Identification of (R)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-1-tosylpiperidine-2-carboxamide, ML277, as a novel, potent and selective K(v)7.1 (KCNQ1) potassium channel activator

A high-throughput screen utilizing a depolarization-triggered thallium influx through KCNQ1 channels was developed and used to screen the MLSMR collection of over 300,000 compounds. An iterative medicinal chemistry approach was initiated and from this effort, ML277 was identified as a potent activator of KCNQ1 channels (EC(50)=260nM). ML277 was shown to be highly selective against other KCNQ channels (>100-fold selectivity versus KCNQ2 and KCNQ4) as well as against the distantly related hERG potassium channel.     

Probing Binding Sites and Mechanisms of Action of an IKs Activator by Computations and Experiments

The slow delayed rectifier (I_Ks) channel is composed of the KCNQ1 channel and KCNE1 auxiliary subunit, and functions to repolarize action potentials in the human heart. I_Ks activators may provide therapeutic efficacy for treating long QT syndromes. Here, we show that a new KCNQ1 activator, ML277, can enhance I_Ks amplitude in adult guinea pig and canine ventricular myocytes. We probe its binding site and mechanism of action by computational analysis based on our recently reported KCNQ1 and KCNQ1/KCNE1 3D models, followed by experimental validation. Results from a pocket analysis and docking exercise suggest that ML277 binds to a side pocket in KCNQ1 and the KCNE1-free side pocket of KCNQ1/KCNE1. Molecular-dynamics (MD) simulations based on the most favorable channel/ML277 docking configurations reveal a well-defined ML277 binding space surrounded by the S2-S3 loop and S4-S5 helix on the intracellular side, and by S4–S6 transmembrane helices on the lateral sides. A detailed analysis of MD trajectories suggests two mechanisms of ML277 action. First, ML277 restricts the conformational dynamics of the KCNQ1 pore, optimizing K⁺ ion coordination in the selectivity filter and increasing current amplitudes. Second, ML277 binding induces global motions in the channel, including regions critical for KCNQ1 gating transitions. We conclude that ML277 activates I_Ks by binding to an intersubunit space and allosterically influencing pore conductance and gating transitions. KCNE1 association protects KCNQ1 from an arrhythmogenic (constitutive current-inducing) effect of ML277, but does not preclude its current-enhancing effect.     

Capturing distinct KCNQ2 channel resting states by metal ion bridges in the voltage-sensor domain

Although crystal structures of various voltage-gated K⁺ (Kv) and Na⁺ channels have provided substantial information on the activated conformation of the voltage-sensing domain (VSD), the topology of the VSD in its resting conformation remains highly debated. Numerous studies have investigated the VSD resting state in the Kv Shaker channel; however, few studies have explored this issue in other Kv channels. Here, we investigated the VSD resting state of KCNQ2, a K⁺ channel subunit belonging to the KCNQ (Kv7) subfamily of Kv channels. KCNQ2 can coassemble with the KCNQ3 subunit to mediate the I_M current that regulates neuronal excitability. In humans, mutations in KCNQ2 are associated with benign neonatal forms of epilepsy or with severe epileptic encephalopathy. We introduced cysteine mutations into the S4 transmembrane segment of the KCNQ2 VSD and determined that external application of Cd²⁺ profoundly reduced the current amplitude of S4 cysteine mutants S195C, R198C, and R201C. Based on reactivity with the externally accessible endogenous cysteine C106 in S1, we infer that each of the above S4 cysteine mutants forms Cd²⁺ bridges to stabilize a channel closed state. Disulfide bonds and metal bridges constrain the S4 residues S195, R198, and R201 near C106 in S1 in the resting state, and experiments using concatenated tetrameric constructs indicate that this occurs within the same VSD. KCNQ2 structural models suggest that three distinct resting channel states have been captured by the formation of different S4–S1 Cd²⁺ bridges. Collectively, this work reveals that residue C106 in S1 can be very close to several N-terminal S4 residues for stabilizing different KCNQ2 resting conformations.     

