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1.
Nat Commun ; 15(1): 3849, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38719838

ABSTRACT

Highly selective for K+ at neutral pH, the TWIK1 channel becomes permeable to Na+ upon acidification. Using molecular dynamics simulations, we identify a network of residues involved in this unique property. Between the open and closed states previously observed by electron microscopy, molecular dynamics simulations show that the channel undergoes conformational changes between pH 7.5-6 involving residues His122, Glu235, Lys246 and Phe109. A complex network of interactions surrounding the selectivity filter at high pH transforms into a simple set of stronger interactions at low pH. In particular, His122 protonated by acidification moves away from Lys246 and engages in a salt bridge with Glu235. In addition, stacking interactions between Phe109 and His122, which stabilize the selectivity filter in its K+-selective state at high pH, disappear upon acidification. This leads to dissociation of the Phe109 aromatic side chain from this network, resulting in the Na+-permeable conformation of the channel.

2.
J Biol Chem ; 298(10): 102447, 2022 10.
Article in English | MEDLINE | ID: mdl-36063992

ABSTRACT

Two-pore domain K+ channels (K2P channels), active as dimers, produce inhibitory currents regulated by a variety of stimuli. Among them, TWIK1-related alkalinization-activated K+ channel 1 (TALK1), TWIK1-related alkalinization-activated K+ channel 2 (TALK2), and TWIK1-related acid-sensitive K+ channel 2 (TASK2) form a subfamily of structurally related K2P channels stimulated by extracellular alkalosis. The human genes encoding these proteins are clustered at chromosomal region 6p21 and coexpressed in multiple tissues, including the pancreas. The question whether these channels form functional heteromers remained open. By analyzing single-cell transcriptomic data, we show that these channels are coexpressed in insulin-secreting pancreatic ß-cells. Using in situ proximity ligation assay and electrophysiology, we show that they form functional heterodimers both upon heterologous expression and under native conditions in human pancreatic ß-cells. We demonstrate that heteromerization of TALK2 with TALK1 or with TASK2 endows TALK2 with sensitivity to extracellular alkalosis in the physiological range. We further show that the association of TASK2 with TALK1 and TALK2 increases their unitary conductance. These results provide a new example of heteromerization in the K2P channel family expanding the range of the potential physiological and pathophysiological roles of TALK1/TALK2/TASK2 channels, not only in insulin-secreting cells but also in the many other tissues in which they are coexpressed.


Subject(s)
Alkalosis , Insulin-Secreting Cells , Potassium Channels, Tandem Pore Domain , Humans , Potassium Channels, Tandem Pore Domain/genetics , Potassium Channels, Tandem Pore Domain/metabolism , Insulin-Secreting Cells/metabolism , Hydrogen-Ion Concentration , Insulin/metabolism , Potassium/metabolism
3.
4.
Front Pharmacol ; 12: 755826, 2021.
Article in English | MEDLINE | ID: mdl-34658895

ABSTRACT

Inhibitory potassium channels of the TREK1/TRAAK family are integrators of multiple stimuli, including temperature, membrane stretch, polyunsaturated fatty acids and pH. How these signals affect the gating of these channels is the subject of intense research. We have previously identified a cytoplasmic domain, pCt, which plays a major role in controlling channel activity. Here, we use pharmacology to show that the effects of pCt, arachidonic acid, and extracellular pH converge to the same gate within the channel. Using a state-dependent inhibitor, fluoxetine, as well as natural and synthetic openers, we provide further evidence that the "up" and "down" conformations identified by crystallography do not correspond to open and closed states of these channels.

5.
J Physiol ; 599(4): 1041-1055, 2021 02.
Article in English | MEDLINE | ID: mdl-33347640

ABSTRACT

Potassium channels form the largest family of ion channels with more than 80 members involved in cell excitability and signalling. Most of them exist as homomeric channels, whereas specific conditions are required to obtain heteromeric channels. It is well established that heteromerization of voltage-gated and inward rectifier potassium channels affects their function, increasing the diversity of the native potassium currents. For potassium channels with two pore domains (K2P ), homomerization has long been considered the rule, their polymodal regulation by a wide diversity of physical and chemical stimuli being responsible for the adaptation of the leak potassium currents to cellular needs. This view has recently evolved with the accumulation of evidence of heteromerization between different K2P subunits. Several functional intragroup and intergroup heteromers have recently been identified, which contribute to the functional heterogeneity of this family. K2P heteromerization is involved in the modulation of channel expression and trafficking, promoting functional and signalling diversity. As illustrated in the Abstract Figure, heteromerization of TREK1 and TRAAK provides the cell with more possibilities of regulation. It is becoming increasingly evident that K2P heteromers contribute to important physiological functions including neuronal and cardiac excitability. Since heteromerization also affects the pharmacology of K2P channels, this understanding helps to establish K2P heteromers as new therapeutic targets for physiopathological conditions.


