Channel Activation Voltage Alone Is Directly Altered in an Isoform-specific Manner by Na<Subscript>v1.4</Subscript> and Na<Subscript>v1.5</Subscript> Cytoplasmic Linkers

The isoform-specific direct role of cytoplasmic loops in the gating of two voltage-gated sodium channel isoforms, the human cardiac channel (Na_v1.5; hH1) and the human adult skeletal muscle channel (Na_v1.4; hSkM1), was investigated. Comparison of biophysical characteristics was made among hSkM1, hH1, and several hSkM1/hH1 chimeras in which the putative cytoplasmic loops that join domain I to II (loop A) and domain II to III (loop B) from one isoform replaced one or both of the analogous loops from the other isoform. For all parameters measured, hSkM1 and hH1 behavior were significantly different. Comparison of hSkM1 and hH1 biophysical characteristics with the function of their respective chimeras indicate that only the half-activation voltage (V_a) is directly and differently altered by the species of cytoplasmic loop such that a channel consisting of one or both hSkM1 loops activates at smaller depolarizations, while a larger depolarization is required for activation of a channel containing one or both of the analogous hH1 loops. When either cardiac channel loop A or B is attached to hSkM1, a 6–7 mV depolarizing shift in V_a is measured, increasing to a nearly 20 mV depolarization when both cardiac-channel loops are attached. The addition of either skeletal muscle-channel loop to hH1 causes a 7 mV hyperpolarization in V_a, which increases to about 10 mV for the double loop chimera. There is no significant difference in either steady-state inactivation or in the recovery from inactivation data between hSkM1 and its chimeras and between hH1 and its chimeras. Data indicate that the cytoplasmic loops contribute directly to the magnitude of the window current, suggesting that channels containing skeletal muscle loops have three times the peak persistent channel activity compared to channels containing the cardiac loops. An electrostatic mechanism, in which surface charge differences among these loops might alter differently the voltage sensed by the gating mechanism of the channel, can not account for the observed isoform-specific effects of these loops only on channel activation voltage. In summary, although the DI-DII and DII-DIII loop structures among isoforms are not well conserved, these data indicate that only one gating parameter, V_a is affected directly and in an isoform-specific manner by these divergent loop structures, creating loop-specific window currents and percentages of persistently active channels at physiological voltages that will likely impact the excitability of the cell.     

Monovalent Cations Contribute to T-type Calcium Channel (Ca<Subscript>V</Subscript>3.1 and Ca<Subscript>V</Subscript>3.2) Selectivity

Low voltage-activated (LVA) Ca²⁺ channels regulate chemical signaling by their ability to select for Ca²⁺. Whereas Ca²⁺ is the main permeating species through Ca²⁺ channels, Ca²⁺ permeation may be modified by abundant intra- and extracellular monovalent cations. Therefore, we explored monovalent cation regulation of LVA Ca²⁺ permeation in the cloned T-type Ca²⁺ channels 1G (Ca_V3.1) and 1H (Ca_V3.2). In physiological [Ca²⁺], the reversal potential in symmetrical Li⁺ was 19 mV in 1G and 18 mV in 1H, in symmetrical Cs⁺ the reversal potential was 36 mV in 1G and 37 mV in 1H, and in the bi-ionic condition with Li⁺ in the bath and Cs⁺ in the pipette, the reversal potential was 46 mV in both 1G and 1H. When Cs⁺ was used in the pipette, replacement of external Cs⁺ with Li⁺ (or Na⁺) shifted the reversal potential positive by 5–6 mV and increased the net inward current in 1G. Taken together the data indicate that in physiological [Ca²⁺], external Li⁺ (or Na⁺) permeates more readily than external Cs⁺, resulting in a positive shift of the reversal potential. We conclude that external monovalent cations dictate T-type Ca²⁺ channel selectivity by permeating through the channel. Similar to Li⁺, we previously reported that external [H⁺] can regulate T-type Ca²⁺ channel selectivity. 1Hs selectivity was more sensitive to external pH changes compared to 1G. When Cs⁺ was used in the pipette and Li⁺ was used in the bath external acidification from pH_o 7.4 to 6.0 caused a negative shift of the reversal by 8 mV in 1H. Replacement of internal Cs⁺ with Li⁺ reduced the pH-induced shift of the reversal potential to 2 mV. We conclude that, similar to other external monovalent cations, H⁺ can modify T-type Ca²⁺ channel selectivity. However, in contrast to external monovalent ions that readily permeate, H⁺ regulate T-type Ca²⁺ channel selectivity by increasing the relative permeability of the internal monovalent cation. Present address: B.P. Delisle, Department of Medicine, The University of Wisconsin, Madison, WI 53706, USA     