KCNQ1 Channels Do Not Undergo Concerted but Sequential Gating Transitions in Both the Absence and the Presence of KCNE1 Protein

The co-assembly of KCNQ1 with KCNE1 produces I_KS, a K⁺ current, crucial for the repolarization of the cardiac action potential. Mutations in these channel subunits lead to life-threatening cardiac arrhythmias. However, very little is known about the gating mechanisms underlying KCNQ1 channel activation. Shaker channels have provided a powerful tool to establish the basic gating mechanisms of voltage-dependent K⁺ channels, implying prior independent movement of all four voltage sensor domains (VSDs) followed by channel opening via a last concerted cooperative transition. To determine the nature of KCNQ1 channel gating, we performed a thermodynamic mutant cycle analysis by constructing a concatenated tetrameric KCNQ1 channel and by introducing separately a gain and a loss of function mutation, R231W and R243W, respectively, into the S4 helix of the VSD of one, two, three, and four subunits. The R231W mutation destabilizes channel closure and produces constitutively open channels, whereas the R243W mutation disrupts channel opening solely in the presence of KCNE1 by right-shifting the voltage dependence of activation. The linearity of the relationship between the shift in the voltage dependence of activation and the number of mutated subunits points to an independence of VSD movements, with each subunit incrementally contributing to channel gating. Contrary to Shaker channels, our work indicates that KCNQ1 channels do not experience a late cooperative concerted opening transition. Our data suggest that KCNQ1 channels in both the absence and the presence of KCNE1 undergo sequential gating transitions leading to channel opening even before all VSDs have moved.     

Altered KCNQ3 Potassium Channel Function Caused by the W309R Pore-Helix Mutation Found in Human Epilepsy

The second tryptophan (W) residue of the conserved WW motif in the pore helix of many K⁺ channel subunit is thought to interact with the tyrosine (Y) residues of the selectivity filter. A missense mutation causing the replacement of the corresponding residues with an arginine (W309R) occurs in KCNQ3 subunits forming part of M-channels. In this study, we examined the functional consequences of the W309R mutation in heterogously expressed KCNQ channels. Homomeric KCNQ3^W309R channels lacked KCNQ currents. Heteromeric KCNQ2/KCNQ3^W309R channels displayed a dominant-negative suppression of current and a significant modification in gating properties when compared with heteromeric KCNQ3/KCNQ2 channels mimicking the M-channels. A three-dimensional homology model in the W309R mutant indicated that the R side chain of pore helices is too far from the Y side chain of the selectivity filter to interact via hydrogen bonds with each other and stabilize the pore structure. Collectively, the present results suggest that the second W residues of pore helices and their chemical interaction with the Y residues of the selectivity filter are essential for normal K⁺ channel function. This pore-helix mutation, if occurs in the brain M channels, could thus lead to a channel dysfunction sufficient to trigger epileptic hyperexcitability.     

Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by serum- and glucocorticoid-regulated kinase-1

The voltage-gated KCNQ2/3 and KCNQ3/5 K(+) channels regulate neuronal excitability. We recently showed that KCNQ2/3 and KCNQ3/5 channels are regulated by the ubiquitin ligase Nedd4-2. Serum- and glucocorticoid-regulated kinase-1 (SGK-1) plays an important role in regulation of epithelial ion transport. SGK-1 phosphorylation of Nedd4-2 decreases the ability of Nedd4-2 to ubiquitinate the epithelial Na(+) channel, which increases the abundance of channel protein in the cell membrane. In this study, we investigated the mechanism(s) of SGK-1 regulation of M-type KCNQ channels expressed in Xenopus oocytes. SGK-1 significantly upregulated the K(+) current amplitudes of KCNQ2/3 and KCNQ3/5 channels approximately 1.4- and approximately 1.7-fold, respectively, whereas the kinase-inactive SGK-1 mutant had no effect. The cell surface levels of KCNQ2-hemagglutinin/3 were also increased by SGK-1. Deletion of the KCNQ3 channel COOH terminus in the presence of SGK-1 did not affect the K(+) current amplitude of KCNQ2/3/5-mediated currents. Coexpression of Nedd4-2 and SGK-1 with KCNQ2/3 or KCNQ3/5 channels did not significantly alter K(+) current amplitudes. Only the Nedd4-2 mutant (S448A)Nedd4-2 exhibited a significant downregulation of the KCNQ2/3/5 K(+) current amplitudes. Taken together, these results demonstrate a potential mechanism for regulation of KCNQ2/3 and KCNQ3/5 channels by SGK-1 regulation of the activity of the ubiquitin ligase Nedd4-2.     

