Determinant Role of Membrane Helices in K<Subscript>ATP</Subscript> Channel Gating

The ATP-sensitive K⁺ (K_ATP) channels couple chemical signals to cellular activity, in which the control of channel opening and closure (i.e., channel gating) is crucial. Transmembrane helices play an important role in channel gating. Here we report that the gating of Kir6.2, the core subunit of pancreatic and cardiac K_ATP channels, can be switched by manipulating the interaction between two residues located in transmembrane domains (TM) 1 and 2 of the channel protein. The Kir6.2 channel is gated by ATP and proton, which inhibit and activate the channel, respectively. The channel gating involves two residues, namely, Thr71 and Cys166, located at the interface of the TM1 and TM2. Creation of electrostatic attraction between these sites reverses the channel gating, which makes the ATP an activator and proton an inhibitor of the channel. Electrostatic repulsion with two acidic residues retains or even enhances the wild-type channel gating. A similar switch of the pH-dependent channel gating was observed in the Kir2.1 channel, which is normally pH- insensitive. Thus, the manner in which the TM1 and TM2 helices interact appears to determine whether the channels are open or closed following ligand binding.^*These authors contributed equally to this work.     

Characteristics of Omega-conotoxin GVI A and MVIIC Binding to Ca<Subscript>v</Subscript> 2.1 and Ca<Subscript>v</Subscript> 2.2 Channels Captured by Anti-Ca<Superscript>2+</Superscript> Channel Peptide Antibodies

A New Binding Method (NBM) was used to investigate the characteristics of the specific binding of ¹²⁵I-omega-conotoxin (ω-CTX) GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels captured from chick brain membranes by antibodies against B1Nt (a peptide sequence in Ca_v2.1 and Ca_v2.2 channels). The results for the NBM were as follows. (1) The ED₅₀ values for specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels were about 68 and 60 pM, respectively, and very similar to those (87 and 35 pM, respectively) to crude membranes from chick brain. (2) The specific ¹²⁵I-ω-CTX GVIA (100 pM) binding was inhibited by ω-CTX GVIA (0.5 nM), dynorphine A (Dyn), gentamicin (Gen), neomycin (Neo) and tobramicin (Tob) (100 μM each), but not by ω-agaconotoxin (Aga) IVA, calciseptine, ω-CTX SVIB, ω-CTX MVIIC (0.5 nM each), PN200-110 (PN), diltiazem (Dil) or verapamil (Ver) (100 μM each). Calmodulin (CaM) inhibited the specific binding in a dose-dependent manner (IC₅₀ value of about 100 μg protein/ml). (3) The specific ¹²⁵I-ω-CTX MVIIC (60 pM) binding was inhibited by ω-CTX MVIIC, ω-CTX GVIA, ω-CTX SVIB (0.5 nM each), Dyn, Neo and Tob (100 μM, each), but not by ω-Aga IVA, calciseptine (0.5 nM each), PN, Dil, Ver (100 μM each) or 100 μg protein/ml CaM. These results suggested that the characteristics of the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels in the NBM were very similar to those to crude membranes from chick brain, although the IC₅₀ values for CaM and free Ca²⁺ of CaM were about 33- and 5000-fold higher, respectively, than those for the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to crude membranes.     

Immunocytochemical Localization of TASK-3 (K<Subscript>2P</Subscript>9.1) Channels in Monoaminergic and Cholinergic Neurons

Monoaminergic and cholinergic systems are important regulators of cortical and subcortical systems, and a variety of vegetative functions are controlled by the respective neurotransmitters. Neuronal excitability and transmitter release of these neurons are strongly regulated by their potassium conductances carried by Kir and K_2P channels. Here we describe the generation and characterization of a polyclonal monospecific antibody against rat TASK-3, a major brain K_2P channel. After removal of cross-reactivities and affinity purification the antibody was characterized by ELISA, immunocytochemistry of TASK-3 transfected cells, and Western blots indicating that the antibody only detects TASK-3 protein, but not its paralogs TASK-1 and TASK-5. Western blot analysis of brain membrane fractions showed a single band around 45 kD, close to the predicted molecular weight of the TASK-3 protein. In addition, specific immunolabeling using the anti-TASK-3 antibody in Western blot analysis and immunocytochemistry was blocked in a concentration dependent manner by its cognate antigen only. Immunocytochemical analysis of rat brain revealed strong expression of TASK-3 channels in serotoninergic neurons of the dorsal and median raphe, noradrenergic neurons of the locus coeruleus, histaminergic neurons of the tuberomammillary nucleus and in the cholinergic neurons of the basal nucleus of Meynert. Immunofluorescence double-labeling experiments with appropriate marker enzymes confirmed the expression of TASK-3 in cholinergic, serotoninergic, and noradrenergic neurons. In the dopaminergic system strong TASK-3 expression was found in the ventral tegmental area, whereas TASK-3 immunoreactivity in the substantia nigra compacta was only weak. All immunocytochemical results were supported by in situ hybridization using TASK-3 specific riboprobes.     

