Robin Y. Kim

Atomic basis for therapeutic activation of neuronal potassium channels

Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2–5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.

Decomposition of slide helix contributions to ATP-dependent inhibition of Kir6.2 channels.

Background: Kir6.2 potassium channels are inhibited by intracellular ATP. Results: A rescue mechanism was applied to loss-of-function channel mutations. Asp-58 mutations in an interfacial helix (the “slide” helix) abolish ATP sensitivity. Conclusion: Residue Asp-58 is essential for coupling Kir6.2 channel cytoplasmic and transmembrane domains. Significance: We describe a novel rescue mechanism to characterize loss-of-function Kir6.2 channel mutants. Regulation of inwardly rectifying potassium channels by intracellular ligands couples cell membrane excitability to important signaling cascades and metabolic pathways. We investigated the molecular mechanisms that link ligand binding to the channel gate in ATP-sensitive Kir6.2 channels. In these channels, the “slide helix” forms an interface between the cytoplasmic (ligand-binding) domain and the transmembrane pore, and many slide helix mutations cause loss of function. Using a novel approach to rescue electrically silent channels, we decomposed the contribution of each interface residue to ATP-dependent gating. We demonstrate that effective inhibition by ATP relies on an essential aspartate at residue 58. Characterization of the functional importance of this conserved aspartate, relative to other residues in the slide helix, has been impossible because of loss-of-function of Asp-58 mutant channels. The Asp-58 position exhibits an extremely stringent requirement for aspartate because even a highly conservative mutation to glutamate is insufficient to restore normal channel function. These findings reveal unrecognized slide helix elements that are required for functional channel expression and control of Kir6.2 gating by intracellular ATP.

Forced gating motions by a substituted titratable side chain at the bundle crossing of a potassium channel

Background: ATP-sensitive potassium (KATP) channels translate cellular metabolism (generation of ATP) in an electrical signal. Results: Mutual repulsion between specific substituted titratable residues in the bundle crossing forces KATP channels to open and changes their apparent ATP sensitivity. Conclusion: ATP-dependent gating involves conformational changes in the bundle crossing region of KATP channels. Significance: This reflects an engineered method for control of ion channel activity by a non-natural mechanism. Numerous inwardly rectifying potassium (Kir) channels possess an aromatic residue in the helix bundle crossing region, forming the narrowest pore constriction in crystal structures. However, the role of the Kir channel bundle crossing as a functional gate remains uncertain. We report a unique phenotype of Kir6.2 channels mutated to encode glutamate at this position (F168E). Despite a prediction of four glutamates in close proximity, Kir6.2(F168E) channels are predominantly closed at physiological pH, whereas alkalization causes rapid and reversible channel activation. These findings suggest that F168E glutamates are uncharged at physiological pH but become deprotonated at alkaline pH, forcing channel opening due to mutual repulsion of nearby negatively charged side chains. The potassium channel pore scaffold likely brings these glutamates close together, causing a significant pKa shift relative to the free side chain (as seen in the KcsA selectivity filter). Alkalization also shifts the apparent ATP sensitivity of the channel, indicating that forced motion of the bundle crossing is coupled to the ATP-binding site and may resemble conformational changes involved in wild-type Kir6.2 gating. The study demonstrates a novel mechanism for engineering extrinsic control of channel gating by pH and shows that conformational changes in the bundle crossing region are involved in ligand-dependent gating of Kir channels.

Use-Dependent Activation of Neuronal Kv1.2 Channel Complexes

In excitable cells, ion channels are frequently challenged by repetitive stimuli, and their responses shape cellular behavior by regulating the duration and termination of bursts of action potentials. We have investigated the behavior of Shaker family voltage-gated potassium (Kv) channels subjected to repetitive stimuli, with a particular focus on Kv1.2. Genetic deletion of this subunit results in complete mortality within 2 weeks of birth in mice, highlighting a critical physiological role for Kv1.2. Kv1.2 channels exhibit a unique property described previously as “prepulse potentiation,” in which activation by a depolarizing step facilitates activation in a subsequent pulse. In this study, we demonstrate that this property enables Kv1.2 channels to exhibit use-dependent activation during trains of very brief depolarizations. Also, Kv subunits usually assemble into heteromeric channels in the central nervous system, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that other Kv1 channel types do not exhibit use-dependent activation, but this property is conferred in heteromeric channel complexes containing even a single Kv1.2 subunit. This regulatory mechanism is observed in mammalian cell lines as well as primary cultures of hippocampal neurons. Our findings illustrate that use-dependent activation is a unique property of Kv1.2 that persists in heteromeric channel complexes and may influence function of hippocampal neurons.

PIP2 mediates functional coupling and pharmacology of neuronal KCNQ channels

Significance Despite the availability of many drugs to treat epilepsy, nearly one-third of patients are not responsive to pharmacotherapy. Retigabine (RTG) is the first approved antiepileptic drug that acts by promoting activation of potassium channels, specifically targeting neuronal KCNQ channels that are regulated by both voltage and the membrane phospholipid PIP2. A deeper understanding of the mechanism of action of RTG will enable future development of this unique drug class. In this study, we combine electrophysiology recordings with fluorometric measurements of KCNQ channel conformation to reveal channel features that contribute to the dramatic effects of RTG. Our findings demonstrate that a PIP2-dependent interaction between the pore-forming and voltage-sensing components of the channel is required for optimal RTG action. Retigabine (RTG) is a first-in-class antiepileptic drug that suppresses neuronal excitability through the activation of voltage-gated KCNQ2–5 potassium channels. Retigabine binds to the pore-forming domain, causing a hyperpolarizing shift in the voltage dependence of channel activation. To elucidate how the retigabine binding site is coupled to changes in voltage sensing, we used voltage-clamp fluorometry to track conformational changes of the KCNQ3 voltage-sensing domains (VSDs) in response to voltage, retigabine, and PIP2. Steady-state ionic conductance and voltage sensor fluorescence closely overlap under basal PIP2 conditions. Retigabine stabilizes the conducting conformation of the pore and the activated voltage sensor conformation, leading to dramatic deceleration of current and fluorescence deactivation, but these effects are attenuated upon disruption of channel:PIP2 interactions. These findings reveal an important role for PIP2 in coupling retigabine binding to altered VSD function. We identify a polybasic motif in the proximal C terminus of retigabine-sensitive KCNQ channels that contributes to VSD–pore coupling via PIP2, and thereby influences the unique gating effects of retigabine.