State-dependent electrostatic interactions of S4 arginines with E1 in S2 during Kv7.1 activation

The voltage-sensing domain of voltage-gated channels is comprised of four transmembrane helices (S1–S4), with conserved positively charged residues in S4 moving across the membrane in response to changes in transmembrane voltage. Although it has been shown that positive charges in S4 interact with negative countercharges in S2 and S3 to facilitate protein maturation, how these electrostatic interactions participate in channel gating remains unclear. We studied a mutation in Kv7.1 (also known as KCNQ1 or KvLQT1) channels associated with long QT syndrome (E1K in S2) and found that reversal of the charge at E1 eliminates macroscopic current without inhibiting protein trafficking to the membrane. Pairing E1R with individual charge reversal mutations of arginines in S4 (R1–R4) can restore current, demonstrating that R1–R4 interact with E1. After mutating E1 to cysteine, we probed E1C with charged methanethiosulfonate (MTS) reagents. MTS reagents could not modify E1C in the absence of KCNE1. With KCNE1, (2-sulfonatoethyl) MTS (MTSES)⁻ could modify E1C, but [2-(trimethylammonium)ethyl] MTS (MTSET)⁺ could not, confirming the presence of a positively charged environment around E1C that allows approach by MTSES⁻ but repels MTSET⁺. We could change the local electrostatic environment of E1C by making charge reversal and/or neutralization mutations of R1 and R4, such that MTSET⁺ modified these constructs depending on activation states of the voltage sensor. Our results confirm the interaction between E1 and the fourth arginine in S4 (R4) predicted from open-state crystal structures of Kv channels and reveal an E1–R1 interaction in the resting state. Thus, E1 engages in electrostatic interactions with arginines in S4 sequentially during the gating movement of S4. These electrostatic interactions contribute energetically to voltage-dependent gating and are important in setting the limits for S4 movement.     

Zinc pyrithione-mediated activation of voltage-gated KCNQ potassium channels rescues epileptogenic mutants

KCNQ potassium channels are activated by changes in transmembrane voltage and play an important role in controlling electrical excitability. Human mutations of KCNQ2 and KCNQ3 potassium channel genes result in reduction or loss of channel activity and cause benign familial neonatal convulsions (BFNCs). Thus, small molecules capable of augmenting KCNQ currents are essential both for understanding the mechanism of channel activity and for developing therapeutics. We performed a high-throughput screen in search for agonistic compounds potentiating KCNQ potassium channels. Here we report identification of a new opener, zinc pyrithione (1), which activates both recombinant and native KCNQ M currents. Interactions with the channel protein cause an increase of single-channel open probability that could fully account for the overall conductance increase. Separate point mutations have been identified that either shift the concentration dependence or affect potentiation efficacy, thereby providing evidence for residues influencing ligand binding and downstream events. Furthermore, zinc pyrithione is capable of rescuing the mutant channels causal to BFNCs.     

