handling in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel?

1994 ◽  
Vol 104 (3) ◽  
pp. 425-447 ◽  
Author(s):  
A R Lindsay ◽  
A Tinker ◽  
A J Williams
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Relative Permeability ◽  
Single Channel ◽  
Divalent Cations ◽  
Monovalent Cations ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Release Channel ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Conduction Pathway

Under appropriate conditions, the interaction of the plant alkaloid ryanodine with a single cardiac sarcoplasmic reticulum Ca(2+)-release channel results in a profound modification of both channel gating and conduction. On modification, the channel undergoes a dramatic increase in open probability and a change in single-channel conductance. In this paper we aim to provide a mechanistic framework for the interpretation of the altered conductance seen after ryanodine binding to the channel protein. To do this we have characterized single-channel conductance with representative members of three classes of permeant cation; group 1a monovalent cations, alkaline earth divalent cations, and organic monovalent cations. We have quantified the change in single-channel conductance induced by ryanodine and have expressed this as a fraction of conductance in the absence of ryanodine. Fractional conductance seen in symmetrical 210 mM solutions is not fixed but varies with the nature of the permeant cation. The group 1a monovalent cations (K+, Na+, Cs+, Li+) have values of fractional conductance in a narrow range (0.60-0.66). With divalent cations fractional conductance is considerably lower (Ba2+, 0.22 and Sr2+, 0.28), whereas values of fractional conductance vary considerably with the organic monovalent cations (ammonia 0.66, ethylamine 0.76, propanolamine 0.65, diethanolamine 0.92, diethylamine 1.2). To establish the mechanisms governing these differences, we have monitored the affinity of the conduction pathway for, and the relative permeability of, representative cations in the ryanodine-modified channel. These parameters have been compared with those obtained in previous studies from this laboratory using the channel in the absence of ryanodine and have been modeled by modifying our existing single-ion, four-barrier three-well rate theory model of conduction in the unmodified channel. Our findings indicate that the high affinity, essentially irreversible, interaction of ryanodine with the cardiac sarcoplasmic reticulum Ca(2+)-release channel produces a conformational alteration of the protein which results in modified ion handling. We suggest that, on modification, the affinity of the channel for the group 1a monovalent cations is increased while the relative permeability of this class of cations remains essentially unaltered. The affinity of the conduction pathway for the alkaline earth divalent cations is also increased, however the relative permeability of this class of cations is reduced compared to the unmodified channel. The influence of modification on the handling by the channel of the organic monovalent cations is determined by both the size and the nature of the cation.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Control of Single Channel Conductance in the Outer Vestibule of the Kv2.1 Potassium Channel

2006 ◽  
Vol 128 (2) ◽  
pp. 231-246 ◽  
Author(s):  
Josef G. Trapani ◽  
Payam Andalib ◽  
Joseph F. Consiglio ◽  
Stephen J. Korn
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Markov Modeling ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Current Magnitude ◽  
Hidden Markov Modeling ◽  
Outer Vestibule ◽  
Dependent Change ◽  
Conduction Pathway

Current magnitude in Kv2.1 potassium channels is modulated by external [K+]. In contrast to behavior expected from the change in electrochemical driving force, outward current through Kv2.1 channels becomes larger when extracellular [K+] is increased within the physiological range. The mechanism that underlies this unusual property involves the opening of Kv2.1 channels into one of two different outer vestibule conformations, which are defined by their sensitivity to TEA. Channels that open into a TEA-sensitive conformation generate larger macroscopic currents, whereas channels that open into a TEA-insensitive conformation generate smaller macroscopic currents. At higher [K+], more channels open into the TEA-sensitive conformation. In this manuscript, we examined the mechanism by which the conformational change produced a change in current magnitude. We started by testing the simplest hypothesis: that each pharmacologically defined channel conformation produces a different single channel conductance, one smaller and one larger, and that the [K+]-dependent change in current magnitude reflects the [K+]-dependent change in the percentage of channels that open into each of the two conformations. Using single channel and macroscopic recordings, as well as hidden Markov modeling, we were able to quantitatively account for [K+]-dependent regulation of macroscopic current with this model. Combined with previously published work, these results support a model whereby an outer vestibule lysine interferes with K+ flux through the channel, and that the [K+]-dependent change in orientation of this lysine alters single channel conductance by changing the level of this interference. Moreover, these results provide an experimental example of single channel conductance being modulated at the outer end of the conduction pathway by a mechanism that involves channel activation into open states with different outer vestibule conformations.

scholarly journals Altered sinoatrial node function and intra-atrial conduction in murine gain-of-function Scn5a+/ΔKPQ hearts suggest an overlap syndrome