MitoK<Subscript>ATP</Subscript> activity in healthy and ischemic hearts

In addition to their role in energy transduction, mitochondria play important non-canonical roles in cell pathophysiology, several of which utilize the mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP). In the normal heart, mitoK_ATP regulates energy transfer through its regulation of intermembrane space volume and is accordingly essential for the inotropic response during periods of high workload. In the ischemic heart, mitoK_ATP is the point of convergence of protective signaling pathways and mediates inhibition of the mitochondrial permeability transition, and thus necrosis. In this review, we outline the experimental evidence that support these roles for mitoK_ATP in health and disease, as well as our hypothesis for the mechanism by which complex cardioprotective signals that originate at plasma membrane receptors traverse the cytosol to reach mitochondria and activate mitoK_ATP.     

Mitochondrial K<Subscript>ATP</Subscript> channels-derived reactive oxygen species activate pro-survival pathway in pravastatin-induced cardioprotection

Reactive oxygen species (ROS) are important intracellular signaling molecules and are implicated in cardioprotective pathways including ischemic preconditioning. Statins have been shown to have cardioprotective effects against ischemia/reperfusion injury, however, the precise mechanisms remain to be elucidated. We hypothesized that ROS-mediated signaling cascade may be involved in pravastatin-induced cardioprotection. Cultured rat cardiomyocytes were exposed to H₂O₂ for 30 min to induce cell injury. Pravastatin significantly suppressed H₂O₂-induced cell death evaluated by propidium iodide staining and the MTT assay. Incubation with pravastatin activated catalase, and prevented a ROS burst induced by H₂O₂, which preserved mitochondrial membrane potential. Protective effects were induced very rapidly within 10 min, which was concordant with the up-regulation of phosphorylated ERK1/2. L-NAME, 5HD, N-acetylcysteine (NAC) and staurosporine inhibited ERK1/2 phosphorylation and also reduced pravastatin-induced cardioprotection, suggesting NO, mitochondrial K_ATP (mitoK_ATP) channels, ROS and PKC should be involved in the cardioprotective signaling. We also demonstrated that pravastatin moderately up-regulated ROS generation in a 5HD-inhibitable manner. In isolated perfused rat heart experiments, pravastatin administered 10 min prior to no-flow global ischemia significantly improved left ventricular functional recovery, and also reduced infarct size, which were attenuated by the treatment with NAC, 5HD, L-NAME or staurosporine. Administration of pravastatin from the beginning of reperfusion also conferred cardioprotection. Pravastatin protected the cardiomyocytes against oxidative stress by preventing the ROS burst and preserving mitochondrial function. Moderately up-regulated ROS production by mitoK_ATP channels opening is involved in the pro-survival signaling cascade activated by pravastatin.     

