Slide Helix Research Articles

Structural Dynamics of the Slide Helix of Inactive/Closed Conformation of KirBac1.1 in Micelles and Membranes: A Fluorescence Approach.

Inward rectifying potassium (Kir) channels play a critical role in maintaining the resting membrane potential and cellular homeostasis. The high-resolution crystal structure of homotetrameric KirBac1.1 in detergent micelles provides a snapshot of the closed state. Similar to micelles, KirBac1.1 is reported to be in the inactive/closed conformation in POPC membranes. The slide helix of KirBac1.1 is an important structural motif that regulates channel gating. Despite the importance of slide helix in lipid-dependent gating, conflicting models have emerged for the location of slide helix and its structural dynamics in membrane mimetics is poorly understood. Here, we monitored the structural dynamics of the slide helix (residues 46-57) of KirBac1.1 in both DM micelles and POPC membranes utilizing various site-directed fluorescence approaches. We show, using ACMA-based liposome-flux assay, the cysteine mutants of the slide helix are not functional, ensuring the inactive/closed conformation in POPC membranes similar to wild-type channel. Time-resolved fluorescence and water accessibility measurements of NBD-labeled single-cysteine mutants of slide-helix residues suggest that the location of the slide helix at the interfacial region might be shallower in membranes compared to micelles. Interestingly, the slide helix of KirBac1.1 is more dynamic in the physiologically relevant membrane environment, which is accompanied by a differential hydration dynamics throughout the slide helix. Further, REES and lifetime distribution analyses suggest significant changes in conformational heterogeneity of the slide helix in membrane mimetics. Overall, our results give an insight into how membrane mimetics affect the organization and dynamics of slide helix of the closed state of KirBac1.1, and highlight the importance of lipid-protein interactions in membranes.

Use of a Molecular Switch Probe to Activate or Inhibit GIRK1 Heteromers In Silico Reveals a Novel Gating Mechanism.

G protein-gated inwardly rectifying K+ (GIRK) channels form highly active heterotetramers in the body, such as in neurons (GIRK1/GIRK2 or GIRK1/2) and heart (GIRK1/GIRK4 or GIRK1/4). Based on three-dimensional atomic resolution structures for GIRK2 homotetramers, we built heterotetrameric GIRK1/2 and GIRK1/4 models in a lipid bilayer environment. By employing a urea-based activator ML297 and its molecular switch, the inhibitor GAT1587, we captured channel gating transitions and K+ ion permeation in sub-microsecond molecular dynamics (MD) simulations. This allowed us to monitor the dynamics of the two channel gates (one transmembrane and one cytosolic) as well as their control by the required phosphatidylinositol bis 4-5-phosphate (PIP2). By comparing differences in the two trajectories, we identify three hydrophobic residues in the transmembrane domain 1 (TM1) of GIRK1, namely, F87, Y91, and W95, which form a hydrophobic wire induced by ML297 and de-induced by GAT1587 to orchestrate channel gating. This includes bending of the TM2 and alignment of a dipole of two acidic GIRK1 residues (E141 and D173) in the permeation pathway to facilitate K+ ion conduction. Moreover, the TM movements drive the movement of the Slide Helix relative to TM1 to adjust interactions of the CD-loop that controls the gating of the cytosolic gate. The simulations reveal that a key basic residue that coordinates PIP2 to stabilize the pre-open and open states of the transmembrane gate flips in the inhibited state to form a direct salt-bridge interaction with the cytosolic gate and destabilize its open state.

