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.     

Carbamazepine as a Novel Small Molecule Corrector of Trafficking-impaired ATP-sensitive Potassium Channels Identified in Congenital Hyperinsulinism

ATP-sensitive potassium (K_ATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant K_ATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct K_ATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited K_ATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the K_ATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant K_ATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore K_ATP channel expression and function in a subset of congenital hyperinsulinism patients.     

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.     

Functional Complementation and Genetic Deletion Studies of KirBac Channels: ACTIVATORY MUTATIONS HIGHLIGHT GATING-SENSITIVE DOMAINS*

The superfamily of prokaryotic inwardly rectifying (KirBac) potassium channels is homologous to mammalian Kir channels. However, relatively little is known about their regulation or about their physiological role in vivo. In this study, we have used random mutagenesis and genetic complementation in K⁺-auxotrophic Escherichia coli and Saccharomyces cerevisiae to identify activatory mutations in a range of different KirBac channels. We also show that the KirBac6.1 gene (slr5078) is necessary for normal growth of the cyanobacterium Synechocystis PCC6803. Functional analysis and molecular dynamics simulations of selected activatory mutations identified regions within the slide helix, transmembrane helices, and C terminus that function as important regulators of KirBac channel activity, as well as a region close to the selectivity filter of KirBac3.1 that may have an effect on gating. In particular, the mutations identified in TM2 favor a model of KirBac channel gating in which opening of the pore at the helix-bundle crossing plays a far more important role than has recently been proposed.     

Regulation of the ATP-sensitive potassium channel subunit, Kir6.2, by a Ca2+-dependent protein kinase C

The activity of ATP-sensitive potassium (K(ATP)) channels is governed by the concentration of intracellular ATP and ADP and is thus responsive to the metabolic status of the cell. Phosphorylation of K(ATP) channels by protein kinase A (PKA) or protein kinase C (PKC) results in the modulation of channel activity and is particularly important in regulating smooth muscle tone. At the molecular level the smooth muscle channel is composed of a sulfonylurea subunit (SUR2B) and a pore-forming subunit Kir6.1 and/or Kir6.2. Previously, Kir6.1/SUR2B channels have been shown to be inhibited by PKC, and Kir6.2/SUR2B channels have been shown to be activated or have no response to PKC. In this study we have examined the modulation of channel complexes formed of the inward rectifier subunit, Kir6.2, and the sulfonylurea subunit, SUR2B. Using a combination of biochemical and electrophysiological techniques we show that this complex can be inhibited by protein kinase C in a Ca(2+)-dependent manner and that this inhibition is likely to be as a result of internalization. We identify a residue in the distal C terminus of Kir6.2 (Ser-372) whose phosphorylation leads to down-regulation of the channel complex. This inhibitory effect is distinct from activation which is seen with low levels of channel activity.     

Distant Cytosolic Residues Mediate a Two-way Molecular Switch That Controls the Modulation of Inwardly Rectifying Potassium (Kir) Channels by Cholesterol and Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2)

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. An emerging feature of several Kir channels is that they are regulated by cholesterol. However, the mechanism by which cholesterol affects channel function is unclear. Here we show that mutations of two distant Kir2.1 cytosolic residues, Leu-222 and Asn-251, form a two-way molecular switch that controls channel modulation by cholesterol and affects critical hydrogen bonding. Notably, these two residues are linked by a residue chain that continues from Asn-251 to connect adjacent subunits. Furthermore, our data indicate that the same switch also regulates the sensitivity of the channels to phosphatidylinositol 4,5-bisphosphate, a phosphoinositide that is required for activation of Kir channels. Thus, although cholesterol and phosphatidylinositol 4,5-bisphosphate do not interact with the same region of Kir2.1, these different modulators induce a common gating pathway of the channel.     

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.     

Acid-sensitive TWIK and TASK Two-pore Domain Potassium Channels Change Ion Selectivity and Become Permeable to Sodium in Extracellular Acidification