Calmodulin Colocalizes with Connexins and Plays a Direct Role in Gap Junction Channel Gating

The direct calmodulin (CaM) role in chemical gating was tested with CaM mutants, expressed in oocytes, and CaM-connexin labeling methods. CaMCC, a CaM mutant with greater Ca-sensitivity obtained by replacing the N-terminal EF hand pair with a duplication of the C-terminal pair, drastically increased the chemical gating sensitivity of Cx32 channels and decreased their V_j sensitivity. This only occurred when CaMCC was expressed before Cx32, suggesting that CaMCC, and by extension CaM, interacts with Cx32 before junction formation. Direct CaM-Cx interaction at junctional and cytoplasmic spots was demonstrated by confocal immunofluorescence microscopy in HeLa cells transfected with Cx32 and in cryosectioned mouse liver. This was confirmed in HeLa cells coexpressing Cx32-GFP (green) and CaM-RFP (red) or Cx32-CFP (cyan) and CaM-YFP (yellow) fusion proteins. Significantly, these cells did not form gap junctions. In contrast, HeLa cells expressing only one of the two fusion proteins (Cx32-GFP, Cx32-CFP, CaM-RFP or CaM-YFP) revealed both junctional and non-junctional fluorescent spots. In these cells, CaM-Cx32 colocalization was demonstrated by secondary immunofluorescent labeling of Cx32 in cells expressing CaM-YFP or CaM in cells expressing Cx32-GFP. CaM-Cx colocalization was further demonstrated at rat liver gap junctions by Freeze-fracture Replica Immunogold Labeling (FRIL).     

The mechano-gated K<Subscript>2P</Subscript> channel TREK-1

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K⁺ channels. More than 80 genes encoding the main K⁺ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K⁺ channels. Mammalian two-pore domain K⁺ channels (K_2P) make up one class of K⁺ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K⁺ channels. Each K_2P channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including “background” or “leak” K⁺ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K_2P channel TREK-1 and will emphasize on the polymodal function of this “unconventional” K⁺ channel. EBSA Satellite meeting: Ion channels, Leeds, July 2007.     

Effects of Permeant Cations on K+ Channel Gating in Nerve Axons Revisited

An increase in extracellular potassium ion concentration, K _o , significantly slows the potassium channel deactivation rate in squid giant axons, as previously shown. Surprisingly, the effect does not occur in all preparations which, coupled with the voltage independence of this result in preparations in which it does occur, suggests that it is mediated at a site outside of the electric field of the channel, and that this site is accessible to potassium ions in some preparations, but not in others. In other words, the effect does not appear to be related to occupancy of the channel by potassium ions. This conclusion is supported by a four-barrier, three-binding site model of single file diffusion through the channel in which one site, at most, is unoccupied by a potassium ion (single-vacancy model). The model is consistent with current-voltage relations with various levels of K _o , and, by definition, with multiple occupancy by K⁺. The model predicts that occupancy of any given site is essentially independent of K _o (or K _i ). The effects of extracellular Rb⁺ and Cs⁺ on gating are strongly voltage dependent, and they were observed in all preparations investigated. Consequently, the mechanism underlying these results would appear to be different from that which underlies the effect of K⁺ on gating. In particular, the effect of Rb⁺ on gating is reduced by strong hyperpolarization, which in the context of the occupancy hypothesis, is consistent with the voltage dependence of the current-voltage relation in the presence of Rb⁺. The primary, novel, finding in this study is that the effects of Cs⁺ are counterintuitive in this regard. Specifically, the slowing of channel deactivation rate by Cs⁺ is also reduced by hyperpolarization, similar to the Rb⁺ results, whereas blockade is enhanced, which is seemingly inconsistent with the concept that occupancy of the channel by Cs⁺ underlies the effect of this ion on gating. This result is further elucidated by barrier modeling of the current-voltage relation in the presence of Cs⁺. Received: 19 December 1995/Revised: 10 June 1996     

Role of the Conserved Lysine Residue in the Middle of the Predicted Extracellular Loop Between M2 and M3 in the GABAA Receptor