Subject(s)
Potassium Channels, Tandem Pore Domain , Neurons/metabolism , Potassium , Potassium Channels, Tandem Pore Domain/genetics , Potassium Channels, Tandem Pore Domain/metabolism , Protein Transport , Signal Transduction
6.
Nat Commun ; 10(1): 787, 2019 02 15.
Article in English | MEDLINE | ID: mdl-30770809

ABSTRACT

Mutations that modulate the activity of ion channels are essential tools to understand the biophysical determinants that control their gating. Here, we reveal the conserved role played by a single amino acid position (TM2.6) located in the second transmembrane domain of two-pore domain potassium (K2P) channels. Mutations of TM2.6 to aspartate or asparagine increase channel activity for all vertebrate K2P channels. Using two-electrode voltage-clamp and single-channel recording techniques, we find that mutation of TM2.6 promotes channel gating via the selectivity filter gate and increases single channel open probability. Furthermore, channel gating can be progressively tuned by using different amino acid substitutions. Finally, we show that the role of TM2.6 was conserved during evolution by rationally designing gain-of-function mutations in four Caenorhabditis elegans K2P channels using CRISPR/Cas9 gene editing. This study thus describes a simple and powerful strategy to systematically manipulate the activity of an entire family of potassium channels.


Subject(s)
Membrane Potentials/physiology , Potassium Channels, Tandem Pore Domain/metabolism , Animals , CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/physiology , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Drosophila , Evolution, Molecular , Humans , Invertebrates , Membrane Potentials/genetics , Mutation/genetics , Potassium Channels, Tandem Pore Domain/genetics , Vertebrates
7.
Front Mol Neurosci ; 11: 301, 2018.
Article in English | MEDLINE | ID: mdl-30233308

ABSTRACT

TREK/TRAAK channels are polymodal K+ channels that convert very diverse stimuli, including bioactive lipids, mechanical stretch and temperature, into electrical signals. The nature of the structural changes that regulate their activity remains an open question. Here, we show that a cytoplasmic domain (the proximal C-ter domain, pCt) exerts antagonistic effects in TREK1 and TRAAK. In basal conditions, pCt favors activity in TREK1 whereas it impairs TRAAK activity. Using the conformation-dependent binding of fluoxetine, we show that TREK1 and TRAAK conformations at rest are different, and under the influence of pCt. Finally, we show that depleting PIP2 in live cells has a more pronounced inhibitory effect on TREK1 than on TRAAK. This differential regulation of TREK1 and TRAAK is related to a previously unrecognized PIP2-binding site (R329, R330, and R331) present within TREK1 pCt, but not in TRAAK pCt. Collectively, these new data point out pCt as a major regulatory domain of these channels and suggest that the binding of PIP2 to the pCt of TREK1 results in the stabilization of the conductive conformation in basal conditions.

8.
Proc Natl Acad Sci U S A ; 113(15): 4200-5, 2016 Apr 12.
Article in English | MEDLINE | ID: mdl-27035965

ABSTRACT

The tandem of pore domain in a weak inwardly rectifying K(+) channel (Twik)-related acid-arachidonic activated K(+) channel (TRAAK) and Twik-related K(+) channels (TREK) 1 and TREK2 are active as homodimers gated by stretch, fatty acids, pH, and G protein-coupled receptors. These two-pore domain potassium (K2P) channels are broadly expressed in the nervous system where they control excitability. TREK/TRAAK KO mice display altered phenotypes related to nociception, neuroprotection afforded by polyunsaturated fatty acids, learning and memory, mood control, and sensitivity to general anesthetics. These channels have emerged as promising targets for the development of new classes of anesthetics, analgesics, antidepressants, neuroprotective agents, and drugs against addiction. Here, we show that the TREK1, TREK2, and TRAAK subunits assemble and form active heterodimeric channels with electrophysiological, regulatory, and pharmacological properties different from those of homodimeric channels. Heteromerization occurs between all TREK variants produced by alternative splicing and alternative translation initiation. These results unveil a previously unexpected diversity of K2P channels that will be challenging to analyze in vivo, but which opens new perspectives for the development of clinically relevant drugs.


