Molecular determinants of ion conduction and inactivation in K+ channels

K+ channel-forming proteins can be grouped into three families that differ by the number of potential membrane-spanning segments. The largest of these families is composed of tetrameric channels with subunits containing six putative membrane-spanning segments (S1-S6). Inward rectifiers comprise a second family of K+ channels with subunits having two transmembrane domains (M1, M2). Monomers in the third family are proteins containing only one membrane-spanning segment, and they give origin to minK+ channels. Joining together segments S5 and S6 in the case of voltage-gated K+ channels and M1 and M2 in inward rectifiers, there is a highly conserved region with a hairpin shape called the H5 or P region. The P region, the loop connecting the S4 and S5 domains and the S6 transmembrane segment in Shaker-type K+ channels and the COOH-terminal in inward rectifiers, appears to play crucial roles in ion conduction. In Shaker K+ channels the NH2-terminal has been identified as responsible for fast inactivation (N-type inactivation). If the fast-inactivation gate is removed, a slower inactivation process persists, and its rate can be altered by mutations of amino acid residues forming part of the region in the neighborhood of the COOH-terminal (C-type inactivation). In this review we discuss the strategies followed to identify the different structures of K+ channels involved in ion conduction and inactivation processes and how they interplay.

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Low molecular weight poly(A)+ mRNA species encode factors that modulate gating of a non-Shaker A-type K+ channel.

The Journal of General Physiology ◽

10.1085/jgp.102.4.713 ◽

1993 ◽

Vol 102 (4) ◽

pp. 713-728 ◽

Cited By ~ 35

Author(s):

L D Chabala ◽

N Bakry ◽

M Covarrubias

Keyword(s):

Molecular Weight ◽

Xenopus Oocytes ◽

Membrane Potentials ◽

K Channel ◽

Low Molecular Weight ◽

K Channels ◽

Fourfold Increase ◽

Mrna Species ◽

Type K

Voltage-dependent K+ channels control repolarization of action potentials and help establish firing patterns in nerve cells. To determine the nature and role of molecular components that modulate K+ channel function in vivo, we coinjected Xenopus oocytes with cRNA encoding a cloned subthreshold A-type K+ channel (mShal1, also referred to as mKv4.1) and a low molecular weight (LMW) fraction (2-4 kb) of poly(A)+ mRNA (both from rodent brain). Coinjected oocytes exhibited a significant (fourfold) increase in the surface expression of mShal1 K+ channels with no change in the open-channel conductance. Coexpression also modified the gating kinetics of mShal1 current in several respects. Macroscopic inactivation of whole oocyte currents was fitted with the sum of two exponential components. Both fast and slow time constants of inactivation were accelerated at all membrane potentials in coinjected oocytes (tau f = 47.2 ms vs 56.5 ms at 0 mV and tau s = 157 ms vs 225 ms at 0 mV), and the corresponding ratios of amplitude terms were shifted toward domination by the fast component (Af/As = 2.71 vs 1.17 at 0 mV). Macroscopic activation was characterized in terms of the time-to-peak current, and it was found to be more rapid at all membrane potentials in coinjected oocytes (9.9 ms vs 13.5 ms at 0 mV). Coexpression also leads to more rapid recovery from inactivation (approximately 2.4-fold faster at -100 mV). The coexpressed K+ currents in oocytes resemble currents expressed in mouse fibroblasts (NIH3T3) transfected only with mShal1 cDNA. These results indicate that mammalian regulatory subunits or enzymes encoded by LMW mRNA species, which are apparently missing or expressed at low levels in Xenopus oocytes, may modulate gating in some native subthreshold A-type K+ channels.

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Castration Induces Down-Regulation of A-Type K+ Channel in Rat Vas Deferens Smooth Muscle

International Journal of Molecular Sciences ◽

10.3390/ijms20174073 ◽

2019 ◽

Vol 20 (17) ◽

pp. 4073 ◽

Cited By ~ 1

Author(s):

Susumu Ohya ◽

Katsunori Ito ◽

Noriyuki Hatano ◽

Akitoshi Ohno ◽

Katsuhiko Muraki ◽

...

