Structure Of Potassium Channel Research Articles

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

Gene Structures and Expression Profiles of Three Human KCND (Kv4) Potassium Channels Mediating A-Type Currents ITO and ISA

The four known members of the KCND/Kv4 channel family encode voltage-gated potassium channels. Recent studies provide evidence that members of the Kv4 channel family are responsible for native, rapidly inactivating (A-type) currents described in heart (ITO) and neurons (ISA). In this study, we cloned the human KCND1 cDNA, localized the KCND1 gene to chromosome Xp11.23–p11.3, and determined the genomic structure and tissue-specific expression of the KCND1, KCND2, and KCND3 genes, respectively. The open reading frame of Kv4.1 is 1941 nucleotides long, predicting a protein of 647 amino acids. The deduced protein sequence of Kv4.1 shows an overall identity of 60% with Kv4.2 and Kv4.3L and corresponds to the common structure of voltage-gated potassium channels. KCND1-specific transcripts were detectable in human brain, heart, liver, kidney, thyroid gland, and pancreas, as revealed by Northern blot and RT-PCR experiments. The comparison of the expression patterns of the known Kv4 family members shows subtype specificity with significant overlaps. The KCND gene structures exhibit an evolutionarily conserved exon pattern with a large first exon containing the intracellular N-terminus and the putative membrane-spanning regions S1 to S5, as well as part of the pore region. The KCND3 gene contains an additional exon of 57 bp, which is not present in the other two KCND genes and gives rise to the C-terminal splice KCND3L variant with an insertion of 19 amino acids.

Localization of the K+ lock-In and the Ba2+ binding sites in a voltage-gated calcium-modulated channel. Implications for survival of K+ permeability.

Using Ba2+ as a probe, we performed a detailed characterization of an external K+ binding site located in the pore of a large conductance Ca2+-activated K+ (BKCa) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba2+ blocks BKCa channels and decreasing external K+ using a K+ chelator, (+)-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba2+-blocked events. Average Ba2+ dwell time changes from 10 s at 10 mM external K+ to 100 ms in the limit of very low [K+]. Using a model where external K+ binds to a site hindering the exit of Ba2+ toward the external side (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-568), we calculated a dissociation constant of 2.7 mircoM for K) at this lock-in site. We also found that BK(Ca) channels enter into a long-lasting nonconductive state when the external [K+] is reduced below 4 microM using the crown ether. Channel activity can be recovered by adding K+, Rb+, Cs+, or NH4+ to the external solution. These results suggest that the BK(Ca) channel stability in solutions of very low [K+] is due to K+ binding to a site having a very high affinity. Occupancy of this site by K+ avoids the channel conductance collapse and the exit of Ba2+ toward the external side. External tetraethylammonium also reduced the Ba2+ off rate and impeded the channel from entering into the long-lasting nonconductive state. This effect requires the presence of external K+. It is explained in terms of a model in which the conduction pore contains Ba2+, K+, and tetraethylammonium simultaneously, with the K+ binding site located internal to the tetraethylammonium site. Altogether, these results and the known potassium channel structure (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) imply that the lock-in site and the Ba2+ sites are the external and internal ion sites of the selectivity filter, respectively.

Dynamic selectivity filters in ion channels.

This work was supported by a Wellcome Trust (UK) International Prize Travelling Fellowship and the NIH (NS-11756).

Pharmacology of voltage-gated and calcium-activated potassium channels

Several important new findings have furthered the development of voltage-gated and calcium-activated potassium channel pharmacology. The molecular constituents of several members of these large ion channel families were identified. Small-molecule modulators of some of these channels were reported, including correolide, the first potent, small-molecule, natural product inhibitor of the Shaker family of voltage-gated potassium channels to be disclosed. The initial crystal structure of a bacterial potassium channel was determined; this work gives a physical basis for understanding the mechanisms of ion selectivity and ion conduction. With the recent molecular characterization of a potassium channel structure and the discovery of new templates for channel modulatory agents, the ability to rationally identify and develop potassium channel agonists and antagonists may become a reality in the near future.

Structure and function of cardiac potassium channels.