A novel small-molecule selective activator of homomeric GIRK4 channels

G protein–sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.e., GIRK2 and GIRK4) or heteromeric (e.g., GIRK1/2, GIRK1/4, and GIRK2/3) tetramers. Activators, such as ML297, ivermectin, and GAT1508, have been shown to activate heteromeric GIRK1/2 channels better than GIRK1/4 channels with varying degrees of selectivity but not homomeric GIRK2 and GIRK4 channels. In addition, VU0529331 was discovered as the first homomeric GIRK channel activator, but it shows weak selectivity for GIRK2 over GIRK4 (or G4) homomeric channels. Here, we report the first highly selective small-molecule activator targeting GIRK4 homomeric channels, 3hi2one-G4 (3-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]-3-hydroxy-1-(1-naphthylmethyl)-1,3-dihydro-2H-indol-2-one). We show that 3hi2one-G4 does not activate GIRK2, GIRK1/2, or GIRK1/4 channels. Using molecular modeling, mutagenesis, and electrophysiology, we analyzed the binding site of 3hi2one-G4 formed by the transmembrane 1, transmembrane 2, and slide helix regions of the GIRK4 channel, near the phosphatidylinositol-4,5-bisphosphate binding site, and show that it causes channel activation by strengthening channel–phosphatidylinositol-4,5-bisphosphate interactions. We also identify slide helix residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as a major determinant of isoform-specific selectivity. We propose that 3hi2one-G4 could serve as a useful pharmaceutical probe in studying GIRK4 channel function and may also be pursued in drug optimization studies to tackle GIRK4-related diseases such as primary aldosteronism and late-onset obesity.

The Novel Small Molecule 3hi2one-G4 Selectively Activates Homomeric GIRK4 Channels

G protein-sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical therapeutic targets related to neuronal, cardiac and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, highly selective modulators are still lacking. GIRK channels function as either homomeric (i.e. GIRK2 and GIRK4) or heteromeric (e.g. GIRK1/2 and GIRK1/4) tetramers. Activators such as, ML297, ivermectin, and GAT1508 selectively activate GIRK1/2 over GIRK1/4 but not homomeric GIRK2 and GIRK4 channels. VU0529331 was discovered as the first homomeric GIRK channel activator, which shows weak selectivity for GIRK2 over GIRK4 homomeric channels. Here we report the first selective small molecule activator targeting GIRK4 homomeric channels that we refer to as 3hi2one-G4. 3hi2one-G4 selectively activates GIRK4 channels, but does not activate GIRK2, GIRK1/2 or GIRK1/4 channels. By using molecular modeling, mutagenesis, and electrophysiology, we report that the compound utilizes a binding site formed by the TM1, TM2 and slide helix regions of the channel, near the PIP2 site. We identify residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as the selectivity determining residue. 3hi2one-G4 shares a similar binding site and activation mechanism to ivermectin, previously identified as a selective activator of GIRK1/2 heteromeric channels. 3hi2one-G4 could be a useful pharmaceutical probe molecule to study GIRK4 channel function. The newly identified binding site could be used in structure-based drug design studies to identify novel modulators of GIRK channels.

Andersen-Tawil Syndrome Is Associated With Impaired PIP2 Regulation of the Potassium Channel Kir2.1.

Andersen–Tawil syndrome (ATS) type-1 is associated with loss-of-function mutations in KCNJ2 gene. KCNJ2 encodes the tetrameric inward-rectifier potassium channel Kir2.1, important to the resting phase of the cardiac action potential. Kir-channels’ activity requires interaction with the agonist phosphatidylinositol-4,5-bisphosphate (PIP2). Two mutations were identified in ATS patients, V77E in the cytosolic N-terminal “slide helix” and M307V in the C-terminal cytoplasmic gate structure “G-loop.” Current recordings in Kir2.1-expressing HEK cells showed that each of the two mutations caused Kir2.1 loss-of-function. Biotinylation and immunostaining showed that protein expression and trafficking of Kir2.1 to the plasma membrane were not affected by the mutations. To test the functional effect of the mutants in a heterozygote set, Kir2.1 dimers were prepared. Each dimer was composed of two Kir2.1 subunits joined with a flexible linker (i.e. WT-WT, WT dimer; WT-V77E and WT-M307V, mutant dimer). A tetrameric assembly of Kir2.1 is expected to include two dimers. The protein expression and the current density of WT dimer were equally reduced to ~25% of the WT monomer. Measurements from HEK cells and Xenopus oocytes showed that the expression of either WT-V77E or WT-M307V yielded currents of only about 20% compared to the WT dimer, supporting a dominant-negative effect of the mutants. Kir2.1 sensitivity to PIP2 was examined by activating the PIP2 specific voltage-sensitive phosphatase (VSP) that induced PIP2 depletion during current recordings, in HEK cells and Xenopus oocytes. PIP2 depletion induced a stronger and faster decay in Kir2.1 mutant dimers current compared to the WT dimer. BGP-15, a drug that has been demonstrated to have an anti-arrhythmic effect in mice, stabilized the Kir2.1 current amplitude following VSP-induced PIP2 depletion in cells expressing WT or mutant dimers. This study underlines the implication of mutations in cytoplasmic regions of Kir2.1. A newly developed calibrated VSP activation protocol enabled a quantitative assessment of changes in PIP2 regulation caused by the mutations. The results suggest an impaired function and a dominant-negative effect of the Kir2.1 variants that involve an impaired regulation by PIP2. This study also demonstrates that BGP-15 may be beneficial in restoring impaired Kir2.1 function and possibly in treating ATS symptoms.