Modulation of synaptic potentials and cell excitability by dendritic K<Subscript>IR</Subscript> and K<Subscript>As</Subscript> channels in nucleus accumbens medium spiny neurons: A computational study

The nucleus accumbens (NAc), a critical structure of the brain reward circuit, is implicated in normal goal-directed behaviour and learning as well as pathological conditions like schizophrenia and addiction. Its major cellular substrates, the medium spiny (MS) neurons, possess a wide variety of dendritic active conductances that may modulate the excitatory post synaptic potentials (EPSPs) and cell excitability. We examine this issue using a biophysically detailed 189-compartment stylized model of the NAc MS neuron, incorporating all the known active conductances. We find that, of all the active channels, inward rectifying K⁺ (K_IR) channels play the primary role in modulating the resting membrane potential (RMP) and EPSPs in the down-state of the neuron. Reduction in the conductance of K_IR channels evokes facilitatory effects on EPSPs accompanied by rises in local input resistance and membrane time constant. At depolarized membrane potentials closer to up-state levels, the slowly inactivating A-type potassium channel (K_As) conductance also plays a strong role in determining synaptic potential parameters and cell excitability. We discuss the implications of our results for the regulation of accumbal MS neuron biophysics and synaptic integration by intrinsic factors and extrinsic agents such as dopamine.     

Senicapoc: Repurposing a Drug to Target Microglia K<Subscript>Ca</Subscript>3.1 in Stroke

Stroke is the leading cause of serious long-term disability and the fifth leading cause of death in the United States. Treatment options for stroke are few in number and limited in efficacy. Neuroinflammation mediated by microglia and infiltrating peripheral immune cells is a major component of stroke pathophysiology. Interfering with the inflammation cascade after stroke holds the promise to modulate stroke outcome. The calcium activated potassium channel K_Ca3.1 is expressed selectively in the injured CNS by microglia. K_Ca3.1 function has been implicated in pro-inflammatory activation of microglia and there is recent literature suggesting that this channel is important in the pathophysiology of ischemia/reperfusion (stroke) related brain injury. Here we describe the potential of repurposing Senicapoc, a K_Ca3.1 inhibitor, to intervene in the inflammation cascade that follows ischemia/reperfusion.     

In vitro fluorescence assay to study the folding of K<Subscript>v</Subscript> ion channels

A fluorescence assay to check the folding of potassium K_v channels expressed in vitro has been developed. For this aim, the fluorescently labeled channel blocker, recombinant agitoxin of yellow scorpion was employed. The level of expression of various K_v channels in vitro has been tested. It has been demonstrated that K_v2 channels form clusters on the cell surface, which are not associated with actin filaments. On the other hand, K_v10 channels form larger clusters, which are associated with actin, indicating the principal differences in the organization of cytoplasmic domains of K_v2 and K_v10 channels.     

Expression and Coexpression of CO<Subscript>2</Subscript>-sensitive Kir Channels in Brainstem Neurons of Rats