Subject(s)
Potassium Channels, Tandem Pore Domain/chemistry , Animals , Dimerization , Dogs , Humans , Madin Darby Canine Kidney Cells , Mice
9.
J Physiol ; 593(12): 2587-603, 2015 Jun 15.
Article in English | MEDLINE | ID: mdl-25530075

ABSTRACT

Potassium channels participate in many biological functions, from ion homeostasis to generation and modulation of the electrical membrane potential. They are involved in a large variety of diseases. In the human genome, 15 genes code for K(+) channels with two pore domains (K2P ). These channels form dimers of pore-forming subunits that produce background conductances finely regulated by a range of natural and chemical effectors, including signalling lipids, temperature, pressure, pH, antidepressants and volatile anaesthetics. Since the cloning of TWIK1, the prototypical member of this family, a lot of work has been carried out on their structure and biology. These studies are still in progress, but data gathered so far show that K2P channels are central players in many processes, including ion homeostasis, hormone secretion, cell development and excitability. A growing number of studies underline their implication in physiopathological mechanisms, such as vascular and pulmonary hypertension, cardiac arrhythmias, nociception, neuroprotection and depression. This review gives a synthetic view of the most noticeable features of these channels.


Subject(s)
Potassium Channels, Tandem Pore Domain , Animals , Humans , Potassium Channels, Tandem Pore Domain/chemistry , Potassium Channels, Tandem Pore Domain/metabolism , Potassium Channels, Tandem Pore Domain/physiology , Protein Subunits/chemistry , Protein Subunits/metabolism , Protein Subunits/physiology , Receptors, G-Protein-Coupled/metabolism
10.
Pflugers Arch ; 467(5): 1121-31, 2015 May.
Article in English | MEDLINE | ID: mdl-25339226

ABSTRACT

Among K2P channels, a few of them turned out to be difficult to express in heterologous systems and were coined "silent subunits". Recent studies have shed light on the mechanisms behind this apparent lack of channel activity at the plasma membrane. For TWIK1 and THIK2 channels, silence is related to a combination of intracellular retention and low intrinsic activity. TWIK1 is constitutively endocytosed from the plasma membrane before being transported to recycling endosomes, whereas THIK2 is restricted to endoplasmic reticulum. These intracellular localizations are related to trafficking signals located in the cytoplasmic parts of the channels. When these motifs are mutated or masked, channels are redistributed at the plasma membrane and produce measurable currents. However, these currents are of modest amplitude. This weak basal activity is due to a hydrophobic barrier in the deep pore that limits water and ions in the conduction pathway. Other silent channels KCNK7, TWIK2, and TASK5 are still under study. Expression and characterization of these K2P channels pave the way for a better understanding of the mechanisms controlling intracellular trafficking of membrane proteins, ion conduction, and channel gating.


Subject(s)
Cell Membrane/metabolism , Cytoplasm/metabolism , Endoplasmic Reticulum/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Protein Transport/physiology , Animals , Endocytosis/physiology , Humans
11.
J Biol Chem ; 289(41): 28202-12, 2014 Oct 10.
Article in English | MEDLINE | ID: mdl-25148687