Life with Bacterial Mechanosensitive Channels, from Discovery to Physiology to Pharmacological Target.

General principles in biology have often been elucidated from the study of bacteria. This is true for the bacterial mechanosensitive channel of large conductance, MscL, the channel highlighted in this review. This channel functions as a last-ditch emergency release valve discharging cytoplasmic solutes upon decreases in osmotic environment. Opening the largest gated pore, MscL passes molecules up to 30 Å in diameter; exaggerated conformational changes yield advantages for study, including in vivo assays. MscL contains structural/functional themes that recur in higher organisms and help elucidate how other, structurally more complex, channels function. These features of MscL include (i) the ability to directly sense, and respond to, biophysical changes in the membrane, (ii) an α helix ("slide helix") or series of charges ("knot in a rope") at the cytoplasmic membrane boundary to guide transmembrane movements, and (iii) important subunit interfaces that, when disrupted, appear to cause the channel to gate inappropriately. MscL may also have medical applications: the modality of the MscL channel can be changed, suggesting its use as a triggered nanovalve in nanodevices, including those for drug targeting. In addition, recent studies have shown that the antibiotic streptomycin opens MscL and uses it as one of the primary paths to the cytoplasm. Moreover, the recent identification and study of novel specific agonist compounds demonstrate that the channel is a valid drug target. Such compounds may serve as novel-acting antibiotics and adjuvants, a way of permeabilizing the bacterial cell membrane and, thus, increasing the potency of commonly used antibiotics.

Single Molecule FRET Reveals Lipid Induced Conformational Changes in Cytoplasmic Domain of Kir2.1

Anionic phospholipids are common regulators of ion channel activity, including Kir2.1, an inward rectifying potassium channel crucial for setting and maintaining resting membrane potential in many tissues, which is activated specifically by PIP2 and phosphatidyl glycerol (PG). Crystal structures indicate that a simple upward translocation of the cytoplasmic Kir domain is induced by PIP2 and PG binding. However, recent computational and single-molecule FRET experiments show more complex intra-domain movements and rotation in the Kir domain of prokaryotic KirBac1.1 upon PIP2 binding. Single molecule FRET measurements on eukaryotic human Kir2.1 indicate similar complex motions that are not detected in static crystal structures. Our measurements indicate dynamic conformational ‘breathing’ throughout the Kir domain, suggesting greater flexibility in the intracellular domain of ion channels than may be apparent in structural studies. Specifically, our data indicate a PIP2 dependent shifts in average single molecule FRET (smFRET) distributions, with outward motions seen prior to the slide helix (residue 59) and an inward motion seen at the base (residues 286 and 288) consistent with a twisting motion of the Kir domain that was previously shown to be associated with opening in KirBac1.1. In the absence of PIP2, smFRET signals are very dynamic with relatively large (>20A) motions at specific residues. PIP2 stabilizes the conformational dynamics of the Kir domain, with less transitions between structural states, and reduction in the occurrence of large conformational transitions.