Several inward rectifier K⁺ (Kir) channels are inhibited by hypercapnic acidosis and may be involved in CO₂ central chemoreception. Among them are Kir1.1, Kir2.3, and Kir4.1. The Kir4.1 is expressed predominantly in the brainstem. Although its CO₂ sensitivity is low, coexpression of Kir4.1 with Kir5.1 in Xenopus oocytes greatly enhances the CO₂/pH sensitivities of the heteromeric channels. If these Kir channels play a part in the central CO₂ chemosensitivity, they should be expressed in neurons of brainstem cardio-respiratory nuclei. To test this hypothesis, we performed in-situ hybridization experiments in which the expression of Kir1.1, Kir2.3, Kir4.1 and Kir5.1, and coexpression of Kir4.1 and Kir5.1 were studied in brainstem neurons using non-radioactive riboprobes. We found that mRNAs of these Kir channels were present in several brainstem nuclei, especially those involved in cardio-respiratory controls. Strong labeling was observed in the locus coeruleus, ventralateral medulla, parabrachial-Kölliker-Fuse nuclei, solitary tract nucleus, and area postrema. Strong expression was also seen in several cranial motor nuclei, including the nucleus of ambiguus, hypoglossal nucleus, facial nucleus and dorsal vagus motor nucleus. In general, the expression of Kir5.1 and Kir4.1 was much more prominent than that of Kir1.1 and Kir2.3 in all the nuclei. Evidence for the coexpression of Kir4.1 and Kir5.1 was found in a good number of neurons in these nuclei. The expression and coexpression of these CO₂/pH-sensitive Kir channels suggest that they are likely to contribute to CO₂ chemosensitivity of the brainstem neurons.     

Gating-Related Molecular Motions in the Extracellular Domain of the I<Subscript>Ks</Subscript> Channel: Implications for I<Subscript>Ks</Subscript> Channelopathy

Cardiac slow delayed rectifier (I_Ks) channel complex consists of KCNQ1 channel and KCNE1 auxiliary subunits. The extracellular juxtamembranous region of KCNE1 is an unstructured loop that contacts multiple KCNQ1 positions in a gating-state-dependent manner. Congenital arrhythmia-related mutations have been identified in the extracellular S1–S2 linker of KCNQ1. These mutations manifest abnormal phenotypes only when coexpressed with KCNE1, pointing to the importance of proper KCNQ1/KCNE1 interactions here in I_Ks channel function. We investigate the interactions between the KCNE1 loop (positions 36–47) and KCNQ1 S1–S2 linker (positions 140–148) by means of disulfide trapping and voltage clamp techniques. During transitions among the resting-state conformations, KCNE1 positions 36–43 make contacts with KCNQ1 positions 144, 145, and 147 in a parallel fashion. During conformational changes in the activated state, KCNE1 position 40 can make contacts with all three KCNQ1 positions, while the neighboring KCNE1 positions (36, 38, 39, and 41) can make contact with KCNQ1 position 147. Furthermore, KCNQ1 positions 143 and 146 are high-impact positions that cannot tolerate cysteine substitution. To maintain the proper I_Ks channel function, position 143 requires a small side chain with a hydroxyl group, and position 146 requires a negatively charged side chain. These data and the proposed molecular motions provide insights into the mechanisms by which mutations in the extracellular juxtamembranous region of the I_Ks channel impair its function.     

ATP-sensitive potassium channel (K<Subscript>ATP</Subscript>channel) expression in the normal canine pancreas and in canine insulinomas

Background

Pancreatic beta cells express ATP-sensitive potassium (K_ATP) channels that are needed for normal insulin secretion and are targets for drugs that modulate insulin secretion. The K_ATP channel is composed of two subunits: a sulfonylurea receptor (SUR 1) and an inward rectifying potassium channel (Kir_6.2). K_ATP channel activity is influenced by the metabolic state of the cell and initiates the ionic events that precede insulin exocytosis. Although drugs that target the K_ATP channel have the expected effects on insulin secretion in dogs, little is known about molecular aspects of this potassium channel. To learn more about canine beta cell K_ATP channels, we studied K_ATP channel expression by the normal canine pancreas and by insulin-secreting tumors of dogs.

Results

Pancreatic tissue from normal dogs and tumor tissue from three dogs with histologically-confirmed insulinomas was examined for expression of K_ATP channel subunits (SUR1 and Kir_6.2) using RT-PCR. Normal canine pancreas expressed SUR1 and Kir_6.2 subunits of the K_ATP channel. The partial nucleotide sequences for SUR1 and Kir_6.2 obtained from the normal pancreas showed a high degree of homology to published sequences for other mammalian species. SUR1 and Kir_6.2 expression was observed in each of the three canine insulinomas examined. Comparison of short sequences from insulinomas with those obtained from normal pancreas did not reveal any mutations in either SUR1 or Kir_6.2 in any of the insulinomas.

Conclusion

Canine pancreatic K_ATP channels have the same subunit composition as those found in the endocrine pancreases of humans, rats, and mice, suggesting that the canine channel is regulated in a similar fashion as in other species. SUR1 and Kir_6.2 expression was found in the three insulinomas examined indicating that unregulated insulin secretion by these tumors does not result from failure to express one or both K_ATP channel subunits.