Keyword(s):

Smooth Muscle ◽

Vas Deferens ◽

Oral Treatment ◽

Functional Expression ◽

Rat Vas Deferens ◽

K Channel ◽

Α Subunit ◽

K Channels ◽

Expression Levels ◽

Type K

A-type K+ channels contribute to regulating the propagation and frequency of action potentials in smooth muscle cells (SMCs). The present study (i) identified the molecular components of A-type K+ channels in rat vas deferens SMs (VDSMs) and (ii) showed the long-term, genomic effects of testosterone on their expression in VDSMs. Transcripts of the A-type K+ channel α subunit, Kv4.3L and its regulatory β subunits, KChIP3, NCS1, and DPP6-S were predominantly expressed in rat VDSMs over the other related subtypes (Kv4.2, KChIP1, KChIP2, KChIP4, and DPP10). A-type K+ current (IA) density in VDSM cells (VDSMCs) was decreased by castration without changes in IA kinetics, and decreased IA density was compensated for by an oral treatment with 17α-methyltestosterone (MET). Correspondingly, in the VDSMs of castrated rats, Kv4.3L and KChIP3 were down-regulated at both the transcript and protein expression levels. Changes in Kv4.3L and KChIP3 expression levels were compensated for by the treatment with MET. These results suggest that testosterone level changes in testosterone disorders and growth processes control the functional expression of A-type K+ channels in VDSMCs.

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A forward genetic screen identifies chaperone CNX-1 as a conserved biogenesis regulator of ERG K+ channels

The Journal of General Physiology ◽

10.1085/jgp.201812025 ◽

2018 ◽

Vol 150 (8) ◽

pp. 1189-1201 ◽

Cited By ~ 4

Author(s):

Xue Bai ◽

Kai Li ◽

Li Yao ◽

Xin-Lei Kang ◽

Shi-Qing Cai

Keyword(s):

Action Potentials ◽

K Channel ◽

Translation System ◽

Loss Of Function ◽

K Channels ◽

Type K ◽

Evolutionarily Conserved ◽

Protein Biogenesis ◽

Free Translation ◽

Current Densities

The human ether-a-go-go–related gene (hERG) encodes a voltage-gated potassium channel that controls repolarization of cardiac action potentials. Accumulating evidence suggests that most disease-related hERG mutations reduce the function of the channel by disrupting protein biogenesis of the channel in the endoplasmic reticulum (ER). However, the molecular mechanism underlying the biogenesis of ERG K+ channels is largely unknown. By forward genetic screening, we identified an ER-located chaperone CNX-1, the worm homologue of mammalian chaperone Calnexin, as a critical regulator for the protein biogenesis of UNC-103, the ERG-type K+ channel in Caenorhabditis elegans. Loss-of-function mutations of cnx-1 decreased the protein level and current density of the UNC-103 K+ channel and suppressed the behavioral defects caused by a gain-of-function mutation in unc-103. Moreover, CNX-1 facilitated tetrameric assembly of UNC-103 channel subunits in a liposome-assisted cell-free translation system. Further studies showed that CNX-1 act in parallel to DNJ-1, another ER-located chaperone known to regulate maturation of UNC-103 channels, on controlling the protein biogenesis of UNC-103. Importantly, Calnexin interacted with hERG proteins in the ER in HEK293T cells. Deletion of calnexin reduced the expression and current densities of endogenous hERG K+ channels in SH-SY5Y cells. Collectively, we reveal an evolutionarily conserved chaperone CNX-1/Calnexin controlling the biogenesis of ERG-type K+ channels.

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Localization and Molecular Determinants of the Hanatoxin Receptors on the Voltage-Sensing Domains of a K+ Channel

The Journal of General Physiology ◽

10.1085/jgp.115.6.673 ◽

2000 ◽

Vol 115 (6) ◽

pp. 673-684 ◽

Cited By ~ 99

Author(s):

Yingying Li-Smerin ◽

Kenton J. Swartz

Keyword(s):

K Channel ◽

Alanine Scanning Mutagenesis ◽

Voltage Sensing ◽

Alanine Scanning ◽

K Channels ◽

Voltage Gated ◽

Physical Location ◽

Molecular Determinants ◽

Central Pore ◽

Pore Domain

Hanatoxin inhibits voltage-gated K+ channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K+ channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1–S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K+ channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K+ channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20–25 Å away from the central pore axis.

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Collapse of Conductance Is Prevented by a Glutamate Residue Conserved in Voltage-Dependent K+ Channels

The Journal of General Physiology ◽

10.1085/jgp.116.2.181 ◽

2000 ◽

Vol 116 (2) ◽

pp. 181-190 ◽

Cited By ~ 28

Author(s):

Patricia Ortega-Sáenz ◽

Ricardo Pardal ◽

Antonio Castellano ◽

José López-Barneo

Keyword(s):

Critical Role ◽

K Channel ◽

Kinetic Properties ◽

K Channels ◽

Charged Amino Acids ◽

Extracellular Loops ◽

Open Conformation ◽

Voltage Dependent ◽

Membrane Spanning ◽

External K

Voltage-dependent K+ channel gating is influenced by the permeating ions. Extracellular K+ determines the occupation of sites in the channels where the cation interferes with the motion of the gates. When external [K+] decreases, some K+ channels open too briefly to allow the conduction of measurable current. Given that extracellular K+ is normally low, we have studied if negatively charged amino acids in the extracellular loops of Shaker K+ channels contribute to increase the local [K+]. Surprisingly, neutralization of the charge of most acidic residues has minor effects on gating. However, a glutamate residue (E418) located at the external end of the membrane spanning segment S5 is absolutely required for keeping channels active at the normal external [K+]. E418 is conserved in all families of voltage-dependent K+ channels. Although the channel mutant E418Q has kinetic properties resembling those produced by removal of K+ from the pore, it seems that E418 is not simply concentrating cations near the channel mouth, but has a direct and critical role in gating. Our data suggest that E418 contributes to stabilize the S4 voltage sensor in the depolarized position, thus permitting maintenance of the channel open conformation.