ABSTRACT

Despite a high level of sequence homology, tandem pore domain halothane-inhibited K(+) channel 1 (THIK1) produces background K(+) currents, whereas THIK2 is silent. This lack of activity is due to a unique combination of intracellular retention and weak basal activity in the plasma membrane. Here, we designed THIK subunits containing dominant negative mutations (THIK1(DN) and THIK2(DN)). THIK2(DN) mutant inhibits THIK1 currents, whereas THIK1(DN) inhibits an activated form of THIK2 (THIK2-A155P-I158D). In situ proximity ligation assays and Förster/fluorescence resonance energy transfer (FRET) experiments support a physical association between THIK1 and THIK2. Next, we expressed covalent tandems of THIK proteins to obtain expression of pure heterodimers. Td-THIK1-THIK2 (where Td indicates tandem) produces K(+) currents of amplitude similar to Td-THIK1-THIK1 but with a noticeable difference in the current kinetics. Unlike Td-THIK2-THIK2 that is mainly detected in the endoplasmic reticulum, Td-THIK1-THIK2 distributes at the plasma membrane, indicating that THIK1 can mask the endoplasmic reticulum retention/retrieval motif of THIK2. Kinetics and unitary conductance of Td-THIK1-THIK2 are intermediate between THIK1 and THIK2. Altogether, these results show that THIK1 and THIK2 form active heteromeric channels, further expanding the known repertoire of K(+) channels.


Subject(s)
Chlorides/chemistry , Potassium Channels, Tandem Pore Domain/chemistry , Potassium/chemistry , Amino Acid Sequence , Animals , Chlorides/metabolism , Dogs , Gene Expression Regulation , HEK293 Cells , Humans , Ion Transport , Kinetics , Madin Darby Canine Kidney Cells , Membrane Potentials , Models, Molecular , Molecular Sequence Data , Mutation , Oocytes/cytology , Oocytes/physiology , Patch-Clamp Techniques , Potassium/metabolism , Potassium Channels, Tandem Pore Domain/genetics , Potassium Channels, Tandem Pore Domain/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Xenopus laevis
12.
J Biol Chem ; 288(49): 35081-92, 2013 Dec 06.
Article in English | MEDLINE | ID: mdl-24163367

ABSTRACT

The tandem pore domain halothane-inhibited K(+) channel 1 (THIK1) produces background K(+) currents. Despite 62% amino acid identity with THIK1, THIK2 is not active upon heterologous expression. Here, we show that this apparent lack of activity is due to a unique combination of retention in the endoplasmic reticulum and low intrinsic channel activity at the plasma membrane. A THIK2 mutant containing a proline residue (THIK2-A155P) in its second inner helix (M2) produces K(+)-selective currents with properties similar to THIK1, including inhibition by halothane and insensitivity to extracellular pH variations. Another mutation in the M2 helix (I158D) further increases channel activity and affects current kinetics. We also show that the cytoplasmic amino-terminal region of THIK2 (Nt-THIK2) contains an arginine-rich motif (RRSRRR) that acts as a retention/retrieval signal. Mutation of this motif in THIK2 induces a relocation of the channel to the plasma membrane, resulting in measurable currents, even in the absence of mutations in the M2 helix. Cell surface delivery of a Nt-THIK2-CD161 chimera is increased by mutating the arginines of the retention motif but also by converting the serine embedded in this motif to aspartate, suggesting a phosphorylation-dependent regulation of THIK2 trafficking.


Subject(s)
Potassium Channels, Tandem Pore Domain/genetics , Potassium Channels, Tandem Pore Domain/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Membrane/metabolism , Dogs , Endoplasmic Reticulum/metabolism , Female , Gene Silencing , Humans , Intracellular Space/metabolism , Madin Darby Canine Kidney Cells , Membrane Potentials , Molecular Sequence Data , Mutagenesis, Site-Directed , Oocytes/metabolism , Phosphorylation , Potassium Channels, Tandem Pore Domain/chemistry , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Xenopus laevis
13.
Proc Natl Acad Sci U S A ; 109(14): 5499-504, 2012 Apr 03.
Article in English | MEDLINE | ID: mdl-22431633

ABSTRACT

TWIK1 belongs to the family of background K(+) channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K(+) selective at neutral pH, it becomes permeable to Na(+) at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1(-/-) pancreatic ß cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.


