Runying Yang

Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains

Conserved acidic and aromatic residues in the four sodium channel voltage-sensor domains make domain-specific functional contributions.

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.

Sequence determinants of subtype‐specific actions of KCNQ channel openers

Retigabine is a KCNQ voltage‐gated potassium channel opener that was recently approved as an add‐on therapeutic for patients with drug‐resistant epilepsy. Retigabine exhibits very little specificity between most KCNQ channel subtypes, and there is interest in generating more potent and specific KCNQ channel openers. The present study describes the marked specificity of ICA069673 for KCNQ2 vs. KCNQ3, and exploits this property to investigate determinants of KCNQ subtype specificity. ICA069673 acts on a binding site in the voltage‐sensing domain that is distinct from the putative retigabine site in the channel pore. ICA069673 has two separable effects on KCNQ channel activity. We identify two channel residues required for subtype specificity of KCNQ channel openers and show that these are sufficient to generate ICA069673 sensitivity in KCNQ3.

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.

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.

Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels

ABSTRACT Voltage-gated potassium channels are important regulators of electrical excitation in many tissues, with Kv1.2 standing out as an essential contributor in the CNS. Genetic deletion of Kv1.2 invariably leads to early lethality in mice. In humans, mutations affecting Kv1.2 function are linked to epileptic encephalopathy and movement disorders. We have demonstrated that Kv1.2 is subject to a unique regulatory mechanism in which repetitive stimulation leads to dramatic potentiation of current. In this study, we explore the properties and molecular determinants of this use-dependent potentiation/activation. First, we examine how alterations in duty cycle (depolarization and repolarization/recovery times) affect the onset and extent of use-dependent activation. Also, we use trains of repetitive depolarizations to test the effects of a variety of Thr252 (S2-S3 linker) mutations on use-dependent activation. Substitutions of Thr with some sterically similar amino acids (Ser, Val, and Met, but not Cys) retain use-dependent activation, while bulky or charged amino acid substitutions eliminate use-dependence. Introduction of Thr at the equivalent position in other Kv1 channels (1.1, 1.3, 1.4), was not sufficient to transfer the phenotype. We hypothesize that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulator that binds preferentially to the channel closed state, with Thr252 being necessary but not sufficient for this interaction to alter channel function. These findings extend the conclusions of our recent demonstration of use-dependent activation of Kv1.2-containing channels in hippocampal neurons, by adding new details about the molecular mechanism underlying this effect.

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.