Abstract : In α1, β2, and γ2 subunits of the γ-aminobutyric acid A (GABA_A) receptor, a conserved lysine residue occupies the position in the middle of the predicted extracellular loop between the transmembrane M2 and M3 regions. In all three subunits, this residue was mutated to alanine. Whereas the mutation in α1 and β2 subunits results each in about a sixfold shift of the concentration-response curve for GABA to higher concentrations, no significant effect by mutation in the γ subunit was detected. The affinity for the competitive inhibitor bicuculline methiodide was not affected by the mutations in either the α1 subunit or the β2 subunit. Concentration-response curves for channel activation by pentobarbital were also shifted to higher concentrations by the mutation in the α and β subunits. Binding of [³H]Ro 15-1788 was unaffected by the mutation in the α subunit, whereas the binding of [³H]muscimol was shifted to lower affinity. Mutation of the residue in the α1 subunit to E, Q, or R resulted in an about eight-, 10-, or fivefold shift, respectively, to higher concentrations of the concentration-response curve for GABA. From these observations, it is concluded that the corresponding residues on the α1 and β2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.     

Modulation of the reaction cycle of the Na<Superscript>+</Superscript>:Ca<Superscript>2+</Superscript>, K<Superscript>+</Superscript> exchanger

Ca²⁺ concentration in retinal photoreceptor rod outer segment (OS) strongly affects the generator potential kinetics and the receptor light adaptation. The response to intense light stimuli delivered in the dark produce potential changes exceeding 40 mV: since the Ca²⁺ extrusion in the OS is entirely controlled by the Na⁺:Ca²⁺, K⁺ exchanger, it is important to assess how the exchanger ion transport rate is affected by the voltage and, in general, by intracellular factors. It is indeed known that the cardiac Na⁺:Ca²⁺ exchanger is regulated by Mg-ATP via a still unknown metabolic pathway. In the present work, the Na⁺:Ca²⁺, K⁺ exchanger regulation was investigated in isolated OS, recorded in whole-cell configuration, using ionic conditions that activated maximally the exchanger in both forward and reverse mode. In all species examined (amphibia: Rana esculenta and Ambystoma mexicanum; reptilia: Gecko gecko), the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. Since hyperpolarisation increases Ca²⁺ extrusion rate, the recovery of the dark level of Ca²⁺ (and, in turn, of the generator potential) after intense light stimuli results accelerated. Mg-ATP increased the size of forward and reverse exchange current by a factor of ∼2.3 and ∼2.6, respectively, without modifying their voltage dependence. This indicates that Mg-ATP regulates the number of active exchanger sites and/or the exchanger turnover number, although via an unknown mechanism. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

K<Subscript>ATP</Subscript> channel polymorphism is associated with left ventricular size in hypertensive individuals: a large-scale community-based study

ATP-sensitive K⁺ (K_ATP) channel mutations have been identified in individuals with dilated cardiomyopathy and overt heart failure. Here, a common E23K functional polymorphism in the Kir6.2 channel pore versus cardiac phenotype was studied in a cross-sectional community-based cohort (n = 2,031). The KK genotype was associated with greater left ventricular size among subjects with increased stress load due to hypertension. These findings implicate Kir6.2 K23 as a risk factor for adverse subclinical myocardial remodeling, and underscore the significance of cardiac K_ATP channels within the population.     

Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in the Potassium Channel from Streptomyces lividans (KcsA)

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp⁶⁷–Glu⁷¹–Asp⁸⁰ inactivation triad within the channel structure and its bearing on the selectivity filter.     

Gating of the expressed Cav3.1 calcium channel

Intramembrane charge movement originating from Cav3.1 (T-type) channel expressed in HEK 293 cells was investigated. Ion current was blocked by 1 mM La3+. Charge movement was detectable for depolarizations above approximately -70 mV and saturated above +60 mV. The voltage dependence of charge movement followed a single Boltzmann function with half-maximal activation voltage +12.9 mV and +12.3 mV and with slopes of 22.4 mV and 18.1 mV for the ON- and OFF-charge movement, respectively. Inactivation of I(Ca) by prolonged depolarization pulse did not immobilize intramembrane charge movement in the Cav3.1 channel.     