Subject(s)
Potassium Channels/metabolism , Amino Acid Sequence , Animals , Hydrogen-Ion Concentration , Models, Molecular , Molecular Sequence Data , Potassium Channels/chemistry , Sequence Homology, Amino Acid , Xenopus
14.
Pflugers Arch ; 460(3): 571-81, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20490539

ABSTRACT

Mechano-gated ion channels play a key physiological role in cardiac, arterial, and skeletal myocytes. For instance, opening of the non-selective stretch-activated cation channels in smooth muscle cells is involved in the pressure-dependent myogenic constriction of resistance arteries. These channels are also implicated in major pathologies, including cardiac hypertrophy or Duchenne muscular dystrophy. Seminal work in prokaryotes and invertebrates highlighted the role of transient receptor potential (TRP) channels in mechanosensory transduction. In mammals, recent findings have shown that the canonical TRPC1 and TRPC6 channels are key players in muscle mechanotransduction. In the present review, we will focus on the functional properties of TRPC1 and TRPC6 channels, on their mechano-gating, regulation by interacting cytoskeletal and scaffolding proteins, physiological role and implication in associated diseases.


Subject(s)
Mechanotransduction, Cellular , Muscle Cells/physiology , TRPC Cation Channels/physiology , Animals , Arteries/physiology , Cardiomegaly/physiopathology , Cytoskeletal Proteins/metabolism , Humans , Receptors, G-Protein-Coupled/metabolism , TRPP Cation Channels/metabolism
15.
J Mol Cell Cardiol ; 48(1): 83-9, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19345226

ABSTRACT

Despite the central physiological importance of cardiovascular mechanotransduction, the molecular identities of the sensors and the signaling pathways have long remained elusive. Indeed, how pressure is transduced into cellular excitation has only recently started to emerge. In both arterial and cardiac myocytes, the diacylglycerol-sensitive canonical transient receptor potential (TRPC) subunits are proposed to underlie the stretch-activated depolarizing cation channels. An indirect mechanism of activation through a ligand-independent conformational switch of Gq-coupled receptors by mechanical stress is invoked. Such a mechanism involving the angiotensin type 1 receptor and TRPC6 is proposed to trigger the arterial myogenic response to intraluminal pressure. TRPC6 is also involved in load-induced cardiac hypertrophy. In this review, we will focus on the molecular basis of pressure sensing in the cardiovascular system and associated disease states.


Subject(s)
Cardiovascular System/metabolism , Mechanoreceptors/metabolism , TRPC Cation Channels/metabolism , Animals , Cardiomegaly/metabolism , Cardiomegaly/physiopathology , Diglycerides/metabolism , Humans , Mechanoreceptors/chemistry , Mechanotransduction, Cellular/physiology , Models, Biological , TRPC Cation Channels/chemistry
16.
Cell ; 139(3): 587-96, 2009 Oct 30.
Article in English | MEDLINE | ID: mdl-19879844

ABSTRACT

Autosomal-dominant polycystic kidney disease, the most frequent monogenic cause of kidney failure, is induced by mutations in the PKD1 or PKD2 genes, encoding polycystins TRPP1 and TRPP2, respectively. Polycystins are proposed to form a flow-sensitive ion channel complex in the primary cilium of both epithelial and endothelial cells. However, how polycystins contribute to cellular mechanosensitivity remains obscure. Here, we show that TRPP2 inhibits stretch-activated ion channels (SACs). This specific effect is reversed by coexpression with TRPP1, indicating that the TRPP1/TRPP2 ratio regulates pressure sensing. Moreover, deletion of TRPP1 in smooth muscle cells reduces SAC activity and the arterial myogenic tone. Inversely, depletion of TRPP2 in TRPP1-deficient arteries rescues both SAC opening and the myogenic response. Finally, we show that TRPP2 interacts with filamin A and demonstrate that this actin crosslinking protein is critical for SAC regulation. This work uncovers a role for polycystins in regulating pressure sensing.


Subject(s)
Pressure , TRPP Cation Channels/metabolism , Actins/metabolism , Animals , Contractile Proteins/metabolism , Filamins , Mechanotransduction, Cellular , Mice , Microfilament Proteins/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Pressoreceptors/metabolism
17.
Channels (Austin) ; 3(1): 69-72, 2009.
Article in English | MEDLINE | ID: mdl-19221511

ABSTRACT

The annual meeting of the French Ion Channels Society, held on the Mediterranean coast of France, is aimed at gathering the international scientific community working on various aspects of ion channels. In this report of the 19th edition of the meeting, held in September 2008, we summarize selected symposia on aspects of the ion channel field from fundamental to clinical research. The meeting is an opportunity for leading investigators as well as young researchers to present and discuss their recent advances and future challenges in the ion channel field.