Two-pore domain K⁺ channels (K2P) mediate background K⁺ conductance and play a key role in a variety of cellular functions. Among the 15 mammalian K2P isoforms, TWIK-1, TASK-1, and TASK-3 K⁺ channels are sensitive to extracellular acidification. Lowered or acidic extracellular pH (pH_o) strongly inhibits outward currents through these K2P channels. However, the mechanism of how low pH_o affects these acid-sensitive K2P channels is not well understood. Here we show that in Na⁺-based bath solutions with physiological K⁺ gradients, lowered pH_o largely shifts the reversal potential of TWIK-1, TASK-1, and TASK-3 K⁺ channels, which are heterologously expressed in Chinese hamster ovary cells, into the depolarizing direction and significantly increases their Na⁺ to K⁺ relative permeability. Low pH_o-induced inhibitions in these acid-sensitive K2P channels are more profound in Na⁺-based bath solutions than in channel-impermeable N-methyl-d-glucamine-based bath solutions, consistent with increases in the Na⁺ to K⁺ relative permeability and decreases in electrochemical driving forces of outward K⁺ currents of the channels. These findings indicate that TWIK-1, TASK-1, and TASK-3 K⁺ channels change ion selectivity in response to lowered pH_o, provide insights on the understanding of how extracellular acidification modulates acid-sensitive K2P channels, and imply that these acid-sensitive K2P channels may regulate cellular function with dynamic changes in their ion selectivity.     

Changes in Accessibility of Cytoplasmic Substances to the Pore Associated with Activation of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

Opening of the cystic fibrosis transmembrane conductance regulator Cl⁻ channel is dependent both on phosphorylation and on ATP binding and hydrolysis. However, the mechanisms by which these cytoplasmic regulatory factors open the Cl⁻ channel pore are not known. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining sixth transmembrane region (TM6) of a cysteine-less variant of cystic fibrosis transmembrane conductance regulator. We find that methanethiosulfonate (MTS) reagents modify irreversibly cysteines substituted for TM6 residues Phe-337, Thr-338, Ser-341, Ile-344, Val-345, Met-348, Ala-349, Arg-352, and Gln-353 when applied to the cytoplasmic side of open channels. However, the apparent rate of modification by internal [2-sulfonatoethyl] methanethiosulfonate (MTSES), a negatively charged MTS reagent, is dependent on the activation state of the channels. In particular, cysteines introduced far along the axis of TM6 from the inside (T338C, S341C, I344C) showed no evidence of significant modification even after prolonged pretreatment of non-activated channels with internal MTSES. In contrast, cysteines introduced closer to the inside of TM6 (V345C, M348C) were readily modified in both activated and non-activated channels. Access of a permeant anion, Au(CN)₂⁻, to T338C was similarly dependent upon channel activation state. The pattern of MTS modification we observe allows us to designate different pore-lining amino acid side chains to distinct functional regions of the channel pore. One logical interpretation of these findings is that cytoplasmic access to residues at the narrowest region of the pore changes concomitant with activation.     

Stabilization of the Conductive Conformation of a Voltage-gated K+ (Kv) Channel: THE LID MECHANISM*

Voltage-gated K⁺ (Kv) channels are molecular switches that sense membrane potential and in response open to allow K⁺ ions to diffuse out of the cell. In these proteins, sensor and pore belong to two distinct structural modules. We previously showed that the pore module alone is a robust yet dynamic structural unit in lipid membranes and that it senses potential and gates open to conduct K⁺ with unchanged fidelity. The implication is that the voltage sensitivity of K⁺ channels is not solely encoded in the sensor. Given that the coupling between sensor and pore remains elusive, we asked whether it is then possible to convert a pore module characterized by brief openings into a conductor with a prolonged lifetime in the open state. The strategy involves selected probes targeted to the filter gate of the channel aiming to modulate the probability of the channel being open assayed by single channel recordings from the sensorless pore module reconstituted in lipid bilayers. Here we show that the premature closing of the pore is bypassed by association of the filter gate with two novel open conformation stabilizers: an antidepressant and a peptide toxin known to act selectively on Kv channels. Such stabilization of the conductive conformation of the channel is faithfully mimicked by the covalent attachment of fluorescein at a cysteine residue selectively introduced near the filter gate. This modulation prolongs the occupancy of permeant ions at the gate. It is this longer embrace between ion and gate that we conjecture underlies the observed stabilization of the conductive conformation. This study provides a new way of thinking about gating.     