Roles of alternative splicing in the functional properties of inner ear-specific KCNQ4 channels

The function of the KCNQ4 channel in the auditory setting is crucial to hearing, underpinned by the finding that mutations of the channel result in an autosomal dominant form of nonsyndromic progressive high frequency hearing loss. The precise function of KCNQ4 in the inner ear has not been established. However, recently we demonstrated that there is differential expression among four splice variants of KCNQ4 (KCNQ4_v1-v4) along the tonotopic axis of the cochlea. Alternative splicing specifies the outcome of functional channels by modifying the amino acid sequences within the C terminus at a site designated as the membrane proximal region. We show that variations within the C terminus of splice variants produce profound differences in the voltage-dependent phenotype and functional expression of the channel. KCNQ4_v4 lacks exons 9-11, resulting in deletion of 54 amino acid residues adjacent to the S6 domain compared with KCNQ4_v1. Consequently, the voltage-dependent activation of KCNQ4_v4 is shifted leftward by approximately 20 mV, and the number of functional channels is increased severalfold compared with KCNQ4_v1. The properties of KCNQ4_v2 and KCNQ4_v3 fall between KCNQ4_v1 and KCNQ4_v4. Because of variations in the calmodulin binding domains of the splice variants, the channels are differentially modulated by calmodulin. Co-expression of these splice variants yielded current magnitudes suggesting that the channels are composed of heterotetramers. Indeed, a dominant negative mutant of KCNQ4_v1 cripples the currents of the entire KCNQ4 channel family. Furthermore, the dominant negative KCNQ4 mutant stifles the activity of KCNQ2-5, raising the possibility of a global disruption of KCNQ channel activity and the ensuing auditory phenotype.     

The role of S4 charges in voltage-dependent and voltage-independent KCNQ1 potassium channel complexes

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 alpha subunits to generate the voltage-dependent I(Ks) channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium "leak" currents in gastrointestinal epithelia. Other Kv alpha subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2.     

Direct observation of a preinactivated, open state in BK channels with beta2 subunits

Proteins arising from the Slo family assemble into homotetramers to form functional large-conductance, Ca2+- and voltage-activated K+ channels, or BK channels. These channels are also found in association with accessory beta subunits, which modulate several aspects of channel gating and expression. Coexpression with either of two such subunits, beta2 or beta3b, confers time-dependent inactivation onto BK currents. mSlo1+beta3b channels display inactivation that is very rapid but incomplete. Previous studies involving macroscopic recordings from these channels have argued for the existence of a second, short-lived conducting state in rapid equilibrium with the nonconducting, inactivated conformation. This state has been termed "pre-inactivated," or O*. beta2-mediated inactivation, in contrast, occurs more slowly but is virtually complete at steady state. Here we demonstrate, using both macroscopic and single channel current recordings, that a preinactivated state is also a property of mSlo1+beta2 channels. Detection of this state is enhanced by a mutation (W4E) within the initial beta2 NH2-terminal segment critical for inactivation. This mutation increases the rate of recovery to the preinactivated open state, yielding macroscopic inactivation properties qualitatively more similar to those of beta3b. Furthermore, short-lived openings corresponding to entry into the preinactivated state can be observed directly with single-channel recording. By examining the initial openings after depolarization of a channel containing beta2-W4E, we show that channels can arrive directly at the preinactivated state without passing through the usual long-lived open conformation. This final result suggests that channel opening and inactivation are at least partly separable in this channel. Mechanistically, the preinactivated and inactivated conformations may correspond to binding of the beta subunit NH2 terminus in the vicinity of the cytoplasmic pore mouth, followed by definitive movement of the NH2 terminus into a position of occlusion within the ion-conducting pathway.     

Regulation of KCNQ4 potassium channel prepulse dependence and current amplitude by SGK1 in Xenopus oocytes.