Conserved functional consequences of disease-associated mutations in the slide helix of Kir6.1 and Kir6.2 subunits of the ATP-sensitive potassium channel

Cantu syndrome (CS) is a condition characterized by a range of anatomical defects, including cardiomegaly, hyperflexibility of the joints, hypertrichosis, and craniofacial dysmorphology. CS is associated with multiple missense mutations in the genes encoding the regulatory sulfonylurea receptor 2 (SUR2) subunits of the ATP-sensitive K+ (KATP) channel as well as two mutations (V65M and C176S) in the Kir6.1 (KCNJ8) subunit. Previous analysis of leucine and alanine substitutions at the Val-65-equivalent site (Val-64) in Kir6.2 indicated no major effects on channel function. In this study, we characterized the effects of both valine-to-methionine and valine-to-leucine substitutions at this position in both Kir6.1 and Kir6.2 using ion flux and patch clamp techniques. We report that methionine substitution, but not leucine substitution, results in increased open state stability and hence significantly reduced ATP sensitivity and a marked increase of channel activity in the intact cell irrespective of the identity of the coassembled SUR subunit. Sulfonylurea inhibitors, such as glibenclamide, are potential therapies for CS. However, as a consequence of the increased open state stability, both Kir6.1(V65M) and Kir6.2(V64M) mutations essentially abolish high-affinity sensitivity to the KATP blocker glibenclamide in both intact cells and excised patches. This raises the possibility that, at least for some CS mutations, sulfonylurea therapy may not prove to be successful and highlights the need for detailed pharmacogenomic analyses of CS mutations.

Ivermectin activates GIRK channels in a PIP2 -dependent, Gβγ -independent manner and an amino acid residue at the slide helix governs the activation.

Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channel in parasites. It is known that IVM binds to the transmembrane domains (TMs) of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. We found that the G-protein-gated inwardly rectifying K+ (GIRK) channel, especially GIRK2, is activated by IVM directly in a Gβγ -independent manner, but the activation is dependent on phosphatidylinositol-4,5-biphosphate (PIP2 ). We identified a critical amino acid residue of GIRK2 for activation by IVM, Ile82, located in the slide helix between the TM1 and the N-terminal cytoplasmic tail domain (CTD). The results demonstrate that the TM-CTD interface in GIRK channel, rather than the TMs, governs IVM-mediated activation and provide us with novel insights on the mode of action of IVM in ion channels. Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channel in parasites. It is also known that IVM binds to the transmembrane domains (TMs) of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. In this study, we found that the G-protein-gated inwardly rectifying K+ (GIRK) channel is activated by IVM directly. Electrophysiological recordings in Xenopus oocytes revealed that IVM activates GIRK channel in a phosphatidylinositol-4,5-biphosphate (PIP2 )-dependent manner, and that the IVM-mediated GIRK activation is independent of Gβγ subunits. We found that IVM activates GIRK2 more efficiently than GIRK4. In cultured hippocampal neurons, we also observed that IVM activates native GIRK current. Chimeric and mutagenesis analyses identified an amino acid residue unique to GIRK2 among the GIRK family, Ile82, located in the slide helix between the TM1 and the N-terminal cytoplasmic tail domain (CTD), which is critical for the activation. The results demonstrate that the TM-CTD interface in GIRK channels, rather than the TMs, governs IVM-mediated activation. These findings provide us with novel insights on the mode of action of IVM in ion channels that could lead to identification of new pharmacophores which activate the GIRK channel.

Characterization of Structural Changes via Lipid Regulation of KIR2.1 in a Lipid Environment