     

Effects of the Oral Administration of K<Subscript>2</Subscript>Cr<Subscript>2</Subscript>O<Subscript>7</Subscript> and Na<Subscript>2</Subscript>SeO<Subscript>3</Subscript> on Ca,Mg, Mn,Fe, Cu,and Zn Contents in the Heart,Liver, Spleen,and Kidney of Chickens

This study aimed to investigate the effects of selenium on the ion profiles in the heart, liver, spleen, and kidney through the oral administration of hexavalent chromium. Approximately 22.14 mg/kg b.w. K₂Cr₂O₇ was added to water to establish a chronic poisoning model. Different selenium levels (0.00, 0.31, 0.63, 1.25, 2.50, and 5.00 mg Na₂SeO₃/kg b.w.) around the safe dose were administered to the experimental group model. Ca, Mg, Mn, Fe, Cu, and Zn were detected in the organs through flame atomic absorption spectrometry after these organs were exposed to K₂Cr₂O₇ and Na₂SeO₃ for 14, 28, and 42 days. Results showed that these elements exhibited various changes. Ca contents declined in the heart, liver, and spleen. Ca contents also decreased on the 28th day and increased on the 42nd day in the kidney. Mn contents declined in the heart and spleen but increased in the kidney. Mn contents also decreased on the 28th day and increased on the 42nd day in the liver. Cu contents declined in the heart and spleen. Cu contents increased on the 28th day and decreased on the 42nd day in the liver and kidney. Zn contents declined in the heart and spleen. Zn contents increased on the 28th day and decreased on the 42nd day in the liver and kidney. Fe contents decreased in the heart and liver. Fe contents increased on the 28th day and decreased on the 42nd day in the spleen and kidney. Mg contents did not significantly change in these organs. Appropriate selenium contents enhanced Mn and Zn contents, which were declined by chromium. Conversely, appropriate selenium contents reduced Ca, Fe, and Cu contents, which were increased by chromium. In conclusion, the exposure of chickens to K₂Cr₂O₇ induced changes in different trace elements, and Na₂SeO₃ supplementation could alleviate this condition.     

The F<Subscript>O</Subscript>F<Subscript>1</Subscript> ATP synthase: from atomistic three-dimensional structure to the rotary-chemical function

There are numerous studies describing how growth conditions influence the efficiency of C₄ photosynthesis. However, it remains unclear how changes in the biochemical capacity versus leaf anatomy drives this acclimation. Therefore, the aim of this study was to determine how growth light and nitrogen availability influence leaf anatomy, biochemistry and the efficiency of the CO₂ concentrating mechanism in Miscanthus × giganteus. There was an increase in the mesophyll cell wall surface area but not cell well thickness in the high-light (HL) compared to the low-light (LL) grown plants suggesting a higher mesophyll conductance in the HL plants, which also had greater photosynthetic capacity. Additionally, the HL plants had greater surface area and thickness of bundle-sheath cell walls compared to LL plants, suggesting limited differences in bundle-sheath CO₂ conductance because the increased area was offset by thicker cell walls. The gas exchange estimates of phosphoenolpyruvate carboxylase (PEPc) activity were significantly less than the in vitro PEPc activity, suggesting limited substrate availability in the leaf due to low mesophyll CO₂ conductance. Finally, leakiness was similar across all growth conditions and generally did not change under the different measurement light conditions. However, differences in the stable isotope composition of leaf material did not correlate with leakiness indicating that dry matter isotope measurements are not a good proxy for leakiness. Taken together, these data suggest that the CO₂ concentrating mechanism in Miscanthus is robust under low-light and limited nitrogen growth conditions, and that the observed changes in leaf anatomy and biochemistry likely help to maintain this efficiency.     

