Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block

Background

Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs.

Methodology/Principal Findings

We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T→S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding.

Conclusions/Significance

The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features.     

Identification of a potassium channel site that interacts with G protein betagamma subunits to mediate agonist-induced signaling

Activation of heterotrimeric GTP-binding (G) proteins by their coupled receptors, causes dissociation of the G protein alpha and betagamma subunits. Gbetagamma subunits interact directly with G protein-gated inwardly rectifying K+ (GIRK) channels to stimulate their activity. In addition, free Gbetagamma subunits, resulting from agonist-independent dissociation of G protein subunits, can account for a major component of the basal channel activity. Using a series of chimeric constructs between GIRK4 and a Gbetagamma-insensitive K+ channel, IRK1, we have identified a critical site of interaction of GIRK with Gbetagamma. Mutation of Leu339 to Glu within this site impaired agonist-induced sensitivity and decreased binding to Gbetagamma, without removing the Gbetagamma contribution to basal currents. Mutation of the corresponding residue in GIRK1 (Leu333) resulted in a similar phenotype. Both the GIRK1 and GIRK4 subunits contributed equally to the agonist-induced sensitivity of the heteromultimeric channel. Thus, we have identified a channel site that interacts specifically with Gbetagamma subunits released through receptor stimulation.     

Multiple subunits of a voltage-dependent potassium channel contribute to the binding site for tetraethylammonium.

RNAs encoding a wild-type (RBK1) and a mutant (RBK1(Y379V,V381T); RBK1*) subunit of voltage-dependent potassium channels were injected into Xenopus oocytes. When expressed separately, they made homotetrameric channels that differed about 100-fold in sensitivity to tetraethylammonium (TEA). Mixtures of channels having one, two, or three low affinity subunits were expressed by injecting various proportions of RBK1 and RBK1* RNAs. The affinity for TEA of these three channel species was deduced by fitting concentration-response curves for the inhibition of potassium currents. DNAs were also concatenated to construct a sequence that encoded two connected subunits, and channels that contained four, two, or no TEA-sensitive subunits were expressed. The results suggest that bound TEA interacts simultaneously with all four subunits.     

Molecular dynamics simulation of the cytosolic mouth in Kcv-type potassium channels

The functional effect of mutations near the intracellular mouth of the short viral Kcv potassium channel was studied by molecular dynamics simulations. As a model system we used the analogously mutated and truncated KirBac1.1, a channel with known crystal structure that shares genuine local sequence motifs with Kcv. By a novel simulated annealing methodology for structural averaging, information about the structure and dynamics of the intracellular mouth was extracted and complemented by Poisson-Boltzmann and 3D-RISM (reference interaction site model) integral equation theory for the determination of the K+ free energy surface. Besides the wild-type analogue of Kcv with its experimental reference activity (truncated KirBac1.1), two variants were studied: a deletion mutant where the N-terminus is further truncated by eight amino acids, showing inactivity in the Kcv reference system, and a point mutant where the kink-forming proline at position 13 is substituted by alanine, resulting in hyperactivity. The computations reveal that the change of activity is closely related to a hydrophilic intracellular constriction formed by the C-terminal residues of the monomers. Hyperactivity of the point mutant is correlated with both sterical and electrostatic factors, while inactivity of the deletion mutant is related to a loss of specific salt bridge patterns between the C- and N-terminus at the constriction and to the consequences for ion passage barriers, as revealed by integral equation theory. The cytosolic gate, however, is probably formed by the N-terminal segment up to the proline kink and not by the constriction. The results are compared with design principles found for other channels.     