2012 ◽  
Vol 302 (7) ◽  
pp. H1510-H1523 ◽  
Author(s):  
Jingjing Wu ◽  
Yanmin Zhang ◽  
Xinzhao Zhang ◽  
Longxian Cheng ◽  
Wim J. Lammers ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Sinoatrial Node ◽  
Syndrome Type ◽  
Two Dimensional ◽  
Wild Type ◽  
Long Qt ◽  
Gain Of Function ◽  
Intraventricular Conduction ◽  
Qt Syndrome ◽  
Type 3

Mutations in SCN5A, the gene encoding the pore-forming subunit of cardiac Na+ channels, cause a spectrum of arrhythmic syndromes. Of these, sinoatrial node (SAN) dysfunction occurs in patients with both loss- and gain-of-function SCN5A mutations . We explored for corresponding alterations in SAN function and intracardiac conduction and clarified possible mechanisms underlying these in an established mouse long QT syndrome type 3 model carrying a mutation equivalent to human SCN5A-ΔKPQ. Electrophysiological characterizations of SAN function in living animals and in vitro sinoatrial preparations were compared with cellular SAN and two-dimensional tissue models exploring the consequences of Scn5a+/ΔKPQ mutations. Scn5a+/ΔKPQ mice showed prolonged electrocardiographic QT and corrected QT intervals confirming long QT phenotypes. They showed frequent episodes of sinus bradycardia, sinus pause/arrest, and significantly longer sinus node recovery times, suggesting compromised pacemaker activity compared with wild-type mice. Electrocardiographic waveforms suggested depressed intra-atrial, atrioventricular node, and intraventricular conduction in Scn5a+/ΔKPQ mice . Isolated Scn5a+/ΔKPQ sinoatrial preparations similarly showed lower mean intrinsic heart rates and overall slower conduction through the SAN to the surrounding atrium than did wild-type preparations. Computer simulations of both single SAN cells as well as two-dimensional SAN-atrial models could reproduce the experimental observations of impaired pacemaker and sinoatrial conduction in terms of changes produced by both augmented tail and reduced total Na+ currents, respectively. In conclusion, the gain-of-function long QT syndrome type 3 murine Scn5a+/ΔKPQ cardiac system, in overlap with corresponding features reported in loss-of-function Na+ channel mutations, shows compromised SAN pacemaker and conduction function explicable in modeling studies through a combination of augmented tail and reduced peak Na+ currents.

scholarly journals Biophysical and Molecular Mechanisms Underlying the Modulation of Heteromeric Kir4.1–Kir5.1 Channels by Co2 and Ph

2000 ◽  
Vol 116 (1) ◽  
pp. 33-46 ◽  
Author(s):  
Zhenjiang Yang ◽  
Haoxing Xu ◽  
Ningren Cui ◽  
Zhiqiang Qu ◽  
Sengthong Chanchevalap ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Single Channel ◽  
Ph Sensitivity ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Kir Channels ◽  
Kir Channel ◽  
Open Time ◽  
Excised Patches ◽  
Single Channel Currents

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4.1–Kir5.1 were studied in inside-out patches. These Kir4.1–Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (Popen) ∼ 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of Popen without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at ∼1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1–Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline Popen and reduced channel sensitivity to intracellular protons. In the presence of 10 μM PIP2, the Kir4.1–Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1–Kir5.1. In excised patches, interestingly, the Kir4.1–Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.

scholarly journals The channel-tunnel HI1462 of Haemophilus influenzae reveals differences to Escherichia coli TolC

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1639-1647 ◽  
Author(s):  
Georg Polleichtner ◽  
Christian Andersen
Keyword(s):  
Escherichia Coli ◽  
Single Channel ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Tunnel Entrance

Efflux pumps play a major role in multidrug resistance of pathogenic bacteria. The TolC homologue HI1462 was identified as the single channel-tunnel in Haemophilus influenzae required to form a functional multidrug efflux pump. The outer-membrane protein was expressed in Escherichia coli, purified and reconstituted in black lipid membranes. It exhibited a comparatively small single-channel conductance of 43 pS in 1 M KCl and is the first known TolC homologue which is anion-selective. The HI1462 structure was modelled and an arginine residue lining the tunnel entrance was identified. The channel-tunnel of a mutant with the arginine substituted by an alanine residue was cation-selective and had a sevenfold higher single-channel conductance compared to wild-type. These results confirm that the arginine is responsible for anion selectivity and forms a salt bridge with a glutamate residue of the adjacent monomer, establishing a circular network, which keeps the tunnel entrance in a tightly closed conformation. In in vivo experiments, both the wild-type HI1462 and the mutant were able to substitute for E. coli TolC in the haemolysin secretion system, but not in the AcrAB/TolC multidrug efflux pump. The structure–function relationship of HI1462 is discussed in the context of the well-studied TolC channel-tunnel of E. coli.

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