Synthetic Analogues of the Snail Toxin 6-Bromo-2-mercaptotryptamine Dimer (BrMT) Reveal That Lipid Bilayer Perturbation Does Not Underlie Its Modulation of Voltage-Gated Potassium Channels.

Drugs do not act solely by canonical ligand-receptor binding interactions. Amphiphilic drugs partition into membranes, thereby perturbing bulk lipid bilayer properties and possibly altering the function of membrane proteins. Distinguishing membrane perturbation from more direct protein-ligand interactions is an ongoing challenge in chemical biology. Herein, we present one strategy for doing so, using dimeric 6-bromo-2-mercaptotryptamine (BrMT) and synthetic analogues. BrMT is a chemically unstable marine snail toxin that has unique effects on voltage-gated K+ channel proteins, making it an attractive medicinal chemistry lead. BrMT is amphiphilic and perturbs lipid bilayers, raising the question of whether its action against K+ channels is merely a manifestation of membrane perturbation. To determine whether medicinal chemistry approaches to improve BrMT might be viable, we synthesized BrMT and 11 analogues and determined their activities in parallel assays measuring K+ channel activity and lipid bilayer properties. Structure-activity relationships were determined for modulation of the Kv1.4 channel, bilayer partitioning, and bilayer perturbation. Neither membrane partitioning nor bilayer perturbation correlates with K+ channel modulation. We conclude that BrMT's membrane interactions are not critical for its inhibition of Kv1.4 activation. Further, we found that alkyl or ether linkages can replace the chemically labile disulfide bond in the BrMT pharmacophore, and we identified additional regions of the scaffold that are amenable to chemical modification. Our work demonstrates a strategy for determining if drugs act by specific interactions or bilayer-dependent mechanisms, and chemically stable modulators of Kv1 channels are reported.

Amino Acid Properties of Trafficking Determinants in the Outer Pore-Forming Region of Kv1 Potassium Channels in Cell Lines.

Different classes of Kv1 potassium channels have different trafficking patterns despite having very similar amino acid sequences. Two amino acids responsible for these differences have been identified in the outer pore turret region of Kv1.1 and Kv1.4. Here we tested a series of substitutions at these two determinants on Kv1.4. All P506 substitutions tested resulted in a significant decrease in surface protein, total protein, and protein half-life, indicating that proline is required at 506 to stabilize protein conformation and increase trafficking to the cell surface. All K533 substitutions tested had no effect on total protein, suggesting that the lysine at 533 is not important for maintaining Kv1.4 protein conformation. However, a basic or long polar amino acid, such as K, R, or Q, at this position favored high surface protein and efficient trafficking of Kv1.4, whereas an acidic or short amino acid, such as D, E, S, L, N, or H, at this position induced partial high endoplasmic reticulum-retention. This intracellular retention was not due to protein misfolding. We propose that these four prolines and four lysines in a Kv1.4 homotetramer might provide a binding site for a putative endoplasmic reticulum-export molecule to ensure high cell surface protein expression of the channel.

The episodic ataxia type 1 mutation I262T alters voltage-dependent gating and disrupts protein biosynthesis of human Kv1.1 potassium channels.

Voltage-gated potassium (Kv) channels are essential for setting neuronal membrane excitability. Mutations in human Kv1.1 channels are linked to episodic ataxia type 1 (EA1). The EA1-associated mutation I262T was identified from a patient with atypical phenotypes. Although a previous report has characterized its suppression effect, several key questions regarding the impact of the I262T mutation on Kv1.1 as well as other members of the Kv1 subfamily remain unanswered. Herein we show that the dominant-negative effect of I262T on Kv1.1 current expression is not reversed by co-expression with Kvß1.1 or Kvß2 subunits. Biochemical examinations indicate that I262T displays enhanced protein degradation and impedes membrane trafficking of Kv1.1 wild-type subunits. I262T appears to be the first EA1 mutation directly associated with impaired protein stability. Further functional analyses demonstrate that I262T changes the voltage-dependent activation and Kvß1.1-mediated inactivation, uncouples inactivation from activation gating, and decelerates the kinetics of cumulative inactivation of Kv1.1 channels. I262T also exerts similar dominant effects on the gating of Kv1.2 and Kv1.4 channels. Together our data suggest that I262T confers altered channel gating and reduced functional expression of Kv1 channels, which may account for some of the phenotypes of the EA1 patient.