Ion-Ion interactions at the selectivity filter. Evidence from K(+)-dependent modulation of tetraethylammonium efficacy in Kv2.1 potassium channels

In the Kv2.1 potassium channel, binding of K(+) to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na(+) current, K(+) interacts with the TEA modulation site at concentrations 相似文献   

Separation of heteromeric potassium channel Kcv towards probing subunit composition-regulated ion permeation and gating

The chlorella virus-encoded Kcv can form a homo-tetrameric potassium channel in lipid membranes. This miniature peptide can be synthesized in vitro, and the tetramer purified from the SDS-polyacrylamide gel retains the K⁺ channel functionality. Combining this capability with the mass-tagging method, we propose a simple, straightforward approach that can generically manipulate individual subunits in the tetramer, thereby enabling the detection of contribution from individual subunits to the channel functions. Using this approach, we showed that the structural change in the selectivity filter from only one subunit is sufficient to cause permanent channel inactivation (“all-or-none” mechanism), whereas the mutation near the extracellular entrance additively modifies the ion permeation with the number of mutant subunits in the tetramer (“additive” mechanism).     

The aromatic binding site for tetraethylammonium ion on potassium channels.

K+ channels are quite variable in their sensitivity to the pore-blocking agent tetraethylammonium ion (TEA) when it is applied to the extracellular side of the membrane. A Shaker K+ channel can be made highly sensitive by introducing a tyrosine (or phenylalanine) at residue 449 in each of the four subunits. A shift in the voltage dependence of blockade indicates that TEA senses a smaller fraction of the transmembrane electric field in the highly sensitive channels. There is a linear relationship between the free energy for TEA blockade and the number of subunits (zero, two, or four) containing tyrosine at 449, as if these four residues interact simultaneously with a TEA molecule to produce a high affinity binding site. The temperature dependence of blockade suggests that the interaction is not purely hydrophobic. These findings are consistent with a TEA-binding site formed by a bracelet of pore-lining aromatic residues. The center of the bracelet could bind a TEA molecule through a cation-pi orbital interaction.     

TEA(+)-sensitive KCNQ1 constructs reveal pore-independent access to KCNE1 in assembled I(Ks) channels

I(Ks), a slowly activating delayed rectifier K(+) current through channels formed by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contributes to the control of the cardiac action potential duration. Coassembly of the two subunits is essential in producing the characteristic and physiologically critical kinetics of assembled channels, but it is not yet clear where or how these subunits interact. Previous investigations of external access to the KCNE1 protein in assembled I(Ks) channels relied on occlusion of the pore by extracellular application of TEA(+), despite the very low TEA(+) sensitivity (estimated EC(50) > 100 mM) of channels encoded by coassembly of wild-type KCNQ1 with the wild type (WT) or a series of cysteine-mutated KCNE1 constructs. We have engineered a high affinity TEA(+) binding site into the h-KCNQ1 channel by either a single (V319Y) or double (K318I, V319Y) mutation, and retested it for pore-delimited access to specific sites on coassembled KCNE1 subunits. Coexpression of either KCNQ1 construct with WT KCNE1 in Chinese hamster ovary cells does not alter the TEA(+) sensitivity of the homomeric channels (IC(50) approximately 0.4 mM [TEA(+)](out)), providing evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Coexpression of a cysteine-substituted KCNE1 (F54C) with V319Y significantly increases the sensitivity of channels to external Cd(2+), but neither the extent of nor the kinetics of the onset of (or the recovery from) Cd(2+) block was affected by [TEA(+)](o) at 10x the IC(50) for channel block. These data strongly suggest that access of Cd(2+) to the cysteine-mutated site on KCNE1 is independent of pore occlusion caused by TEA(+) binding to the outer region of the KCNE1/V319Y pore, and that KCNE1 does not reside within the pore region of the assembled channels.     

In vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel Kcv

Chlorella virus-encoded membrane protein Kcv represents a new class of potassium channel. This 94-amino acids miniature K(+) channel consists of two trans-membrane alpha-helix domains intermediated by a pore domain that contains a highly conserved K(+) selectivity filter. Therefore, as an archetypal K(+) channel, the study of Kcv may yield valuable insights into the structure-function relationships underlying this important class of ion channel. Here, we report a series of new properties of Kcv. We first verified Kcv can be synthesized in vitro. By co-synthesis and assembly of wild-type and the tagged version of Kcv, we were able to demonstrate a tetrameric stoichiometry, a molecular structure adopted by all known K(+) channels. Most notably, the tetrameric Kcv complex retains its functional integrity in SDS (strong detergent)-containing solutions, a useful feature that allows for direct purification of protein from polyacrylamide gel. Once purified, the tetramer can form single potassium-selective ion channels in a lipid bilayer with functions consistent to the heterologously expressed Kcv. These finding suggest that the synthetic Kcv can serve as a model of virus-encoded K(+) channels; and its newly identified properties can be applied to the future study on structure-determined mechanisms such as K(+) channel functional stoichiometry.     

Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K⁺ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K⁺ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K⁺, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.     

Multiple residues specify external tetraethylammonium blockade in voltage-gated potassium channels.

We have mapped residues in the carboxyl half of the P region of a voltage-gated K+ channel that influence external tetraethylammonium (TEA) block. Fifteen amino acids were substituted with cysteine and expressed in oocytes from monomeric or heterodimeric cRNAs. From a total of six mutant channels with altered TEA sensitivity, three were susceptible to modification by extracellularly applied charged methanethiosulfonates (MTSX). Another residue did not affect TEA block but was protected from MTSX by TEA. MTSX modification of position Y380C, thought to form the TEA binding site, affected TEA affinity only moderately, and this effect could be reversed by additional charge transfer from an oppositely charged MTSX analog. The results show that TEA block is modulated from multiple sites, including residues located deep in the pore and that several side chains besides that of Y380 are exposed at the TEA receptor.     

A novel potassium channel encoded by Ectocarpus siliculosus virus

Kcv, the first identified viral potassium channel encoded by the green algae Paramecium bursaria chlorella virus (PBCV-1), conducted K(+) selective currents when expressed in heterologous systems. This K(+) channel was proposed to be important for PBCV-1 infection and replication. In the present study, we identified and functionally characterized a novel K(+) channel Kesv, encoded by Ectocarpus siliculosus virus that infects filamentous marine brown algae. Kesv encodes a protein of 124 amino acids and is 21.8% identical and 37.1% homologous to Kcv. Membrane topology programs predicted that Kesv consists of three transmembrane domains. When expressed in Xenopus oocytes, Kesv induced largely instantaneous, K(+) selective currents that were sensitive to block by Ba(2+) and amantadine. Thus, Kesv along with Kcv, constitutes an emerging family of viral potassium channels, which may play important roles in the life cycle of viruses.     

A cation-pi interaction between extracellular TEA and an aromatic residue in potassium channels

Open-channel blockers such as tetraethylammonium (TEA) have a long history as probes of the permeation pathway of ion channels. High affinity blockade by extracellular TEA requires the presence of an aromatic amino acid at a position that sits at the external entrance of the permeation pathway (residue 449 in the eukaryotic voltage-gated potassium channel Shaker). We investigated whether a cation-pi interaction between TEA and such an aromatic residue contributes to TEA block using the in vivo nonsense suppression method to incorporate a series of increasingly fluorinated Phe side chains at position 449. Fluorination, which is known to decrease the cation-pi binding ability of an aromatic ring, progressively increased the inhibitory constant K(i) for the TEA block of Shaker. A larger increase in K(i) was observed when the benzene ring of Phe449 was substituted by nonaromatic cyclohexane. These results support a strong cation-pi component to the TEA block. The data provide an empirical basis for choosing between Shaker models that are based on two classes of reported crystal structures for the bacterial channel KcsA, showing residue Tyr82 in orientations either compatible or incompatible with a cation-pi mechanism. We propose that the aromatic residue at this position in Shaker is favorably oriented for a cation-pi interaction with the permeation pathway. This choice is supported by high level ab initio calculations of the predicted effects of Phe modifications on TEA binding energy.     