Regulation of ion channel activity occurs through various mechanisms, via small molecule regulators, membrane potential, and specific interaction with lipid molecules. Inward rectifying potassium (Kir) channels conduction is controlled both by small molecules, such as polyamines blocking ion conduction, and lipid molecules that stabilize a conductive state. Advancing molecular understanding of this lipid regulation has been difficult as many structural techniques rely on detergent solubilized channels, which removes specific channel:lipid interaction. FRET, both macroscopically and in single molecules, can help surmount this issue, providing measurements of structural and dynamic changes induced by lipid regulation in a membrane environment. We have used macroscopic FRET to measure lipid specific structural changes in Kir2.1 in liposomes of defined composition. We have optimized purification and fluorophore labeling of the human Kir2.1 channel, which are reconstituted into liposomes of various lipid composition. We have measured PIP2-dependent FRET changes at numerous sites, which indicate structural movements of the intracellular domain of Kir2.1 upon PIP2 binding. This work specifically investigates how movements of the slide helix contribute to activation of the channel. Further efforts will help to define the molecular details of bulk anionic lipid and PIP2-dependent structural dynamics of Kir2.1 and help to understand more generally how lipid-protein interactions regulate membrane protein function.

Structural dynamics of potassium-channel gating revealed by single-molecule FRET.

Crystallography has provided invaluable insights to ion channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites, and examined structural dynamics within lipid membranes by single molecule FRET. We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and for cation channels in general.

Identification of the Conformational transition pathway in PIP2 Opening Kir Channels.

The gating of Kir channels depends critically on phosphatidylinositol 4,5-bisphosphate (PIP2), but the detailed mechanism by which PIP2 regulates Kir channels remains obscure. Here, we performed a series of Targeted molecular dynamics simulations on the full-length Kir2.1 channel and, for the first time, were able to achieve the transition from the closed to the open state. Our data show that with the upward motion of the cytoplasmic domain (CTD) the structure of the C-Linker changes from a loop to a helix. The twisting of the C-linker triggers the rotation of the CTD, which induces a small downward movement of the CTD and an upward motion of the slide helix toward the membrane that pulls the inner helix gate open. At the same time, the rotation of the CTD breaks the interaction between the CD- and G-loops thus releasing the G-loop. The G-loop then bounces away from the CD-loop, which leads to the opening of the G-loop gate and the full opening of the pore. We identified a series of interaction networks, between the N-terminus, CD loop, C linker and G loop one by one, which exquisitely regulates the global conformational changes during the opening of Kir channels by PIP2.

Studying the Gating Mechanism of Mammalian Kir Channels using in Silico Mutations

Inward rectifier potassium (Kir) channels are essential membrane proteins, controlling the permeation of K+ ions across membranes. They are found in eukaryotes as well as bacteria. These channels are expressed in multiple tissues and responsible for a wide range of physiological processes i.e. shaping the action potential, insulin decrease and neurotransmitter response. Crystal structures have provided major insights into the architecture of these channels. So far, all mammalian structures have been captured in a closed state. Recently, the first putative open structure of a bacterial Kir channel was obtained using a gating mutant (S129R) close to the helix bundle crossing region (HBC) (Bavro et al., 2012). Thus, we reasoned that introducing such a mutant in a mammalian Kir channel might enable investigating the open state.Therefore, we introduced in silico mutations in all for subunits at gating sensitive regions and conducted up to 1μs molecular dynamics simulations.Intriguingly, during the mutant simulations major conformational changes were observed. In all simulations, widening at the HBC gate occurred. These changes enabled continuous water flux through this gate and access for hydrated potassium ions to the cavity. A major difference to native mammalian Kir channels is the PIP2 dependence. In contrast to the wild type channels, our charged mutant channels do not need PIP2 to induce opening. This is likely due to the strong repulsive effects of the negative charges. Importantly, changes in the lipid interactions of the slide helix reveal a movement of this helix towards the lipid head groups during opening, in agreement with experimentally observed lipid interaction changes (Lee et al., 2013).It cannot be excluded that mutant channels stabilize rarely populated open states, as previously reported for selected bacterial Kir channel mutants (Zubcevic et al. 2014).