The degree of N-glycosylation affects the trafficking and cell surface expression levels of Kv1.4 potassium channels.

Kv1.4 potassium channels are heavily glycosylated proteins involved in shaping action potentials and in neuronal excitability and plasticity. Kv1.4 N354Q, without an N-glycan, exhibited decreased protein stability and trafficking to the cell surface (Watanabe et al. in J Biol Chem 279:8879-8885, 2004). Here we investigated whether the composition of the N-glycan affected Kv1.4 cell surface expression. Kv1.4 proteins carrying N-glycans with different compositions were generated by adding glycosidase inhibitors or using N-glycosylation-deficient mutant cell lines. We found that oligomannose-type, hybrid-type, or incomplete complex-type N-glycans had a negative effect on surface protein expression of Kv1.4 compared with complex-type N-glycans. The decrease in surface protein level of Kv1.4 was mainly due to a reduction in total protein level, induced by altered N-glycan composition. Kv1.4 in CSTP-treated cells carried a unique oligomannose-type N-glycan that contains three glucose residues. This N-glycan had the most negative effect on cell surface expression of Kv1.4. It decreased Kv1.4 surface protein level by a combined mechanism of reducing total protein level and increasing ER-retention. Our data suggest that composition of the N-glycan plays an important role in protein stability and trafficking, and a sialylated complex-type N-glycan promoted high cell surface expression of Kv1.4.

Synaptotagmin I delays the fast inactivation of Kv1.4 channel through interaction with its N-terminus.

BACKGROUND: The voltage-gated potassium channel Kv1.4 is an important A-type potassium channel and modulates the excitability of neurons in central nervous system. Analysis of the interaction between Kv1.4 and its interacting proteins is helpful to elucidate the function and mechanism of the channel. RESULTS: In the present research, synaptotagmin I was for the first time demonstrated to be an interacting protein of Kv1.4 and its interaction with Kv1.4 channel did not require the mediation of other synaptic proteins. Using patch-clamp technique, synaptotagmin I was found to delay the inactivation of Kv1.4 in HEK293T cells in a Ca2+-dependent manner, and this interaction was proven to have specificity. Mutagenesis experiments indicated that synaptotagmin I interacted with the N-terminus of Kv1.4 and thus delayed its N-type fast inactivation. CONCLUSION: These data suggest that synaptotagmin I is an interacting protein of Kv1.4 channel and, as a negative modulator, may play an important role in regulating neuronal excitability and synaptic efficacy.

Heme impairs the ball-and-chain inactivation of potassium channels.

Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.

Properties of Slo1 K+ channels with and without the gating ring.

High-conductance Ca(2+)- and voltage-activated K(+) (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca(2+) and Mg(2+) sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca(2+)- and Mg(2+)-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca(2+) and Mg(2+) activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.

Effect of inserting charged peptide at NH(2)-terminal on N-type inactivation of Kv1.4 channel.

Rapid inactivation of voltage-gated potassium channel plays an important role in shaping the electrical signaling in neurons and other excitable cells. N-type ("ball and chain") inactivation, as the most extensively studied inactivation model, is assumed to be the inactivation mechanism of Kv1.4 channel. The inactivation ball inactivates the channel by interacting with the hydrophobic wall of inner pore and occluding it. Recently, we have proved that the electrostatic interaction between two charged segments in the NH(2)-termainal plays an important role through promoting the inactivation process of the Kv1.4 channel. This study investigates the effect of inserting negatively or positively charged short peptides at NH(2)-terminal on the inactivation of Kv1.4 channel. The results that inserting negatively-charged peptide (either myc or D-peptide) at different sites of NH(2)-terminal, deceleraes inactivation process of Kv1.4 channel to a different extent with inserting site changing and that the mutant Kv1.4-D50 exhibits a more slower inactivation rate than Kv1.4-K50 further identified the role of electrostatic interactions in the "ball and chain" inactivation mechanism.