Recent advances in molecular biology have had a major impact on our understanding of the biophysical and molecular properties of ion channels. This review is focused on cardiac potassium channels which, in general, serve to control and limit cardiac excitability. Approximately 60 K+ channel subunits have been cloned to date. The (evolutionary) oldest potassium channel subunits consist of two transmembrane (Tm) segments with an intervening pore-loop (P). Channels formed by four 2Tm-1P subunits generally function as inwardly rectifying K(+)-selective channels (KirX.Y): they conduct substantial current near the resting potential but carry little or no current at depolarized potentials. The inward rectifier IK1 and the ligand-gated KATP and KACh channels are composed of such subunits. The second major class of K+ channel subunits consists of six transmembrane segments (S1-S6). The S5-P-S6 section resembles the 2Tm-1P subunit, and the additional membrane-spanning segments (especially the charged S4 segment) endow these 6Tm-1P channels with voltage-dependent gating. For both major families, four subunits assemble into a homo- or heterotetrameric channel, subject to specific subunit-subunit interactions. The 6Tm-1P channels are closed at the resting potential, but activate at different rates upon depolarization to carry sustained or transient outward currents (the latter due to inactivation by different mechanisms). Cardiac cells typically display at least one transient outward current and several delayed rectifiers to control the duration of the action potential. The molecular basis for each of these currents is formed by subunits that belong to different Kvx.y subfamilies and alternative splicing can contribute further to the diversity in native cells. These subunits display distinct pharmacological properties and drug-binding sites have been identified. Additional subunits have evolved by concatenation of two 2Tm-1P subunits (4Tm-2P); dimers of such subunits yield voltage-independent leak channels. A special class of 6Tm-1P subunits encodes the 'funny' pacemaker current which activates upon hyperpolarization and carries both Na+ and K+ ions. The regional heterogeneity of K+ currents and action potential duration is explained by the heterogeneity of subunit expression, and significant changes in expression occur in cardiac disease, most frequently a reduction. This electrical remodelling may also be important for novel antiarrhythmic therapeutic strategies. The recent crystallization of a 2Tm-1P channel enhances the outlook for more refined molecular approaches.

Identification and biochemical characterization of a novel nortriterpene inhibitor of the human lymphocyte voltage-gated potassium channel, Kv1.3.

A novel nortriterpene, termed correolide, purified from the tree Spachea correae, inhibits Kv1.3, a Shaker-type delayed rectifier potassium channel present in human T lymphocytes. Correolide inhibits 86Rb+ efflux through Kv1.3 channels expressed in CHO cells (IC50 86 nM; Hill coefficient 1) and displays a defined structure-activity relationship. Potency in this assay increases with preincubation time and with time after channel opening. Correolide displays marked selectivity against numerous receptors and voltage- and ligand-gated ion channels. Although correolide is most potent as a Kv1.3 inhibitor, it blocks all other members of the Kv1 family with 4-14-fold lower potency. C20-29-[3H]dihydrocorreolide (diTC) was prepared and shown to bind in a specific, saturable, and reversible fashion (Kd = 11 nM) to a single class of sites in membranes prepared from CHO/Kv1.3 cells. The molecular pharmacology and stoichiometry of this binding reaction suggest that one diTC site is present per Kv1.3 channel tetramer. This site is allosterically coupled to peptide and potassium binding sites in the pore of the channel. DiTC binding to human brain synaptic membranes identifies channels composed of other Kv1 family members. Correolide depolarizes human T cells to the same extent as peptidyl inhibitors of Kv1.3, suggesting that it is a candidate for development as an immunosuppressant. Correolide is the first potent, small molecule inhibitor of Kv1 series channels to be identified from a natural product source and will be useful as a probe for studying potassium channel structure and the physiological role of such channels in target tissues of interest.