The KCNQ gene family comprises voltage-gated potassium channels expressed in epithelial tissues (KCNQ1, KCNQ5), inner ear structures (KCNQ1, KCNQ4) and the brain (KCNQ2-5). KCNQ4 is expressed in inner and outer hair cells of the inner ear where it determines electrical excitability. Accordingly, loss of function mutations of the KCNQ4 gene cause hearing loss. Several K+ channels including the closely related KCNQ1/KCNE1 channel are regulated by the serum- and glucocorticoid-inducible kinase (SGK) family. The present study utilized the Xenopus oocyte system to explore effects of SGK isoforms on KCNQ4 mediated K(+)-currents: KCNQ4 channels activated in a voltage dependent manner with half maximal activation at -10 mV. The peak channel activity was significantly increased by prepulsing. Coexpression of wild type SGK1 but not coexpression of the inactive mutant (K127N)SGK1 significantly increased current amplitudes (by 67 %) and significantly increased the resting potential of KCNQ4 expressing oocytes. Here we describe for the first time a prepulse dependence of KCNQ4 channels with increased currents after hyperpolarizing prepulses. Coexpression of SGK1 significantly attenuated the effect of prepulsing on peak currents. Mutation of Ser to Asp or Ala in the putative phosphorylation consensus sequence in KCNQ4 significantly decreased the sensitivity to SGK1-coexpression. In conclusion, SGK1 regulates current amplitudes and kinetic properties of KCNQ4 channel activity, an effect sensitive to mutations in the SGK1 consensus sequence of the channel.     

Discovery of a Novel Activator of KCNQ1-KCNE1 K+ Channel Complexes

KCNQ1 voltage-gated K⁺ channels (Kv7.1) associate with the family of five KCNE peptides to form complexes with diverse gating properties and pharmacological sensitivities. The varied gating properties of the different KCNQ1-KCNE complexes enables the same K⁺ channel to function in both excitable and non excitable tissues. Small molecule activators would be valuable tools for dissecting the gating mechanisms of KCNQ1-KCNE complexes; however, there are very few known activators of KCNQ1 channels and most are ineffective on the physiologically relevant KCNQ1-KCNE complexes. Here we show that a simple boronic acid, phenylboronic acid (PBA), activates KCNQ1/KCNE1 complexes co-expressed in Xenopus oocytes at millimolar concentrations. PBA shifts the voltage sensitivity of KCNQ1 channel complexes to favor the open state at negative potentials. Analysis of different-sized charge carriers revealed that PBA also targets the permeation pathway of KCNQ1 channels. Activation by the boronic acid moiety has some specificity for the Kv7 family members (KCNQ1, KCNQ2/3, and KCNQ4) since PBA does not activate Shaker or hERG channels. Furthermore, the commercial availability of numerous PBA derivatives provides a large class of compounds to investigate the gating mechanisms of KCNQ1-KCNE complexes.     

Downregulation of KCNQ4 by Janus Kinase 2

Janus kinase-2 (JAK2) participates in the signaling of several hormones, growth factors and cytokines. Further stimulators of JAK2 include osmotic cell shrinkage, and the kinase activates the cell volume regulatory Na⁺/H⁺ exchanger. The kinase may thus participate in cell volume regulation. Cell shrinkage is known to inhibit K⁺ channels. Volume-regulatory K⁺ channels include the voltage-gated K⁺ channel KCNQ4. The present study explored the effect of JAK2 on KCNQ4 channel activity. KCNQ4 was expressed in Xenopus oocytes with or without wild-type JAK2, constitutively active ^V617FJAK2 or inactive ^K882EJAK2; and cell membrane conductance was determined by dual-electrode voltage clamp. Expression of KCNQ4 was followed by the appearance of voltage-gated K⁺ conductance. Coexpression of JAK2 or of ^V617FJAK2, but not of ^K882EJAK2, resulted in a significant decrease in conductance. Treatment of KCNQ4 and JAK2 coexpressing oocytes with the JAK2 inhibitor AG490 (40 μM) was followed by an increase in conductance. Treatment of KCNQ4 expressing oocytes with brefeldin A (5 μM) was followed by a decrease in conductance, which was similar in oocytes expressing KCNQ4 together with JAK2 as in oocytes expressing KCNQ4 alone. Thus, JAK2 apparently does not accelerate channel protein retrieval from the cell membrane. In conclusion, JAK2 downregulates KCNQ4 activity and thus counteracts K⁺ exit, an effect which may contribute to cell volume regulation.     