Identification and functional characterization of Kir2.6 mutations associated with non-familial hypokalemic periodic paralysis

Hypokalemic periodic paralysis (hypoKPP) is characterized by episodic flaccid paralysis of muscle and acute hypokalemia during attacks. Familial forms of hypoKPP are predominantly caused by mutations of either voltage-gated Ca(2+) or Na(+) channels. The pathogenic gene mutation in non-familial hypoKPP, consisting mainly of thyrotoxic periodic paralysis (TPP) and sporadic periodic paralysis (SPP), is largely unknown. Recently, mutations in KCNJ18, which encodes a skeletal muscle-specific inwardly rectifying K(+) channel Kir2.6, were reported in some TPP patients. Whether mutations of Kir2.6 occur in other patients with non-familial hypoKPP and how mutations of the channel predispose patients to paralysis are unknown. Here, we report one conserved heterozygous mutation in KCNJ18 in two TPP patients and two separate heterozygous mutations in two SPP patients. These mutations result in V168M, R43C, and A200P amino acid substitution of Kir2.6, respectively. Compared with the wild type channel, whole-cell currents of R43C and V168M mutants were reduced by ～78 and 43%, respectively. No current was detected for the A200P mutant. Single channel conductance and open probability were reduced for R43C and V168M, respectively. Biotinylation assays showed reduced cell surface abundance for R43C and A200P. All three mutants exerted dominant negative inhibition on wild type Kir2.6 as well as wild type Kir2.1, another Kir channel expressed in the skeletal muscle. Thus, mutations of Kir2.6 are associated with SPP as well as TPP. We suggest that decreased outward K(+) current from hypofunction of Kir2.6 predisposes the sarcolemma to hypokalemia-induced paradoxical depolarization during attacks, which in turn leads to Na(+) channel inactivation and inexcitability of muscles.     

Crystal Structure of a Voltage-gated K+ Channel Pore Module in a Closed State in Lipid Membranes

Voltage-gated K⁺ channels underlie the electrical excitability of cells. Each subunit of the functional tetramer consists of the tandem fusion of two modules, an N-terminal voltage-sensor and a C-terminal pore. To investigate how sensor coupling to the pore generates voltage-dependent channel opening, we solved the crystal structure and characterized the function of a voltage-gated K⁺ channel pore in a lipid membrane. The structure of a functional channel in a membrane environment at 3.1 Å resolution establishes an unprecedented connection between channel structure and function. The structure is unique in delineating an ion-occupied ready to conduct selectivity filter, a confined aqueous cavity, and a closed activation gate, embodying a dynamic entity trapped in an unstable closed state.     

Probing Pore Constriction in a Ligand-gated Ion Channel by Trapping a Metal Ion in the Pore upon Agonist Dissociation

Eukaryotic pentameric ligand-gated ion channels (pLGICs) are receptors activated by neurotransmitters to rapidly transport ions across cell membranes, down their electrochemical gradients. Recent crystal structures of two prokaryotic pLGICs were interpreted to imply that the extracellular side of the transmembrane pore constricts to close the channel (Hilf, R. J., and Dutzler, R. (2009) Nature 457, 115–118; Bocquet, N., Nury, H., Baaden, M., Le Poupon, C., Changeux, J. P., Delarue, M., and Corringer, P. J. (2009) Nature 457, 111–114). Here, we utilized a eukaryotic acetylcholine (ACh)-serotonin chimeric pLGIC that was engineered with histidines to coordinate a metal ion within the channel pore, at its cytoplasmic side. In a previous study, the access of Zn²⁺ ions to the engineered histidines had been explored when the channel was either at rest (closed) or active (open) (Paas, Y., Gibor, G., Grailhe, R., Savatier-Duclert, N., Dufresne, V., Sunesen, M., de Carvalho, L. P., Changeux, J. P., and Attali, B. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 15877–15882). In this study, the interactions of Zn²⁺ with the pore were probed upon agonist (ACh) dissociation that triggers the transition of the receptor from the active conformation to the resting conformation (i.e. during deactivation). Application of Zn²⁺ onto ACh-bound open receptors obstructed their pore and prevented ionic flow. Removing ACh from its extracellular binding sites to trigger deactivation while Zn²⁺ is still bound led to tight trapping of Zn²⁺ within the pore. Together with single-channel recordings, made to explore single pore-blocking events, we show that dissociation of ACh causes the gate to shut on a Zn²⁺ ion that effectively acts as a “foot in the door.” We infer that, upon deactivation, the cytoplasmic side of the pore of the ACh-serotonin receptor chimera constricts to close the channel.     