The SK3 subunit of small conductance Ca2+-activated K+ channels interacts with both SK1 and SK2 subunits in a heterologous expression system

The aim of this study was to determine whether functional heteromeric channels can be formed by co-assembly of rat SK3 (rSK3) potassium channel subunits with either SK1 or SK2 subunits. First, to determine whether rSK3 could co-assemble with rSK2 we created rSK3VK (an SK3 mutant insensitive to block by UCL 1848). When rSK3VK was co-expressed with rSK2 the resulting currents had an intermediate sensitivity to UCL 1848 (IC50 of approximately 5 nM compared with 120 pM for rSK2 and >300 nM for rSK3VK), suggesting that rSK3 and rSK2 can form functional heteromeric channels. To detect co-assembly of SK3 with SK1, we initially used a dominant negative construct of the human SK1 subunit (hSK1YP). hSK1YP dramatically reduced the SK3 current, supporting the idea that SK3 and SK1 subunits also interact. To determine whether these assemblies were functional we created rSK3VF, an rSK3 mutant with an enhanced affinity for tetraethylammonium chloride (TEA) (IC50 of 0.3 mM). Co-transfection of rSK3VF and hSK1 produced currents with a sensitivity to TEA not different from that of hSK1 alone (IC50 approximately 15 mM). These results suggest that hSK1 does not produce functional cell-surface assemblies with SK3. Antibody-staining experiments suggested that hSK1 may reduce the number of functional SK3 subunits reaching the cell surface. Additional experiments showed that co-expression of the rat SK1 gene with SK3 also dramatically suppressed SK current. The pharmacology of the residual current was consistent with that of homomeric SK3 assemblies. These results demonstrate interactions that cause changes in protein trafficking, cell surface expression, and channel pharmacology and strongly suggest heteromeric assembly of SK3 with the other SK channel subunits.     

Mechanisms of tetraethylammonium ion block in the KcsA potassium channel

We report results from automated docking and microscopic molecular dynamics simulations of the tetraethylammonium (TEA) complexes with KcsA. Binding modes and energies for TEA binding at the external and internal sides of the channel pore are examined utilising the linear interaction energy method. Effects of the channel ion occupancy (based on our previous results for the ion permeation mechanisms) on the binding energies are considered. Calculations show that TEA forms stable complexes at both the external and internal entrances of the selectivity filter. Furthermore, the effects of the Y82V mutation are evaluated and the results show, in agreement with experimental data, that the mutant has a significantly reduced binding affinity for TEA at the external binding site, which is attributed to stabilising hydrophobic interactions between the ligand and the tyrosines.     

Interaction between tetraethylammonium and amino acid residues in the pore of cloned voltage-dependent potassium channels

Extracellular tetraethylammonium (TEA) inhibits currents in Xenopus oocytes that have been injected with mRNAs encoding voltage-dependent potassium channels. Concentration-response curves were used to measure the affinity of TEA; this differed up to 700-fold among channels RBK1 (KD 0.3 mM), RGK5 (KD 11 mM), and RBK2 (KD greater than 200 mM). Studies in which chimeric channels were expressed localized TEA binding to the putative extracellular loop between trans-membrane domains S5 and S6. Site-directed mutagenesis of residues in this region identified the residue Tyr379 of RBK1 as a crucial determinant of TEA sensitivity; substitution of Tyr in the equivalent positions of RBK2 (Val381) and RGK5 (His401) made these channels as sensitive to TEA as RBK1. Nonionic forces are involved in TEA binding because (i) substitution of the Phe for Tyr379 in RBK1 increased its affinity, (ii) protonation of His401 in RGK5 selectively reduced its affinity, and (iii) the affinity of TEA was unaffected by changes in ionic strength. The results suggest an explanation for the marked differences in TEA sensitivity that have been observed among naturally occurring and cloned potassium channels and indicate that the amino acid corresponding to residue 379 in RBK1 lies within the external mouth of the ion channel.     