Interaction of the S6 proline hinge with N-type and C-type inactivation in Kv1.4 channels.

Several voltage-gated channels share a proline-valine-proline (proline hinge) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels, which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine or alanine: P558A, P558G. These mutations were studied in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N- and C-type inactivation. Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed by more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement play a significant role in inactivation and recovery. Computer modeling suggests that both P558G and P558A mutations modify early voltage-dependent steps and make a final voltage-insensitive step that is rate limiting at positive potentials.

Electrostatic interaction between inactivation ball and T1-S1 linker region of Kv1.4 channel.

Inactivation of potassium channels plays an important role in shaping the electrical signaling properties of nerve and muscle cells. The rapid inactivation of Kv1.4 has been assumed to be controlled by a "ball and chain" inactivation mechanism. Besides hydrophobic interaction between inactivation ball and the channel's inner pore, the electrostatic interaction has also been proved to participate in the "ball and chain" inactivation process of Kv1.4 channel. Based on the crystal structure of Kv1.2 channel, the acidic T1-S1 linker is indicated to be a candidate interacting with the positively charged hydrophilic region of the inactivation domain. In this study, through mutating the charged residues to amino acids of opposite polar, we identified the electrostatic interaction between the inactivation ball and the T1-S1 linker region of Kv1.4 channel. Inserting negatively charged peptide at the amino terminal of Kv1.4 channel further confirmed the electrostatic interaction between the two regions.

Episodic ataxia type 1 mutations affect fast inactivation of K+ channels by a reduction in either subunit surface expression or affinity for inactivation domain.

Episodic ataxia type 1 (EA1) is an autosomal dominant disorder characterized by continuous myokymia and episodic attacks of ataxia. Mutations in the gene KCNA1 that encodes the voltage-gated potassium channel Kv1.1 are responsible for EA1. In several brain areas, Kv1.1 coassembles with Kv1.4, which confers N-type inactivating properties to heteromeric channels. It is therefore likely that the rate of inactivation will be determined by the number of Kv1.4 inactivation particles, as set by the precise subunit stoichiometry. We propose that EA1 mutations affect the rate of N-type inactivation either by reduced subunit surface expression, giving rise to a reduced number of Kv1.1 subunits in heterotetramer Kv1.1-Kv1.4 channels, or by reduced affinity for the Kv1.4 inactivation domain. To test this hypothesis, quantified amounts of mRNA for Kv1.4 or Kv1.1 containing selected EA1 mutations either in the inner vestibule of Kv1.1 on S6 or in the transmembrane regions were injected into Xenopus laevis oocytes and the relative rates of inactivation and stoichiometry were determined. The S6 mutations, V404I and V408A, which had normal surface expression, reduced the rate of inactivation by a decreased affinity for the inactivation domain while the mutations I177N in S1 and E325D in S5, which had reduced subunit surface expression, increased the rate of N-type inactivation due to a stoichiometric increase in the number of Kv1.4 subunits.

Electrostatic interaction in the NH(2)-terminus accelerates inactivation of the Kv1.4 channel.

Inactivation of potassium channels plays an important role in shaping the electrical signalling properties of nerve and muscle cells. While it has been assumed that the rapid inactivation of the Kv1.4 channel is controlled by a "ball and chain" inactivation mechanism, the chain structure of the channel has not been well defined. Here, by conducting electrophysiological studies on variants containing mutations of the positively charged and negatively charged segments of the NH(2)-terminal of the channel protein, we show that neutralization or deletion of the positively charged segment (residues 83-98) significantly slowed the inactivation process. Replacement of this positively charged segment with the negatively charged segment (residues 123-137), and vice versa, so that both segments were simultaneously positively or negatively charged, also slowed the inactivation process. Furthermore, the inactivation process was not changed when the positively charged and the negatively charged segments were interchanged. In contrast, the voltage dependence of activation and inactivation of the channels was not significantly altered by these mutants. These results indicate that the electrostatic interaction between the positively and negatively charged segments plays a critical role in the inactivation process of the Kv1.4 channel. Taken together, we propose that the electrostatic interaction accelerates the inactivation of the Kv1.4 channel by making it easier for the inactivation ball to access its binding site.

N-glycans modulate K(v)1.5 gating but have no effect on K(v)1.4 gating.

Nerve and muscle action potential repolarization are produced and modulated by the regulated expression and activity of several types of voltage-gated K(+) (K(v)) channels. Here, we show that sialylated N-glycans uniquely impact gating of a mammalian Shaker family K(v) channel isoform, K(v)1.5, but have no effect on gating of a second Shaker isoform, K(v)1.4. Each isoform contains one potential N-glycosylation site located along the S1-S2 linker; immunoblot analyses verified that K(v)1.4 and K(v)1.5 were N-glycosylated. The conductance-voltage (G-V) relationships and channel activation rates for two glycosylation-site deficient K(v)1.5 mutants, K(v)1.5(N290Q) and K(v)1.5(S292A), and for wild-type K(v)1.5 expressed under conditions of reduced sialylation, were each shifted linearly by a depolarizing approximately 18 mV compared to wild-type K(v)1.5 activation. External divalent cation screening experiments suggested that K(v)1.5 sialic acids contribute to an external surface potential that modulates K(v)1.5 activation. Channel availability was unaffected by changes in K(v)1.5 glycosylation or sialylation. The data indicate that sialic acid residues attached to N-glycans act through electrostatic mechanisms to modulate K(v)1.5 activation. The sialic acids fully account for effects of N-glycans on K(v)1.5 gating. Conversely, K(v)1.4 gating was unaffected by changes in channel sialylation or following mutagenesis to remove the N-glycosylation site. Each phenomenon is unique for K(v)1 channel isoforms, indicating that sialylated N-glycans modulate gating of homologous K(v)1 channels through isoform-specific mechanisms. Such modulation is relevant to changes in action potential repolarization that occur as ion channel expression and glycosylation are regulated.

NMR assignments of a 48 kDa tetramer of the T1 domain of the mammalian voltage gated potassium channel Kv1.4.

The N-terminal cytosolic T1 domain of the mammalian voltage gated potassium channel Kv1.4 is strongly involved in the tetramerization of the Kv1.4 subunit that is required for forming a functional ion channel. The T1 domain forms a stable tetramer of 48 kDa in solution that cannot be dissociated into monomers. In spite of the high molecular mass it was possible to completely assign the backbone and part of the side chain resonances by multidimensional NMR spectroscopy on uniformly (2)H, (13)C, (15)N enriched protein. The secondary structure analysis derived from the chemical shifts is in line with the expectations from X-ray structures of related proteins.

Identification of an evolutionarily conserved extracellular threonine residue critical for surface expression and its potential coupling of adjacent voltage-sensing and gating domains in voltage-gated potassium channels.

The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.

Ginsenoside Rg3 inhibits human Kv1.4 channel currents by interacting with the Lys531 residue.

We have demonstrated previously that the 20(S) but not the 20(R) form of ginsenoside Rg(3) inhibited K(+) currents flowing through Kv1.4 (hKv1.4) channels expressed in Xenopus laevis oocytes, pointing to the presence of specific interaction site(s) for Rg(3) in the hKv1.4 channel. In the current study, we sought to identify this site(s). To this end, we first assessed how point mutations of various amino acid residues of the hKv1.4 channel affected inhibition by 20(S)-ginsenoside Rg(3) (Rg(3)). Lys531 residue is known to be a key site for K(+) activation and to be part of the extracellular tetraethylammonium (TEA) binding site; the mutation K531Y abolished the Rg(3) effect and made the Kv1.4 channel sensitive to TEA applied to the extracellular side of the membrane. Mutations of many other residues, including the pH sensitive-site (H507Q), were without any significant effect. We next examined whether K(+) and TEA could alter the effect of Rg(3) and vice versa. We found that 1) raising [K(+)](o) reduced the inhibitory effect of Rg(3) on hKv1.4 channel currents, whereas Rg(3) shifted the K(+) activation curve to the right, and 2) TEA caused a rightward shift of the Rg(3) concentration-response curve of wild-type hKv1.4 channel currents, whereas Rg(3) caused a rightward shift of the TEA concentration-response curve of K531Y mutant channel currents. The docked modeling revealed that Lys531 plays a key role in forming hydrogen bonds between Rg(3) and hKv1.4 channels. These results indicate that Rg(3) inhibits the hKv1.4 channel current by interacting with residue Lys531.

Genetically encoding unnatural amino acids for cellular and neuronal studies.

Proteins participate in various biological processes and can be harnessed to probe and control biological events selectively and reproducibly, but the genetic code limits the building block to 20 common amino acids for protein manipulation in living cells. The genetic encoding of unnatural amino acids will remove this restriction and enable new chemical and physical properties to be precisely introduced into proteins. Here we present new strategies for generating orthogonal tRNA-synthetase pairs, which made possible the genetic encoding of diverse unnatural amino acids in different mammalian cells and primary neurons. Using this new methodology, we incorporated unnatural amino acids with extended side chains into the K+ channel Kv1.4, and found that the bulkiness of residues in the inactivation peptide is essential for fast channel inactivation, a finding that had not been possible using conventional mutagenesis. This technique will stimulate and facilitate new molecular studies using tailored unnatural amino acids for cell biology and neurobiology.

Kv1 potassium channel C-terminus constant HRETE region: arginine substitution affects surface protein level and conductance level of subfamily members differentially.

We have shown previously that truncating all of the variable cytoplasmic C-terminus of Kv1.1 potassium channels to G421stop had only a small inhibitory effect on their cell surface conductance density levels and cell surface protein levels. Here we investigated the role of a highly conserved cytoplasmic C-terminal charged region of five amino acids (HRETE) of the S6 transmembrane domain in the protein and conductance expression of Kv1.1, Kv1.2, and Kv1.4 channels. For Kv1.1 we found that E420stop, T419stop, and E418stop showed cell surface conductance densities and cell surface protein levels similar to full length control, whereas R417stop and H416stop exhibited essentially no conductance but their surface protein levels were similar to full length control. A bulky non-negatively charged hydrophilic amino acid at position 417 appeared to be critical for wild type gating of Kv1.1 because R417K and R417Q rescued conductance levels whereas R417A or R417E did not. The R417A mutation in the full length Kv1.1 also exhibited surface protein levels similar to control but it did not exhibit significant conductance. In contrast, mutation of the equivalent arginine to alanine in full length Kv1.2 and Kv1.4 appeared to have little or no effect on channel conductance but rather decreased cell surface protein levels by inducing partial high ER retention. These findings are consistent with the notion that the arginine amino acid in the HRETE region plays a different role in affecting conductance levels or cell surface protein levels of very closely related Kv1 potassium channels.

Modelling the pH-dependent properties of Kv1 potassium channels.

It is known that the pH dependence of conductance for the rat potassium channel Kv1.4 is susbstantially reduced upon mutation of either H508 or K532. These residues lie in the extracellular mouth of the channel pore. We have used continuum electrostatics to investigate their interactions with K(+) sites in the pore. The predicted scale of interactions between H508/K532 and potassium sites is sufficient to significantly alter potassium occupancy and thus channel function. We interpret the effect of K532 mutation as indicating that the pH-dependent effect requires not only an ionisable group with a suitable pK(a) value (i.e. histidine), but also that other charged groups set the potential profile at a threshold level. This hypothesis is examined in the context of pH dependence for other members of the Kv1 family, and may represent a general tool with which to study potassium channels.

Clustering assay for studying the interaction of membrane proteins with PDZ domain proteins.

Some membrane proteins must be clustered at target sites to efficiently perform their functions. PDZ domain-containing scaffold proteins bind to the tails of target membrane proteins and promote their localization and clustering on the cell surface. This chapter describes the experimental details of the clustering assay, using the interaction between potassium channels and PSD-95, an abundant PDZ domain protein in neuronal synapses, as a model.