Determinants within the turret and pore-loop domains of KCNQ3 K+ channels governing functional activity

KCNQ1-5 (Kv7.1-7.5) subunits assemble to form a variety of functional K⁺ channels in the nervous system, heart, and epithelia. KCNQ1 and KCNQ4 homomers and KCNQ2/3 heteromers yield large currents, whereas KCNQ2 and KCNQ3 homomers yield small currents. Since the unitary conductance of KCNQ3 is five- to 10-fold greater than that of KCNQ4 or KCNQ1, these differences are even more striking. To test for differential membrane protein expression, we performed biotinylation and total internal reflection fluorescence imaging assays; however, both revealed only small differences among the channels, leading us to investigate other mechanisms at work. We probed the molecular determinants governing macroscopic current amplitudes, with focus on the turret and pore-loop domains of KCNQ1 and KCNQ3. Elimination of the putative N289 glycosylation site in KCNQ1 reduced current density by ∼56%. A chimera consisting of KCNQ3 with the turret domain (TD) of KCNQ1 increased current density by about threefold. Replacement of the proximal half of the TD in KCNQ3 with that of KCNQ1 increased current density by fivefold. A triple chimera containing the TD of KCNQ1 and the carboxy terminus of KCNQ4 yielded current density 10- or sixfold larger than wild-type KCNQ3 or KCNQ1, respectively, suggesting that the effects on current amplitudes of the TD and the carboxy-terminus are additive. Critical was the role of the intracellular TEA⁺-binding site. The KCNQ3 (A315T) swap increased current density by 10-fold, and the converse KCNQ1 (T311A) swap reduced it by 10-fold. KCNQ3 (A315S) also yielded greatly increased current amplitudes, whereas currents from mutant A315V channels were very small. The KCNQ3 (A315T) mutation increased the sensitivity of the channels to external Ba²⁺ block by eight- to 28-fold, consistent with this mutation altering the structure of the selectivity filter. To investigate a structural hypothesis for the effects of these mutations, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels, using KvAP as a template. The modeling suggests a critical stabilizing interaction between the pore helix and the selectivity filter that is absent in wild-type KCNQ3 and the A315V mutant, but present in the A315T and A315S mutants. We conclude that KCNQ3 homomers are well expressed at the plasma membrane, but that most wild-type channels are functionally silent, with rearrangements of the pore-loop architecture induced by the presence of a hydroxyl-containing residue at the 315 position “unlocking” the channels into a conductive conformation.     

Charges in the Cytoplasmic Pore Control Intrinsic Inward Rectification and Single-Channel Properties in Kir1.1 and Kir2.1 Channels

An E224G mutation of the Kir2.1 channel generates intrinsic inward rectification and single-channel fluctuations in the absence of intracellular blockers. In this study, we showed that positively charged residues H226, R228 and R260, near site 224, regulated the intrinsic inward rectification and single-channel properties of the E224G mutant. By carrying out systematic mutations, we found that the charge effect on the intrinsic inward rectification and single-channel conductance is consistent with a long-range electrostatic mechanism. A Kir1.1 channel where the site equivalent to E224 in the Kir2.1 channel is a glycine residue does not show inward rectification or single-channel fluctuations. The G223K and N259R mutations of the Kir1.1 channel induced intrinsic inward rectification and reduced the single-channel conductance but did not generate large open-channel fluctuations. Substituting the cytoplasmic pore of the E224G mutant into the Kir1.1 channel induced open-channel fluctuations and intrinsic inward rectification. The single-channel conductance of the E224G mutant showed inward rectification. Also, a voltage-dependent gating mechanism decreased open probability during depolarization and contributed to the intrinsic inward rectification in the E224G mutant. In addition to an electrostatic effect, a close interaction of K⁺ with channel pore may be required for generating open-channel fluctuations in the E224G mutant.     