Structural basis for toxin resistance of beta4-associated calcium-activated potassium (BK) channels

The functional diversity of large conductance Ca(2+)- and voltage-dependent K(+) (BK) channels arises mainly from co-assembly of the pore-forming mSlo alpha subunits with four tissue-enriched auxiliary beta subunits. The structural basis of the interaction between alpha subunits with beta subunits is not well understood. Using computational and experimental methods, we demonstrated that four mSlo turrets decentralized distally from the channel pore to provide a wide open conformation and that the mSlo and hbeta4 subunits together formed a "helmet" containing three basic residues (Lys-120, Arg-121, and Lys-125), which impeded the entry of charybdotoxin (ChTX) by both the electrostatic interaction and limited space. In addition, the tyrosine insert mutant (in100Y) showed 56% inhibition, with a K(d) = 17 nm, suggesting that the hbeta4 lacks an external ChTX-binding site (Tyr-100). We also found that mSlo had an internal binding site (Tyr-294) in the alpha subunits that could "permanently" block 15% of mSlo+hbeta4 currents in the presence of 100 nm ChTX. These findings provide a better understanding of the diverse interactions between alpha and beta subunits and will improve the design of channel inhibitors.     

Competition for block of a Ca2(+)-activated K+ channel by charybdotoxin and tetraethylammonium

Single high-conductance Ca2(+)-activated K+ channels were incorporated into planar lipid bilayers, and the discrete block by charybdotoxin (CTX), a protein inhibitor of this channel, was studied. In particular, the effect of externally added tetraethylammonium (TEA) on CTX blocking kinetics was investigated. TEA decreases the on-rate of CTX in exact proportion to its blocking of the single-channel current. The CTX off-rate is unaffected by TEA. The results demonstrate that TEA and CTX are mutually exclusive in their binding to the channel. Since the site of TEA binding is known to be located on the external side of the conduction pore, this result further strengthens the proposal that the CTX binding site is located in the external mouth of the channel.     

Evidence for a tRNA/rRNA interaction site within the peptidyltransferase center of the Escherichia coli ribosome

A nine-base oligodeoxyribonucleotide complementary to bases 2497-2505 of 23S rRNA was hybridized to both 50S subunits and 70S ribosomes. The binding of the probe to the ribosome or ribosomal subunits was assayed by nitrocellulose filtration and by sucrose gradient centrifugation techniques. The location of the hybridization site was determined by digestion of the rRNA/cDNA heteroduplex with ribonuclease H and gel electrophoresis of the digestion products, followed by the isolation and sequencing of the smaller digestion fragment. The cDNA probe was found to interact specifically with its rRNA target site. The effects on probe hybridization to both 50S and 70S ribosomes as a result of binding deacylated tRNA(Phe) were investigated. The binding of deacylated tRNA(Phe), either with or without the addition of poly(uridylic acid), caused attenuation of probe binding to both 50S and 70S ribosomes. Probe hybridization to 23S rRNA was decreased by about 75% in both 50S subunits and 70S ribosomes. These results suggest that bases within the 2497-2505 site may participate in a deacylated tRNA/rRNA interaction.     

Long distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins

Kcv is a 94-amino acid protein encoded by chlorella virus PBCV-1 that corresponds to the pore module of K(+) channels. Therefore, Kcv can be a model for studying the protein design of K(+) channel pores. We analyzed the molecular diversity generated by approximately 1 billion years of evolution on kcv genes isolated from 40 additional chlorella viruses. Because the channel is apparently required for virus replication, the Kcv variants are all functional and contain multiple and dispersed substitutions that represent a repertoire of allowed sets of amino acid substitutions (from 4 to 12 amino acids). Correlations between amino acid substitutions and the new properties displayed by these channels guided site-directed mutations that revealed synergistic amino acid interactions within the protein as well as previously unknown interactions between distant channel domains. The effects of these multiple changes were not predictable from a priori structural knowledge of the channel pore.