Separate Gating Mechanisms Mediate the Regulation of K2P Potassium Channel TASK-2 by Intra- and Extracellular pH

TASK-2 (KCNK5 or K_2P5.1) is a background K⁺ channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pH_o) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pH_i) is independent from that under the command of pH_o. It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pH_o had normal pH_i-dependent gating. Increasing extracellular K⁺ concentration acid shifts pH_o activity curve of TASK-2 yet did not affect pH_i gating of TASK-2. pH_o modulation of TASK-2 is voltage-dependent, whereas pH_i gating was not altered by membrane potential. These results suggest that pH_o, which controls a selectivity filter external gate, and pH_i act at different gating processes to open and close TASK-2 channels. We speculate that pH_i regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys²⁴⁵) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pH_i. We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pH_i-dependence curve of TASK-2 as expected from its lower pK_a. We conclude that intracellular pH, together with pH_o, is a critical determinant of TASK-2 activity and therefore of its physiological function.     

An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH

Proton-gated TASK-3 K⁺ channel belongs to the K_2P family of proteins that underlie the K⁺ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H⁺]. Use of recently solved K_2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K⁺ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K⁺ ions modulate the pK_a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K⁺ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH_o sensors.     

Role of Derlin-1 protein in proteostasis regulation of ATP-sensitive potassium channels

ATP-sensitive potassium (K(ATP)) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2 regulate insulin secretion by linking glucose metabolism with membrane potential. The number of K(ATP) channels in the plasma membrane affects the sensitivity of β-cells to glucose. Aberrant surface channel expression leads to insulin secretion disease. Previously, we have shown that K(ATP) channel proteins undergo endoplasmic reticulum (ER)-associated degradation (ERAD) via the ubiquitin-proteasome pathway, and inhibition of proteasome function results in an increase in channel surface expression. Here, we investigated whether Derlin-1, a protein involved in retrotranslocation of misfolded or misassembled proteins across the ER membrane for degradation by cytosolic proteasomes, plays a role in ERAD and, in turn, biogenesis efficiency of K(ATP) channels. We show that both SUR1 and Kir6.2 form a complex with Derlin-1 and an associated AAA-ATPase, p97. Overexpression of Derlin-1 led to a decrease in the biogenesis efficiency and surface expression of K(ATP) channels. Conversely, knockdown of Derlin-1 by RNA interference resulted in increased processing of SUR1 and a corresponding increase in surface expression of K(ATP) channels. Importantly, knockdown of Derlin-1 increased the abundance of disease-causing misfolded SUR1 or Kir6.2 proteins and even partially rescued surface expression in a mutant channel. We conclude that Derlin-1, by being involved in ERAD of SUR1 and Kir6.2, has a role in modulating the biogenesis efficiency and surface expression of K(ATP) channels. The results suggest that physiological or pathological changes in Derlin-1 expression levels may affect glucose-stimulated insulin secretion by altering surface expression of K(ATP) channels.     

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca²⁺ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca²⁺ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca²⁺ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca²⁺ that will enable it to act as a Ca²⁺ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca²⁺] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca²⁺ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca²⁺ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.     

RNA Editing in the Central Cavity as a Mechanism to Regulate Surface Expression of the Voltage-gated Potassium Channel Kv1.1

Voltage-gated potassium (Kv) 1.1 channels undergo a specific enzymatic RNA deamination, generating a channel with a single amino acid exchange located in the inner pore cavity (Kv1.1^I400V). We studied I400V-edited Kv1.1 channels in more detail and found that Kv1.1^I400V gave rise to much smaller whole-cell currents than Kv1.1. To elucidate the mechanism behind this current reduction, we conducted electrophysiological recordings on single-channel level and did not find any differences. Next we examined channel surface expression in Xenopus oocytes and HeLa cells using a chemiluminescence assay and found the edited channels to be less readily expressed at the surface membrane. This reduction in surface expression was verified by fluorescence imaging experiments. Western blot analysis for comparison of protein abundances and glycosylation patterns did not show any difference between Kv1.1 and Kv1.1^I400V, further indicating that changed trafficking of Kv1.1^I400V is causing the current reduction. Block of endocytosis by dynasore or AP180C did not abolish the differences in current amplitudes between Kv1.1 and Kv1.1^I400V, suggesting that backward trafficking is not affected. Therefore, our data suggest that I400V RNA editing of Kv1.1 leads to a reduced current size by a decreased forward trafficking of the channel to the surface membrane. This effect is specific for Kv1.1 because coexpression of Kv1.4 channel subunits with Kv1.1^I400V abolishes these trafficking effects. Taken together, we identified RNA editing as a novel mechanism to regulate homomeric Kv1.1 channel trafficking. Fine-tuning of Kv1.1 surface expression by RNA editing might contribute to the complexity of neuronal Kv channel regulation.     

State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3

The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 × 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.