An ERG channel inhibitor from the scorpion Buthus eupeus

The isolation of the peptide inhibitor of M-type K(+) current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K(+) channels was also studied. BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.     

A residue at the cytoplasmic entrance of BK-type channels regulating single-channel opening by its hydrophobicity

Single large-conductance calcium-activated K⁺ (BK) channels encoded by the mSlo gene usually have synchronous gating, but a Drosophila dSlo (A2/C2/E2/G5/10) splice variant (dSlo1A) exhibits very flickery openings. To probe this difference in gating, we constructed a mutant I323T. This channel exhibits four subconductance levels similar to those of dSlo1A. Rectification of the single-channel current-voltage relation of I323T decreased as [Ca²⁺ ]_in increased from 10 to 300 μM. Mutagenesis suggests that the hydrophobicity of the residue at the position is important for the wild-type gating; i.e., increasing hydrophobicity prolongs open duration. Molecular dynamics simulation suggests that four hydrophobic pore-lining residues at position 323 of mSlo act cooperatively in a “shutter-like” mechanism gating the permeation of K⁺ ions. Rate-equilibrium free energy relations analysis shows that the four I323 residues in an mSlo channel have a conformation 65% similar to the closed conformation during gating. Based on these observations, we suggest that the appearance of rectification and substates of BK-type channels arise from a reduction of the cooperativity among these four residues and a lower probability of being open.     

Dynamic interaction of S5 and S6 during voltage-controlled gating in a potassium channel

A gain-of-function mutation in the Caenorhabditis elegans exp-2 K(+)-channel gene is caused by a cysteine-to-tyrosine change (C480Y) in the sixth transmembrane segment of the channel (Davis, M.W., R. Fleischhauer, J.A. Dent, R.H. Joho, and L. Avery. 1999. Science. 286:2501-2504). In contrast to wild-type EXP-2 channels, homotetrameric C480Y mutant channels are open even at -160 mV, explaining the lethality of the homozygous mutant. We modeled the structure of EXP-2 on the 3-D scaffold of the K(+) channel KcsA. In the C480Y mutant, tyrosine 480 protrudes from S6 to near S5, suggesting that the bulky side chain may provide steric hindrance to the rotation of S6 that has been proposed to accompany the open-closed state transitions (Perozo, E., D.M. Cortes, and L.G. Cuello. 1999. Science. 285:73-78). We tested the hypothesis that only small side chains at position 480 allow the channel to close, but that bulky side chains trap the channel in the open state. Mutants with small side chain substitutions (Gly and Ser) behave like wild type; in contrast, bulky side chain substitutions (Trp, Phe, Leu, Ile, Val, and His) generate channels that conduct K(+) ions at potentials as negative as -120 mV. The side chain at position 480 in S6 in the pore model is close to and may interact with a conserved glycine (G421) in S5. Replacement of G421 with bulky side chains also leads to channels that are trapped in an active state, suggesting that S5 and S6 interact with each other during voltage-dependent open-closed state transitions, and that bulky side chains prevent the dynamic changes necessary for permanent channel closing. Single-channel recordings show that mutant channels open frequently at negative membrane potentials indicating that they fail to reach long-lasting, i.e., stable, closed states. Our data support a "two-gate model" with a pore gate responsible for the brief, voltage-independent openings and a separately located, voltage-activated gate (Liu, Y., and R.H. Joho. 1998. Pflügers Arch. 435:654-661).     

Inhibition of the heterotetrameric K+ channel KCNQ1/KCNE1 by the AMP-activated protein kinase

Abstract

The heterotetrameric K⁺-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na⁺ channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), of the kinase dead mutant ^αK45RAMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.