Probing the structure and function of potassium channels with α-K toxin blockers

This review examines recent work aimed at revealing the molecular structures of the potassium channel vestibules using the α-K toxin (α-KTx) peptide blockers from the venoms of scorpions. The three subfamilies of α-K toxins are discussed in terms of their specificity for voltage-gated potassium (Kv) channels and for the large-conductance calcium-activated (maxi-K) channel. Among the α-KTx subfamilies, the three-dimensional solution structures all share a common β sheet/helix motif. However, there are differences in the toxin electrostatic structures and subtle differences in their α-carbon backbone structures that may underlie potassium channel specificity. The binding of these α-KTx's to the extracellular potassium channel pore is modulated by electrostatic interactions and by the channel gating conformation. Thus, these toxins are exceptional sensors of the electrostatic environment of the channel vestibule. Changes in toxin binding free energy, resulting from site-specific toxin mutants, have revealed a low resolution image of the α-KTx receptor surface and consequently the K channel vestibule. Interactions between specific residues on the toxin and on the channel were revealed by applying the principle of additivity of binding free energy to identify pairwise toxin:channel contacts. These interactions provide structural information about amino acids that line the Shaker potassium channel pore.

Two-dimensional crystallization and projection structure of KcsA potassium channel

Potassium channels are integral membrane proteins that play a crucial role in regulating diverse cell functions in both electrically excitable and non-excitable cells. Molecular cloning has revealed a diverse family of genes that encode these proteins, and a variety of experimental strategies have defined functional domains. We have cloned, over-expressed and purified the KcsA potassium channel to homogeneity and reconstituted this channel protein with phospholipids to form two-dimensional crystals. The crystals belong to plane group p4 and have unit cell dimensions of a = b = 48 Å. A projection map at 6 Å resolution has been obtained by electron crystallography. The map shows that the protein is a homotetramer, having a low-density region on the 4-fold axis that is the site of the ion conduction pathway. Each monomer contains density features that are consistent with the molecular model of a truncated form of KcsA recently determined by X-ray crystallography.

Molecular structure and expression of shaker type potassium channels in glial cells of trout CNS.

Two shaker-related potassium channel transcripts (tsha1, tsha2) were identified in glial cells of trout central nervous system (CNS) by polymerase chain reaction (PCR)-cloning and sequencing. While tsha1 was highly similar to the mammalian Kv1.2 subtype of potassium channels, tsha2 did not show a preferential sequence homology with a particular subtype of shaker, but exhibited uniform similarity with mammalian Kv1.1, Kv1.2, and Kv1.3, respectively. Transcripts for the shaw and shal subfamilies of voltage-gated potassium channels were detected in whole brain tissue only but not in freshly dissociated glial cells. Using mRNA extracted from different glial cell types in combination with sequence specific PCR primers, tshal was found restricted to oligodendrocytes and their progenitor cells, while tsha2 was common to astrocytes, as well.

Molecular structure and expression of shaker type potassium channels in glial cells of trout CNS

Molecular structure and expression of shaker type potassium channels in glial cells of trout CNS

Solution structure and proposed binding mechanism of a novel potassium channel toxin kappa-conotoxin PVIIA.

kappa-PVIIA is a 27-residue polypeptide isolated from the venom of Conus purpurascens and is the first member of a new class of conotoxins that block potassium channels. By comparison to other ion channels of eukaryotic cell membranes, voltage-sensitive potassium channels are relatively simple and methodology has been developed for mapping their interactions with small-peptide toxins. PVIIA, therefore, is a valuable new probe of potassium channel structure. This study of the solution structure and mode of channel binding of PVIIA forms the basis for mapping the interacting residues at the conotoxin-ion channel interface. The three-dimensional structure of PVIIA resembles the triple-stranded beta sheet/cystine-knot motif formed by a number of toxic and inhibitory peptides. Subtle structural differences, predominantly in loops 2 and 4, are observed between PVIIA and other conotoxins with similar structural frameworks, however. Electrophysiological binding data suggest that PVIIA blocks channel currents by binding in a voltage-sensitive manner to the external vestibule and occluding the pore. Comparison of the electrostatic surface of PVIIA with that of the well-characterised potassium channel blocker charybdotoxin suggests a likely binding orientation for PVIIA. Although the structure of PVIIA is considerably different to that of the alphaK scorpion toxins, it has a similar mechanism of channel blockade. On the basis of a comparison of the structures of PVIIA and charybdotoxin, we suggest that Lys19 of PVIIA is the residue which is responsible for physically occluding the pore of the potassium channel.

Structure and function of cardiac sodium and potassium channels.