An Amino-Acid Substitution in the Influenza-B NB Protein Affects Ion-Channel Gating

The effects of site-directed mutations in NB, a protein encoded by the influenza B virus that has been shown to form cation-selective ion channels at pH 6.0, were studied on ion channel characteristics in artificial lipid bilayers. It was thought that the residues in the hydrophobic region of NB we selected for mutation might be involved in the transport of cations across the channel and that changes in these residues might affect channel properties such as gating and ion-selectivity. Serine residues at positions 20 and 28, threonine at position 24 and cysteine at position 26 were replaced by alanine. We found that the mutation S20A gave channels that did not gate and that remained open most of the time. Proton permeability of NB channels, as detected by fluorescence quenching, was also altered by the mutation S20A: channels were no longer proton-permeable. The other mutations, S28A, T24A and C26A, did not have any detectable effect on the activity or proton permeability of channels formed by NB. The results indicate that serine 20 may have an important role in normal function of NB channels. Present address for G.D. Ewart: Biotron, LPO Box A315, Australian National University, ACT 2601, Australia     

Differential gene expression during apoptosis induced by a serum factor: Role of mitochondrial F<Subscript>0</Subscript>-F<Subscript>1</Subscript> ATP synthase complex

The number of genes that are up regulated or down regulated during apoptosis is large and still increasing. In an attempt to characterize differential gene expression during serum factor induced apoptosis in AK-5 cells (a rat histiocytoma), we found subunit 6 and subunit 8 of the transmembrane proton channel and subunit alpha of the catalytic core of the mitochondrial F₀-F₁ ATP synthase complex to be up regulated during apoptosis. The increase in the expression levels of these subunits was concomitant with a transient increase in the intracellular ATP levels, suggesting that the increase in cellular ATP content is a result of the increase in the expression of ATP synthase subunits' gene and de novo protein synthesis. Depleting the cellular ATP levels with oligomycin inhibited apoptosis significantly, pointing to the requirement of ATP during apoptosis. Caspase 1 and caspase 3 activity and the loss of mitochondrial membrane potential were also inhibited by oligomycin during apoptosis in these cells, suggesting that the oligomycin induced inhibition of apoptosis could be due to inhibition of caspase activity and inhibition of mitochondrial depolarization. However, cytochrome C release during apoptosis was found to be completely independent of intracellular ATP content. Besides the ATP synthase complex genes, other mitochondrial genes like cytochrome C oxidase subunit II and III also showed elevated levels of expression during apoptosis. This kind of a mitochondrial gene expression profile suggests that in AK-5 cells, these genes are upregulated in a time-linked manner to ensure sufficient intracellular ATP levels and an efficient functioning of the mitochondrial respiratory chain for successful completion of the apoptotic pathway.     

pH-Sensitive Gating Kinetics of the Maxi-K Channel in the Tonoplast of Chara australis

The most frequently observed K⁺ channel in the tonoplast of Characean giant internodal cells with a large conductance (ca. 170 pS; Lühring, 1986; Laver & Walker, 1987) behaves, although inwardly rectifying, like animal maxi-K channels. This channel is accessible for patch–clamp techniques by preparation of cytoplasmic droplets, where the tonoplast forms the membrane delineating the droplet. Lowering the pH of the bathing solution, that virtually mimicks the vacuolar environment, from an almost neutral level to values below pH 7, induced a significant but reversible decrease in channel activity, whereas channel conductance remained largely unaffected. Acidification (pH 5) on both sides of the membrane decreased open probability from a maximum of 80% to less than 20%. Decreasing pH at the cytosolic side inhibited channel activity cooperatively with a slope of 2.05 and a pK _a 6.56. In addition, low pH at the vacuolar face shifted the activating voltage into a positive direction by almost 100 mV. This is the first report about an effect of extraplasmatic pH on gating of a maxi-K channel. It is suggested that the Chara maxi-K channel possesses an S4-like voltage sensor and negatively charged residues in neighboring transmembrane domains whose S4-stabilizing function may be altered by protonation. It was previously shown that gating kinetics of this channel respond to cytosolic Ca²⁺ (Laver & Walker, 1991). With regard to natural conditions, pH effects are discussed as contributing mainly to channel regulation at the vacuolar membrane face, whereas at the cytosolic side Ca²⁺ affects the channel. An attempt was made to ascribe structural mechanisms to different states of a presumptive gating reaction scheme. Received: 8 May 1998/Revised: 18 September 1998     