Atom‐by‐atom engineering of voltage‐gated ion channels: Magnified insights into function and pharmacology

Unnatural amino acid incorporation into ion channels has proven to be a valuable approach to interrogate detailed hypotheses arising from atomic resolution structures. In this short review, we provide a brief overview of some of the basic principles and methods for incorporation of unnatural amino acids into proteins. We also review insights into the function and pharmacology of voltage‐gated ion channels that have emerged from unnatural amino acid mutagenesis approaches.

A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels.

Background: Kir6.2 potassium channels are regulated by ATP. Results: We measured Kir6.2 gating kinetics in response to rapid ATP concentration jumps. Mutations to Trp-68 and Lys-170 dramatically decelerate gating. Conclusion: Trp-68 and Lys-170 interact to form a cluster that enables rapid gating transitions. Significance: The Trp-68/Lys-170 cluster is highly conserved and may play a similar role in other Kir channels. ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

One drug-sensitive subunit is sufficient for a near-maximal retigabine effect in KCNQ channels

Retigabine is an antiepileptic drug and the first voltage-gated potassium (Kv) channel opener to be approved for human therapeutic use. Retigabine is thought to interact with a conserved Trp side chain in the pore of KCNQ2–5 (Kv7.2–7.5) channels, causing a pronounced hyperpolarizing shift in the voltage dependence of activation. In this study, we investigate the functional stoichiometry of retigabine actions by manipulating the number of retigabine-sensitive subunits in concatenated KCNQ3 channel tetramers. We demonstrate that intermediate retigabine concentrations cause channels to exhibit biphasic conductance–voltage relationships rather than progressive concentration-dependent shifts. This suggests that retigabine can exert its effects in a nearly “all-or-none” manner, such that channels exhibit either fully shifted or unshifted behavior. Supporting this notion, concatenated channels containing only a single retigabine-sensitive subunit exhibit a nearly maximal retigabine effect. Also, rapid solution exchange experiments reveal delayed kinetics during channel closure, as retigabine dissociates from channels with multiple drug-sensitive subunits. Collectively, these data suggest that a single retigabine-sensitive subunit can generate a large shift of the KCNQ3 conductance–voltage relationship. In a companion study (Wang et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201812014), we contrast these findings with the stoichiometry of a voltage sensor-targeted KCNQ channel opener (ICA-069673), which requires four drug-sensitive subunits for maximal effect.

Site-Directed Unnatural Amino Acid Mutagenesis to Investigate Potassium Channel Pharmacology in Xenopus laevis Oocytes

Unnatural amino acid mutagenesis is a useful tool enabling detailed investigation of ion channel structure-function relationships and pharmacology. Methods have been developed to apply this technique to different heterologous systems for electrophysiological studies, with each system offering unique advantages and limitations. Synthesis of aminoacylated-tRNA followed by injection into Xenopus laevis oocytes is beneficial because it allows for the incorporation of a wide range of unnatural sidechains, including amino acids with subtle structural differences. Here, we describe a protocol for unnatural amino acid mutagenesis implemented in our lab to study the pharmacology of KCNQ voltage-gated potassium channel opener compounds. This protocol should be applicable to other ion channels and receptor types amenable for functional studies in Xenopus laevis oocytes.

Engineered pH-Dependence at the Kir6.2 Helix Bundle Crossing

The hallmark functional property of KATP (ATP-sensitive potassium) channels is inhibition by intracellular ATP, which binds to a well-defined binding site on Kir6.x subunits and stabilizes the closed conformation of a gate in the channel pore. Numerous inwardly-rectifying potassium (Kir) channels possess an aromatic residue in the ‘helix bundle crossing’ region, forming the narrowest pore constriction in crystal structures of Kir channels, indicating an important role in channel gating. We have identified a remarkable phenotype of mutant channels carrying a glutamate at this position (F168E). Despite the structural prediction of four glutamates in close proximity, F168E channels are predominantly closed at physiological pH. However, intracellular alkalinization causes rapid and reversible channel activation. These findings suggest that F168E glutamates are uncharged at physiological pH but become deprotonated with a pKa∼9, resulting in opening due to mutual repulsion of multiple nearby glutamate sidechains. The K-channel pore scaffold likely brings these glutamates into close proximity, stabilizing the protonated (uncharged) form of the glutamate sidechain, and resulting in a dramatic pKa shift relative to free glutamate. Only at more alkaline pH do the glutamates deprotonate, with their mutual repulsion driving channel opening. Consistent with a role in ATP-mediated channel closure, alkalinization also affects channel sensitivity to ATP. Taken together, these findings demonstrate an engineered (not intrinsic) mechanism of channel gating by pH, and suggest that ATP-mediated gating of Kir6.2 involves conformational rearrangement of the bundle crossing region.