The application of patch-clamp and molecular approaches has resulted in an increasingly refined understanding of the molecular entities underlying cardiac sodium and potassium currents. The sodium current results from expression of a single large alpha-subunit, whereas multiple potassium currents and potassium channel alpha-subunits have been identified. Recapitulation of some ion currents in heterologous expression systems requires not only expression of alpha-subunits but also ancillary (beta) subunits. Domains common to functions such as activation, inactivation, and drug block are now being identified in alpha- and beta-gene products. Variability in the expression or function of individual ion-channel genes is an increasingly recognized source of variability in the ion currents recorded in heart cells under physiological conditions (e.g. during development) as well as in disease.

Primary structure and functional expression of a cGMP-gated potassium channel.

Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcn1) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcn1 also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcn1 represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.

CDNA Sequence, Gene Structure, and Chromosomal Localization of the Human ATP-Sensitive Potassium Channel, uKATP-1, Gene (KCNJ8)

ATP-sensitive K+(KATP) channels play a crucial role in coupling metabolic energy to the membrane potential of cells. Recently, we have isolated a KATPchannel cDNA (uKATP-1) that is expressed ubiquitously in rat tissues including pancreatic islets, pituitary, skeletal muscle, and heart. Here, we report cloning of the human cDNA and gene encoding uKATP-1. Human uKATP-1 is a protein of 424 amino acids exhibiting 98% identity with rat uKATP-1. The human gene encoding uKATP-1, designated KCNJ8, is approximately 9.7 kb in length and is composed of three exons. KCNJ8 was mapped to chromosome 12p11.23 using fluorescencein situhybridization.

The structure, regulation and pathophysiology of potassium channels.

Potassium channels are membrane proteins that allow the passive diffusion of potassium down its electrochemical gradient. They play a critical role in many cellular processes and have, therefore, been extensively studied. Over the past several years, the molecular structures of several types of potassium channels have been elucidated. This review investigates the most recent findings regarding the structure, regulation and pathophysiology of potassium channels.

Potassium Channel Structure and Function as Reported by a Single Glycosylation Sequon

Inwardly rectifying K+ channels (IRKs) are highly K(+)-selective, integral membrane proteins that help maintain resting the membrane potential and cell volume. Integral membrane proteins as a class are frequently N-glycosylated with the attached carbohydrate being extracellular and perhaps modulating function. However, dynamic effects of glycosylation have yet to be demonstrated at the molecular level. ROMK1, a member of the IRK family is particularly suited to the study of glycosylation because it has a single N-glycosylation consensus sequence (Ho, K., Nichols, C. G., Lederer, W. J., Lytton, J., Vassilev, P. M., Kanazirska, M. V., and Herbert, S. C. (1993) Nature 362, 31-38). We show that ROMK1 is expressed in a functional state in the plasmalemma of an insect cell line (Spodoptera frugiperda, Sf9) and has two structures, glycosylated and unglycosylated. To test functionality, glycosylation was abolished by an N117Q mutation or by treatment with tunicamycin. Whole cell currents were greatly reduced in both of the unglycosylated forms compared to wild-type. Single channel currents revealed a dramatic decrease in opening probability, po, as the causative factor. Thus we have shown biochemically that the N-glycosylation sequon is extracellular, a result consistent with present topological models of IRKs, and we conclude that sequon occupancy by carbohydrate stabilizes the open state of ROMK1.

Pharmacology and structure of high conductance calcium-activated potassium channels

Pharmacology and structure of high conductance calcium-activated potassium channels

Intimations of potassium channel structure from a complete functional map of the molecular surface of charybdotoxin

The external vestibules of many K+ channels carry a high-affinity receptor for charybdotoxin, a peptide of known structure. Point mutations of a recombinant toxin identified the residues directly involved in the interaction with a Ca(2+)-activated K+ channel. The interaction surface is formed from 8 of the 37 residues and covers about 25% of the peptide's molecular surface. The shape of the toxin permits a deduced picture of the complementary receptor site in the external vestibule of the K+ channel.

Structure and functional expression of the voltage-gated potassium channels cloned from NG108-15 neuroblastoma-glioma hybrid cells

Structure and functional expression of the voltage-gated potassium channels cloned from NG108-15 neuroblastoma-glioma hybrid cells