Structural Dynamics Underlying Pip2 Modulation of Kir Channels Revealed by Single Molecule FRET

Ion channels are gated by a multitude of electrical, chemical and mechanical stimuli, and thereby play key roles in many physiological processes. Static crystal structures and dynamic electrophysiological studies have generated invaluable molecular insights into channel gating, but the time trajectory of conformational changes has been unattainable. KirBac1.1 is a model for one of the three main K channel families. In the present study, we have successfully (1) labeled purified tetrameric protein molecules with single FRET donor and acceptor fluorophore pairs at specific locations; (2) functionally reconstituted the proteins into liposomes; (3) examined the dynamics of channel structure in liposomes with single molecule FRET techniques. Our results indicate that the amphipathic 'slide helices' of KirBac1.1 that surround the channel gate immediately below the membrane, move toward each other to narrow the pore in the presence of PIP2, which closes the channel; the cytoplasmic domain resides at two major structural states, but demonstrates an overall shift away from the pore axis upon PIP2 inhibition; the extracellular loop of KirBac1.1, adjacent to the selectivity filter, is structurally rigid and does not move during PIP2 gating. Both slide helix and cytoplasmic domain structures become less dynamic when the channel is inhibited by PIP2, which implies that PIP2 acts as a 'lock' to confine the structural fluctuations of the channel protein. We further probed the relative motions of the KirBac1.1 transmembrane domain versus the cytoplasmic domain. The results indicate that the entire cytoplasmic domain moves away from the transmembrane domain during PIP2 inhibition, which may result from a rigid body motion coupled to TM2 bending. In summary, we provide direct observations of dynamic structures of an ion channel within a lipid membrane environment, revealing dynamic structural rearrangements KirBac1.1 upon PIP2 inhibition.

Control of KirBac3.1 Potassium Channel Gating at the Interface between Cytoplasmic Domains

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating.

Rescue Mechanisms for Loss of Function Mutations Highlight Essential Residues at the Kir6.2 Channel Domain Interface

Inspection of the architecture of Kir channels reveals a non-covalent interface between the 'ligand-sensing' C-terminal domain (CTD) and the canonical pore-forming 'gating domain' formed by the TM1 and TM2 transmembrane helices. This interface is involved in transduction of ligand binding, and intact coupling between the CTD and TMD of Kir6.2 channels has been proposed to be crucial for propagation of gating effects induced by intracellular ligands. However, many mutations in this interfacial region prevent channel function and thereby preclude functional studies to interrogate the domain interface. We have developed a novel rescue mechanism that circumvents this problem, and we have applied this method to scan the functional contributions of residues in the CTD-TMD interface of Kir6.2 channels. Overall, residues cluster into three distinct functional types, based on their impact on ATP sensitivity and gating kinetics of the F168E background mutation used for functional rescue. Most loss-of-function mutations are classified as 'efficiently coupled', and their ATP sensitivity is only modestly affected by introduction of mutations. A second group of mutations comprises residues in the ATP binding site, which are 'efficiently coupled' but are profoundly insensitive to ATP. Most interestingly, our studies reveal a subset of 'uncoupling' mutations that form a network between the C-linker, G-loop, C-D loop, and slide helix of the channel. These residues exhibit an unusual combination of low open probability (or complete loss of function) combined with profound ATP insensitivity. The identification of mutations that uncouple the TMD and CTD distinguishes unique gating roles for residues that are required for channel function, and highlights the importance of a rescue approach to understand the impact of loss of function mutations.

Decomposition of Slide Helix Contributions to ATP-dependent Inhibition of Kir6.2 Channels

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.

Non dominant-negative KCNJ2 gene mutations leading to Andersen-Tawil syndrome with an isolated cardiac phenotype

Andersen-Tawil syndrome (ATS) is characterized by dysmorphic features, periodic paralyses and abnormal ventricular repolarization. After genotyping a large set of patients with congenital long-QT syndrome, we identified two novel, heterozygous KCNJ2 mutations (p.N318S, p.W322C) located in the C-terminus of the Kir2.1 subunit. These mutations have a different localization than classical ATS mutations which are mostly located at a potential interaction face with the slide helix or at the interface between the C-termini. Mutation carriers were without the key features of ATS, causing an isolated cardiac phenotype. While the N318S mutants regularly reached the plasma membrane, W322C mutants primarily resided in late endosomes. Co-expression of N318S or W322C with wild-type Kir2.1 reduced current amplitudes only by 20-25%. This mild loss-of-function for the heteromeric channels resulted from defective channel trafficking (W322C) or gating (N318S). Strikingly, and in contrast to the majority of ATS mutations, neither mutant caused a dominant-negative suppression of wild-type Kir2.1, Kir2.2 and Kir2.3 currents. Thus, a mild reduction of native Kir2.x currents by non dominant-negative mutants may cause ATS with an isolated cardiac phenotype.