Subject(s)
Cell Membrane/metabolism , Ion Channels/metabolism , Alzheimer Disease/metabolism , Andersen Syndrome/metabolism , Animals , Epithelial Cells/metabolism , Humans , Ion Channels/genetics , Muscle, Smooth/metabolism , Protein Transport , Signal Transduction
18.
Pflugers Arch ; 455(6): 1097-103, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17957383

ABSTRACT

This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-activated mechano-sensitive Ca(2+) permeable cation channel (MscCa). We have transiently expressed TRPC1 and TRPC6 in African green monkey kidney (COS) or Chinese hamster ovary (CHO) cells and monitored the activity of the stretch-activated channels using a fast pressure clamp system. Although both TRPC1 and TRPC6 are highly expressed at the protein level, the amplitude of the mechano-sensitive current is not significantly altered by overexpression of these subunits. In conclusion, although several TRPC channel members, including TRPC1 and TRPC6, have been recently proposed to form MscCa in vertebrate cells, the functional expression of these TRPC subunits in heterologous systems remains problematic.


Subject(s)
Mechanoreceptors/physiology , TRPC Cation Channels/physiology , Animals , CHO Cells , COS Cells , Cell Membrane/metabolism , Chlorocebus aethiops , Cricetinae , Cricetulus , Humans , Microscopy, Confocal , Patch-Clamp Techniques , Plasmids , TRPC Cation Channels/biosynthesis , TRPC Cation Channels/genetics , TRPC6 Cation Channel , Transfection
19.
Trends Cardiovasc Med ; 16(8): 292-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17055386

ABSTRACT

Mutations in the PKD1 and PKD2 polycystin genes are responsible for autosomal dominant polycystic kidney disease (ADPKD), one of the most prevalent genetic kidney disorders. ADPKD is a multisystem disease characterized by the formation of numerous fluid-filled cysts in the kidneys, the pancreas, and the liver. Moreover, major cardiovascular manifestations are common complications in ADPKD. Intracranial aneurysms and arterial hypertension are among the leading causes of mortality in this disease. In the present review, we summarize our current understanding of the role of polycystins in the development, maintenance, and function of the cardiovascular system.


Subject(s)
Cardiovascular Diseases/etiology , Polycystic Kidney, Autosomal Dominant/etiology , TRPP Cation Channels , Animals , Disease Models, Animal , Humans , Mice , Mice, Transgenic
20.
Proc Natl Acad Sci U S A ; 103(39): 14361-6, 2006 Sep 26.
Article in English | MEDLINE | ID: mdl-16983068

ABSTRACT

Potassium channels are necessary for a number of essential biological tasks such as the generation of action potentials and setting the resting membrane potential in cells, both of which require that these channels selectively permit the passage of potassium ions while suppressing the flow of other ions. Generally, this selectivity is attributed to a narrow stretch of the channel known as the selectivity filter. Over this stretch ions are dehydrated, and the backbone oxygen atoms of the protein mimic the ion's loss of coordination by water. However, channels are long pores with spatially distinct ion-binding sites that all must be traversed during ion permeation. We have shown that selectivity of mutant Kir3.2 (GIRK2) channels can be substantially amplified by introducing acidic residues into the cavity, a binding site below the selectivity filter. Here, we carry out electrostatic calculations on homology models to quantify the degree of stabilization that these mutations have on ions in the cavity. We then construct a multiion model of ion permeation to calculate the channel's permeability to potassium relative to sodium. This kinetic model uses rates derived from the electrostatic calculations and demonstrates that nonselective electrostatic stabilization of cations in the cavity can amplify channel selectivity independently of the selectivity filter. This nonintuitive result highlights the dependence of channel properties on the entire channel architecture and suggests that selectivity may not be fully understood by focusing solely on thermodynamic considerations of ion dehydration and the energetics of the selectivity filter.


Subject(s)
G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism , Ion Channel Gating , Amino Acids/chemistry , Animals , Cell Membrane Permeability , G Protein-Coupled Inwardly-Rectifying Potassium Channels/chemistry , Kinetics , Models, Biological , Mutant Proteins/metabolism , Mutation/genetics , Oocytes , Potassium/metabolism , Sodium/metabolism , Static Electricity , Structure-Activity Relationship , Substrate Specificity , Thermodynamics , Xenopus laevis
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