Targeting pCO<Subscript>2</Subscript> in Asthma: Pilot Evaluation of a Capnometry-Assisted Breathing Training

Objectives This pilot study aimed to evaluate the feasibility and potential benefits of a novel biofeedback breathing training for achieving sustained increases in pCO₂ levels. Methods Twelve asthma patients were randomly assigned to an immediate 4-week treatment group or waiting list control. Patients were instructed to modify their respiration in order to change levels of end-tidal pCO₂ using a hand-held capnometer. Treatment outcome was assessed in frequency and distress of symptoms, asthma control, lung function, and variability of peak expiratory flow (PEF). Results We found stable increases in pCO₂ and reductions in respiration rate during treatment and 2-month follow-up. Mean pCO₂ levels rose from a hypocapnic to a normocapnic range at follow-up. Frequency and distress of symptoms was reduced and reported asthma control increased. In addition, mean PEF variability decreased significantly in the treatment group. Conclusions Our pilot intervention provided evidence for the feasibility of pCO₂-biofeedback training in asthma patients.     

Baclofen and Adenosine Inhibition of Synaptic Transmission at CA3-CA1 Synapses Display Differential Sensitivity to K<Superscript>+</Superscript> Channel Blockade

The metabotropic GABA_B and adenosine A₁ receptors mediate presynaptic inhibition through regulation of voltage-dependent Ca²⁺ channels, whereas K⁺ channel regulation is believed to have no role at the CA3-CA1 synapse. We show here that the inhibitory effect of baclofen (20 μM) and adenosine (300 μM) on field EPSPs are differentially sensitive to Cs⁺ (3.5 mM) and Ba²⁺ (200 μM), but not 4-aminopyridine (100 μM). Barium had no effect on paired-pulse facilitation (PPF) in itself, but gave significant reduction (14 ± 5%) when applied in the presence of baclofen, but not adenosine, suggesting that the effect is presynaptic and selective on the GABA_B receptor-mediated response. The effect of Ba²⁺ on PPF was not mimicked by tertiapin (30 nM), indicating that the underlying mechanism does not involve GIRK channels. Barium did not affect PPF in slices from young rats (P7–P8), suggesting developmental regulation. The above effects of Ba²⁺ on adult tissue were reproduced when measuring evoked whole-cell EPSCs from CA1 pyramidal neurons: PPF was reduced by 22 ± 3% in the presence of baclofen and unaltered in adenosine. In contrast, Ba²⁺ caused no significant change in frequency or amplitude of miniature EPSCs. The Ba²⁺-induced reduction of PPF was antagonized by LY341495, suggesting metabotropic glutamate receptor involvement. We propose that these novel effects of Ba²⁺ and Cs⁺ are exerted through blockade of inwardly rectifying K⁺ channels in glial cells, which are functionally interacting with the GABA_B receptor-dependent glutamate release that generates heterosynaptic depression.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Root Plasma Membrane Lipid Changes in Relation to Water Transport in Pepper: a Response to NaCl and CaCl<Subscript>2</Subscript> Treatment

Seeds ofCapsicum annuum were grown hydroponically in a nutrient medium with or without NaCI and with supplemented Ca²⁺. Plasma membranes were isolated from roots using a two-phase aqueous polymer technique. The lipid composition (fatty acids, phospholipids and sterols) of the purified plasma membrane was determined. In the presence of NaCI, changes in lipid composition were shown, driving the membrane to a more rigid state. This was accomplished by an increase of (i) the saturation of fatty acids, (ii) the content of stearic acid versus palmitic acid, and (iii) the sterols concentration in the membrane. The changes in the phospholipid composition were also related to NaCI, which reverted when Ca²⁺ was also present in the nutrient solution. Furthermore, the alterations of plasma membrane lipid composition under salinity and calcium can be related to water transport properties of the membrane, but other physiological responses have to be taken into account.     

Intramolecular G-quadruplexes formed by d(GT)<Subscript>12</Subscript> microsatellite sequence in the presence of K<Superscript>+</Superscript>

Polymorphic d(GT) _n microsatellite sequences are known to dramatically affect the regulation of gene expression. CD spectroscopy, UV melting, fluorescence polarization of ethidium bromide (EtBr), and FRET allowed the detection of a new G-quartet fold formed by the d(GT)₁₂ oligonucleotide in 0.01 M Na-phosphate (pH 8.0) in the presence of 0.1 M KCl. The monomolecular type of the structure was verified by measuring the rotational relaxation time (ρ = 28 ± 0.5 ns) of the EtBr: d(GT)₁₂ complex. CD spectra supported the G-quartet formation. FRET was used to estimate the distance between intercalated EtBr and FITC covalently attached to the 5′ end of d(GT)₁₂ (R ≤ 17 Å). The experimental data agree with the self-folding of d(GT)₁₂ in a G-quartet structure.