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Expression of inward rectifying K+ channels (KIR2.1, KIR2.2, KIR2.3) and the small – conductance CA2+- activated K+ channel SK2 in normal versus atherosclerotic human radial artery bypass grafts

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-2008-1038018 ◽

2008 ◽

Vol 56 (S 1) ◽

Author(s):

S Wildhirt ◽

M Benz ◽

J Grammer ◽

R Lange

Keyword(s):

Radial Artery ◽

Artery Bypass ◽

K Channel ◽

Bypass Grafts ◽

K Channels ◽

Small Conductance

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Enhanced role of K+ channels in relaxations of hypercholesterolemic rabbit carotid artery to NO

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1995.269.3.h805 ◽

1995 ◽

Vol 269 (3) ◽

pp. H805-H811 ◽

Cited By ~ 16

Author(s):

S. Najibi ◽

R. A. Cohen

Keyword(s):

Carotid Artery ◽

Sodium Nitroprusside ◽

Channel Blocker ◽

Isometric Tension ◽

K Channel ◽

K Channels ◽

Rabbit Carotid Artery ◽

Cyclic Monophosphate ◽

Endothelium Dependent Relaxation

Endothelium-dependent relaxations to acetylcholine remain normal in the carotid artery of hypercholesterolemic rabbits, but unlike endothelium-dependent relaxations of normal rabbits, they are inhibited by charybdotoxin, a specific blocker of Ca(2+)-dependent K+ channels. Because nitric oxide (NO) is the mediator of endothelium-dependent relaxation and can activate Ca(2+)-dependent K+ channels directly or via guanosine 3',5'-cyclic monophosphate, the present study investigated the role of Ca(2+)-dependent K+ channels in relaxations caused by NO, sodium nitroprusside, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-Brc-GMP) in hypercholesterolemic rabbit carotid artery. Isometric tension was measured in rabbit carotid artery denuded of endothelium from normal and hypercholesterolemic rabbits which were fed 0.5% cholesterol for 12 wk. Under control conditions, relaxations to all agents were similar in normal and hypercholesterolemic rabbit arteries. Charybdotoxin had no significant effect on relaxations of normal arteries to NO, sodium nitroprusside, or 8-BrcGMP, but the Ca(2+)-dependent K+ channel blocker significantly inhibited the relaxations caused by each of these agents in the arteries from hypercholesterolemic rabbits. By contrast, relaxations to the calcium channel blocker nifedipine were potentiated to a similar extent by charybdotoxin in both groups. In addition, arteries from hypercholesterolemic rabbits relaxed less than normal to sodium nitroprusside when contracted with depolarizing potassium solution. These results indicate that although nitrovasodilator relaxations are normal in the hypercholesterolemic rabbit carotid artery, they are mediated differently, and to a greater extent, by Ca(2+)-dependent K+ channels. These data also suggest that K+ channel-independent mechanism(s) are impaired in hypercholesterolemia.

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Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel

The Journal of General Physiology ◽

10.1085/jgp.200509457 ◽

2006 ◽

Vol 127 (2) ◽

pp. 159-169 ◽

Cited By ~ 29

Author(s):

Jill Thompson ◽

Ted Begenisich

Keyword(s):

Single Channel ◽

Expression System ◽

Chemical Activation ◽

K Channel ◽

Channel Activity ◽

Single Family ◽

Open Probability ◽

K Channels ◽

Channel Proteins ◽

K Activity

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

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Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

The Journal of General Physiology ◽

10.1085/jgp.110.5.579 ◽

1997 ◽

Vol 110 (5) ◽

pp. 579-589 ◽

Cited By ~ 146

Author(s):

Riccardo Olcese ◽

Ramón Latorre ◽

Ligia Toro ◽

Francisco Bezanilla ◽

Enrico Stefani

Keyword(s):

Time Course ◽

Voltage Dependence ◽

Ionic Current ◽

K Channel ◽

Charge Movement ◽

Slow Inactivation ◽

Gating Currents ◽

Similar Time ◽

K Channels ◽

Recovery From Inactivation

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K+ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

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