Awakening Loss-of-Function KATP Channel Mutants with an Engineered ‘Forced Gating’ Approach

Regulation of inwardly-rectifying potassium channels by intracellular ligands couples membrane excitability to important signaling cascades and metabolic pathways. A significant barrier to understand coupling mechanisms between ligand binding and gating is that many functionally important channel motifs are highly sensitive to mutagenesis, resulting in a loss of function phenotype. We have developed a ‘forced gating’ approach that rescues function in electrically silent channel mutants, enabling characterization of channel motifs that are otherwise intractable to electrophysiological recording. This approach involves substitution of a glutamate in the hydrophobic Kir channel bundle crossing (F168E mutation in Kir6.2), generating channels that are pH sensitive and open upon alkalization, due to mutual repulsion of introduced negatively charged side chains in the channel gate. We have implemented this ‘forced gating’ approach in mutagenic scans of the Kir channel slide helix and G-loop, two motifs proposed to play a role in ligand dependent gating of Kir channels. Both motifs are also highly sensitive to mutagenesis, with alanine mutations causing nearly complete loss-of-function at 7/20 slide helix positions, and 8/13 G-loop positions. Without exception, expression of silent mutants on the Kir6.2[F168E] background permitted activation of functional channels in alkaline pH, and measurement of kinetics and potency of ATP inhibition. Our results highlight an essential ‘aspartate anchor’ (Kir6.2 residue D58) that bridges the slide helix and multiple interacting residues in the cytoplasmic domain. Disruption of the highly conserved ‘aspartate anchor’ uncouples the transmembrane and cytoplasmic domains, reducing ATP sensitivity of Kir6.2 far more than any other G-loop or slide helix mutants. These findings indicate a central role for the ‘aspartate anchor’ in coupling ligand binding to Kir gating, and also emphasize the potential general utility of this ‘forced gating’ method to study loss-of-function channel mutants.

Simulations of the Helix Bundle Crossing Gate Opening in Kir Channels

Inwardly rectifying K+ (Kir) channels are gated by the phospholipid PIP2. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter -SF, the inner helix bundle crossing (HBC), and the intracellular G-loop) may serve as gates to control ion permeation. A crystal structure of a Kir3.1 chimera [Nishida et al., 2007] captured the cytosolic G-loop gate in “closed/constricted” or “open/dilated” conformations. 100 ns Molecular Dynamics (MD) simulations studying the PIP2-driven Kir channel activation of the Kir3.1 chimera led us to propose a molecular mechanism of the G-loop gate opening [Meng et al., 2012]. However, opening of the HBC gate was not observed throughout this simulation. Mutagenesis and single-channel recording studies in our lab showed that a proline mutation on the inner helix of the Kir3.4 channel dramatically increased the open probability of the channel [Jin et al., 2002]. We introduced the corresponding M170P mutation on the Kir3.1 chimera structure and ran 100 ns long simulations of four mutant channel systems: dilated and constricted M170P Kir3.1 chimera in the presence (holo) and absence (apo) of PIP2, using the GROMACS program [Hess et al., 2008]. Three potassium ions present in the SF passed through the HBC gate in the system of the holo dilated M170P Kir3.1 chimera within the 100 ns simulation time. Minimal distance measurements indicated that the HBC gate was able to open only when PIP2 was present and the G-loop gate was stabilized in the open state. Principal component analysis revealed coupled conformational changes in the Slide helix, DE- and LM-loops, possibly related to the opening of the HBC gate. Moreover, unique residue interactions within the transmembrane domains were observed in the dilated holo system. Predictions of these models are being tested experimentally.