Relative Movements of Transmembrane Regions at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Pore during Channel Gating

Multiple transmembrane (TM) segments line the pore of the cystic fibrosis transmembrane conductance regulator Cl(-) channel; however, the relative alignment of these TMs and their relative movements during channel gating are unknown. To gain three-dimensional structural information on the outer pore, we have used patch clamp recording to study the proximity of pairs of cysteine side chains introduced into TMs 6 and 11, using both disulfide cross-linking and Cd(2+) coordination. Following channel activation, disulfide bonds could apparently be formed between three cysteine pairs (of 15 studied): R334C/T1122C, R334C/G1127C, and T338C/S1118C. To examine the state dependence of cross-linking, we combined these cysteine mutations with a nucleotide-binding domain mutation (E1371Q) that stabilizes the channel open state. Investigation of the effects of the E1371Q mutation on disulfide bond formation and Cd(2+) coordination suggests that although R334C/T1122C and T338C/S1118C are closer together in the channel open state, R334C/G1127C are close together and can form disulfide bonds only when the channel is closed. These results provide important new information on the three-dimensional structure of the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore: TMs 6 and 11 are close enough together to form disulfide bonds in both open and closed channels. Moreover, the altered relative locations of residues in open and in closed channels that we infer allow us to propose that channel opening and closing may be associated with a relative translational movement of TMs 6 and 11, with TM6 moving "down" (toward the cytoplasm) during channel opening.     

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.     

K<Subscript>ATP</Subscript> channel Kir6.2 E23K variant overrepresented in human heart failure is associated with impaired exercise stress response

ATP-sensitive K⁺ (K_ATP) channels maintain cardiac homeostasis under stress, as revealed by murine gene knockout models of the KCNJ11-encoded Kir6.2 pore. However, the translational significance of K_ATP channels in human cardiac physiology remains largely unknown. Here, the frequency of the minor K23 allele of the common functional Kir6.2 E23K polymorphism was found overrepresented in 115 subjects with congestive heart failure compared to 2,031 community-based controls (69 vs. 56%, P < 0.001). Moreover, the KK genotype, present in 18% of heart failure patients, was associated with abnormal cardiopulmonary exercise stress testing. In spite of similar baseline heart rates at rest among genotypic subgroups (EE: 72.2 ± 2.3, EK: 75.0 ± 1.8 and KK: 77.1 ± 3.0 bpm), subjects with the KK genotype had a significantly reduced heart rate increase at matched workload (EE: 32.8 ± 2.7%, EK: 28.8 ± 2.1%, KK: 21.7 ± 2.6%, P < 0.05), at 75% of maximum oxygen consumption (EE: 53.9 ± 3.9%, EK: 49.9 ± 3.1%, KK: 36.8 ± 5.3%, P < 0.05), and at peak VO₂ (EE: 82.8 ± 6.0%, EK: 80.5 ± 4.7%, KK: 59.7 ± 8.1%, P < 0.05). Molecular modeling of the tetrameric Kir6.2 pore structure revealed the E23 residue within the functionally relevant intracellular slide helix region. Substitution of the wild-type E residue with an oppositely charged, bulkier K residue would potentially result in a significant structural rearrangement and disrupted interactions with neighboring Kir6.2 subunits, providing a basis for altered high-fidelity K_ATP channel gating, particularly in the homozygous state. Blunted heart rate response during exercise is a risk factor for mortality in patients with heart failure, establishing the clinical relevance of Kir6.2 E23K as a biomarker for impaired stress performance and underscoring the essential role of K_ATP channels in human cardiac physiology.     

Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel

Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 Å. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a ∼3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between β4 and β5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions.