Characterization of the cross-bridge force-generating step using inorganic phosphate and BDM in myofibrils from rabbit skeletal muscles

The inhibitory effects of inorganic phosphate (P_i) on isometric force in striated muscle suggest that in the ATPase reaction P_i release is coupled to force generation. Whether P_i release and the power stroke are synchronous events or force is generated by an isomerization of the quaternary complex of actomyosin and ATPase products (AM.ADP.P_i) prior to the following release of P_i is still controversial. Examination of the dependence of isometric force on [P_i] in rabbit fast (psoas; 5-15 °C) and slow (soleus; 15-20 °C) myofibrils was used to test the two-step hypothesis of force generation and P_i release. Hyperbolic fits of force-[P_i] relations obtained in fast and slow myofibrils at 15 °C produced an apparent asymptote as [P_i]∞ of 0.07 and 0.44 maximal isometric force (i.e. force in the absence of P_i) in psoas and soleus myofibrils, respectively, with an apparent K _d of 4.3 m m in both. In each muscle type, the force-[P_i] relation was independent of temperature. However, 2,3-butanedione 2-monoxime (BDM) decreased the apparent asymptote of force in both muscle types, as expected from its inhibition of the force-generating isomerization. These data lend strong support to models of cross-bridge action in which force is produced by an isomerization of the AM.ADP.P_i complex immediately preceding the P_i release step.     

Influence of ionic strength on the time course of force development and phosphate release by dogfish muscle fibres

We measured the effects of ionic strength (IS), 200 (standard) and 400 mmol l⁻¹ (high), on force and ATP hydrolysis during isometric contractions of permeabilized white fibres from dogfish myotomal muscle at their physiological temperature, 12°C. One goal was to test the validity of our kinetic scheme that accounts for energy release, work production and ATP hydrolysis. Fibres were activated by flash photolysis of the P ³-1-(2 nitrophenyl) ethyl ester of ATP (NPE-caged ATP), and time-resolved phosphate (P_i) release was detected with the fluorescent protein MDCC-PBP, N -(2[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3-carboxamide phosphate binding protein. High IS slowed the transition from rest to contraction, but as the fibres approached the isometric force plateau they showed little IS sensitivity. By 0.5 s of contraction, the force and the rate of P_i release at standard and high IS values were not significantly different. A five-step reaction mechanism was used to account for the observed time courses of force and P_i release in all conditions explored here. Only the rate constants for reactions of ATP, ADP and P_i with the contractile proteins varied with IS, thus suggesting that the actin–myosin interactions are largely non-ionic. Our reaction scheme also fits previous results for intact fibres.     

Role of phosphate and calcium stores in muscle fatigue

Intensive activity of muscles causes a decline in performance, known as fatigue, that is thought to be caused by the effects of metabolic changes on either the contractile machinery or the activation processes. The concentration of inorganic phosphate (P_i) in the myoplasm ([P_i]_myo) increases substantially during fatigue and affects both the myofibrillar proteins and the activation processes. It is known that a failure of sarcoplasmic reticulum (SR) Ca²⁺ release contributes to fatigue and in this review we consider how raised [P_i]_myo contributes to this process. Initial evidence came from the observation that increasing [P_i]_myo causes reduced SR Ca²⁺ release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca²⁺ in the SR declines mirroring the decline in SR Ca²⁺ release. In muscle fibres with inoperative creatine kinase the rise of [P_i]_myo is absent during fatigue and the failure of SR Ca²⁺ release is delayed. These results can all be explained if inorganic phosphate can move from the myoplasm into the SR during fatigue and cause precipitation of CaP_i within the SR. The relevance of this mechanism in different types of fatigue in humans is considered.     

Muscle [phosphocreatine] dynamics following the onset of exercise in humans: the influence of baseline work-rate

The kinetics of pulmonary O₂ uptake is known to be substantially slower when exercise is initiated from a baseline of lower-intensity exercise rather than from rest. However, it is not known whether putative intracellular regulators of mitochondrial respiration (and in particular the phosphocreatine concentration, [PCr]) show similar non-linearities in their response dynamics. The purpose of this study was therefore to investigate the influence of baseline metabolic rate on muscle [PCr] kinetics (as assessed using ³¹P-magnetic resonance spectroscopy) following the onset of exercise. Seven male subjects completed 'step' tests to heavy-intensity exercise (80% of peak work-rate) from a resting baseline and also from a baseline of moderate-intensity exercise (40% of peak work-rate) using a single-leg knee-extensor ergometer situated inside the bore of a 1.5 T super-conducting magnet. The time constant describing the kinetics of the initial exponential-like fall in [PCr] was significantly different between rest-to-moderate (25 ± 14 s), rest-to-heavy (48 ± 11 s) and moderate-to-heavy exercise (95 ± 40 s) ( P < 0.05 for all comparisons). A delayed-onset 'slow component' in the [PCr] response was observed in all subjects during rest-to-heavy exercise, but was attenuated in the moderate-to-heavy exercise condition. These data indicate that muscle [PCr] kinetics does not conform to 'linear, first-order' behaviour during dynamic exercise, and thus have implications for understanding the regulation of muscle oxidative metabolism.     

Investigation of the contribution of total creatine to the CEST Z‐spectrum of brain using a knockout mouse model

The current study aims to assign and estimate the total creatine (tCr) signal contribution to the Z ‐spectrum in mouse brain at 11.7 T. Creatine (Cr), phosphocreatine (PCr) and protein phantoms were used to confirm the presence of a guanidinium resonance at this field strength. Wild‐type (WT) and knockout mice with guanidinoacetate N ‐methyltransferase deficiency (GAMT?/?), which have low Cr and PCr concentrations in the brain, were used to assign the tCr contribution to the Z ‐spectrum. To estimate the total guanidinium concentrations, two pools for the Z ‐spectrum around 2 ppm were assumed: (i) a Lorentzian function representing the guanidinium chemical exchange saturation transfer (CEST) at 1.95 ppm in the 11.7‐T Z ‐spectrum; and (ii) a background signal that can be fitted by a polynomial function. Comparison between the WT and GAMT?/? mice provided strong evidence for three types of contribution to the peak in the Z ‐spectrum at 1.95 ppm, namely proteins, Cr and PCr, the latter fitted as tCr. A ratio of 20 ± 7% (protein) and 80 ± 7% tCr was found in brain at 2 μT and 2 s saturation. Based on phantom experiments, the tCr peak was estimated to consist of about 83 ± 5% Cr and 17 ± 5% PCr. Maps for tCr of mouse brain were generated based on the peak at 1.95 ppm after concentration calibration with in vivo magnetic resonance spectroscopy.     

The Smooth Muscle Myosin Seven Amino Acid Heavy Chain Insert's Kinetic Role in the Crossbridge Cycle for Mouse Bladder

The seven amino acid insert in the smooth muscle myosin heavy chain is thought to regulate the kinetics of contraction, contributing to the differences between fast and slow smooth muscle. The effects of this insert on force and stiffness were determined in bladder tissue of a transgenic mouse line expressing the insert SMB at one of three levels: an SMB wild type (+/+), an SMA homozygous type (−/−) and a heterozygous type (+/−). For skinned muscle, an increase in MgADP or inorganic phosphate (P_i) should shift the distribution of crossbridges in the actomyosin ATPase (AMATPase) to increase the relative population of the crossbridge state prior to ADP release and P_i release, respectively. Exogenous ADP increased force and stiffness in a manner consistent with increasing the Ca²⁺ concentration in both the +/+ and +/− mouse types. However, the −/− type showed a significantly greater increase in force than in stiffness suggesting that immediately prior to ADP release, the AMATPase either has an additional force producing isomerization state or a slower ADP dissociation rate for the −/− type compared to the +/+ or +/− types. Exogenous P_i led to a significantly greater decrease in stiffness than in force for all three mouse types suggesting that there is a force producing state prior to P_i release. In addition, the increase in P_i showed similar changes in the +/+ and −/− types whereas in the +/− type the decreases in both force and stiffness were greater than the other two mouse types indicating that the insert can affect the cooperativity between myosin heads. In conclusion, the seven amino acid insert modulates the kinetics and/or states of the AMATPase, which could lead to differences in the kinetics of contraction between fast and slow smooth muscle.     

Gated dynamic 31P MRS shows reduced contractile phosphocreatine breakdown in mice deficient in cytosolic creatine kinase and adenylate kinase

We developed a new dedicated measurement protocol for dynamic ³¹P MRS analysis in contracting calf muscles of the mouse, using minimally invasive assessment of the contractile force combined with the acquisition of spectroscopic data gated to muscle contraction and determination of phosphocreatine (PCr) recovery rate and ATP contractile cost. This protocol was applied in a comparative study of six wild type (WT) mice and six mice deficient in cytosolic creatine kinase and adenylate kinase isoform 1 (MAK^?/? mice) using 70 repeated tetanic contractions at two contractions per minute. Force levels during single contractions, and metabolite levels and tissue pH during resting conditions were similar in muscles of MAK^?/? and WT mice. Strikingly, muscle relaxation after contraction was significantly delayed in MAK^?/? mice, but during repeated contractions, the decrease in the force was similar in both mouse types. Gated data acquisition showed a negligible PCr breakdown in MAK^?/? immediately after contraction, without a concomitant decrease in ATP or tissue pH. This protocol enabled the determination of rapid PCr changes that would otherwise go unnoticed due to intrinsic low signal‐to‐noise ratio (SNR) in mouse skeletal muscles combined with an assessment of the PCr recovery rate. Our results suggest that MAK^?/? mice use alternative energy sources to maintain force during repeated contractions when PCr breakdown is reduced. Furthermore, the absence of large increases in adenosine diphosphate (ADP) or differences in force compared to WT mice in our low‐intensity protocol indicate that creatine kinase (CK) and adenylate kinase (AK) are especially important in facilitating energy metabolism during very high energy demands. Copyright © 2009 John Wiley & Sons, Ltd.     

Mutant mice deficient in NOS-1 exhibit attenuated long-term facilitation and short-term potentiation in breathing

The objective of the present study is to examine the potential role of nitric oxide (NO) in short-term potentiation (STP) and long-term facilitation (LTF) of breathing. Experiments were performed in wild-type (WT) and mutant mice deficient in nitric oxide synthase-1 (NOS-1), as well as in WT mice administered the NOS-1 inhibitor 7-nitroindazole (7-NI; 50 mg kg⁻¹; i.p. ). Respiratory responses following either single or recurrent episodes of hypoxia (7 % O₂, balance N₂) were analysed in unanaesthetised animals by body plethysmography along with rate of O₂ consumption (_O2) and CO₂ production (_CO2). After a single hypoxic challenge, respiration in WT mice remained elevated for 5 min, suggesting STP in ventilation. Following termination of three consecutive hypoxic challenges, respiration remained elevated during normoxia for as long as 30 min, indicating LTF in breathing under awake conditions. STP and LTF were significantly attenuated or absent in WT mice after 7-NI. A similar attenuation or absence of STP and LTF was also seen in NOS-1 mutant mice. Changes in _O2 and _CO2 were comparable among mice during the post-hypoxic period, suggesting that the absence of STP and LTF was not due to alterations in body metabolism. These results suggest endogenous NO is an important physiological modulator of ventilatory STP and LTF.     

α5 subunit-containing GABAA receptors affect the dynamic range of mouse hippocampal kainate-induced gamma frequency oscillations in vitro

Though all in vitro models of gamma frequency network oscillations are critically dependent on GABA_A receptor-mediated synaptic transmission little is known about the specific role played by different subtypes of GABA_A receptor. Strong expression of the α5 subunit of the GABA_A receptor is restricted to few brain regions, amongst them the hippocampal dendritic layers. Receptors containing this subunit may be expressed on the extrasynaptic membrane of principal cells and can mediate a tonic GABA_A conductance. Using hippocampal slices of wild-type (WT) and α5−/− mice we investigated the role of α5 subunits in the generation of kainate-induced gamma frequency oscillations (20–80 Hz). The change in power of the oscillations evoked in CA3 by increasing network drive (kainate, 50–400 n m ) was significantly greater in α5−/− than in WT slices. However, the change in frequency of gamma oscillations with increasing network drive seen in WT slices was absent in α5−/− slices. Raising the concentration of extracellular GABA by bathing slices in the GABA transaminase inhibitor vigabatrin and blocking uptake with tiagabine reduced the power of gamma oscillations more in WT slices than α5−/− slices (43% versus 15%). The data suggest that loss of this GABA_A receptor subunit alters the dynamic profile of gamma oscillations to changes in network drive, possibly via actions of GABA at extrasynaptic receptors.     

Binding of Helix Pomatia A Hemagglutinin to Human Erythrocytes and their Cells. Influence of Multivalent Interaction on Affinity

The binding of ¹²⁵I-labelled intact (hexavalent) and partially reduced (divalent) Helix pomatia A hemagglutinin to human A.₁, A.₂, A.₃, A₁B and A₂B erythrocytes, human lymphocytes, human lymphoblastoid and other tumor cell lines was investigated. The essential finding was that the association constants calculated for the interaction between hemagglutinin and A-erythrocytes were many orders of magnitude higher than the intrinsic association constant for the interaction between the hemagglutinin and the blood group A determinant. The latter value was 5–10³1/mole. The K-values for intact hemagglutinin and A and AB erythrocytes were in the order of 10¹⁰1/mole at 18–22 °C and pH 7.3. For partially reduced hemagglutinin the K-values were in the order of 5–10⁷ 1/mole. Multivalent interaction would seem to be the essential factor responsible for the high K-values in the cell binding experiments. Intact hemagglutinin reacted against A₁ and A₂ erythrocytes or against a human osteogenic sarcoma cell tine (2T) gave homogeneous binding curves in Scatchard's plot. Human lymphocytes and most lymphoblastoid cell lines lacked hemagglutinin receptors.     

Enhanced production of platelet activating factor by peripheral granulocytes from children with asthma

Schauer U, Koch B, Michl U, Jäger R, Rieger CHL. Enhanced production of platelet activating factor by peripheral granulocytes from children with asthma.
Granulocytes from 23 asthmatic children aged 4–15 years and 32 age-matched healthy children were studied. Cells were purified by Dextran sedimentation and Percoll gradient centrifugation from heparinized blood. After in vitro stimulation by ionophore A23187 the amount of newly synthesized PAF and LTC₄ was assessed by radio receptor assay or radioimmunoassay respectively. Eight patients had symptoms of asthma within the last 3 weeks before examination. Granulocytes from the symptomatic patients showed a significantly higher PAF generation (median 125 ng/10⁶ cells, range 7–189 ng/10⁶ cells) when compared to asymptomatic patients ( p < 0.001. median 14 ng/10⁶cells, range 6–33 ng/10⁶ cells) or controls ( p < 0.001, median 11 ng/10⁶ cells, range 3–26 ng/10⁶ cells). In contrast, LTC₄ generation was increased in both patient groups. The results suggest a regulatory role of PAF in the exacerbation of asthma.     

Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres

In the preceding paper, we reported that flexor digitorum brevis (FDB) muscle fibres from S100A1 knock-out (KO) mice exhibit a selective suppression of the delayed, steeply voltage-dependent component of intra-membrane charge movement current termed Q _γ. Here, we use 50 μ m of the Ca²⁺ indicator fluo-4 in the whole cell patch clamp pipette, in addition to 20 m m EGTA and other constituents included for the charge movement studies, and calculate the SR Ca²⁺ release flux from the fluo-4 signals during voltage clamp depolarizations. Ca²⁺ release flux is decreased in amplitude by the same fraction at all voltages in fibres from S100A1 KO mice compared to fibres from wild-type (WT) littermates, but unchanged in time course at each pulse membrane potential. There is a strong correlation between the time course and magnitude of release flux and the development of Q _γ. The decreased Ca²⁺ release in KO fibres is likely to account for the suppression of Q _γ in these fibres. Consistent with this interpretation, 4-chloro- m -cresol (4–CMC; 100 μ m ) increases the rate of Ca²⁺ release and restores Q _γ at intermediate depolarizations in fibres from KO mice, but does not increase Ca²⁺ release or restore Q _γ at large depolarizations. Our findings are consistent with similar activation kinetics for SR Ca²⁺ channels in both WT and KO fibres, but decreased Ca²⁺ release in the KO fibres possibly due to shorter SR channel open times. The decreased Ca²⁺ release at each voltage is insufficient to activate Q _γ in fibres lacking S100A1.     

Local T-Cell Proliferation and Differentiation in Inflammatory Myopathies

Our objective was to investigate the patterns of proliferation and differentiation of infiltrating cells in inflammatory myopathies. Immunohistochemical staining was performed on muscle biopsy specimens from 18 patients with inclusion body myositis, polymyositis and dermatomyositis using monoclonal and polyclonal antibodies.
An abundance of cells were TNF-α⁺ (4–8%), ICAM-1⁺ (7–65%). IFN-γ⁺ (3–6%), and Ki-67⁺ (4–8%). It was shown that 70% of the Ki-67⁺ cells were Ki-67⁺CD3⁺ cells. Very few mononuclear cells were IL-2R⁺. MHC-I expression was found on nearly all muscle fibres in all cases, while MHC-II expression was found on occasional muscle fibres in 1/3 of cases. Analysis of repeated biopsies from four IBM patients after prednisolone treatment showed no change in the proportions of TNF-α, ICAM-1, IFN-γ or Ki-67 positive cells. In inflammatory myopathies there is an intense proliferation and differentiation of inflammatory cells in situ , indicating a local stimulation of the inflammatory process.     

PROLIFERATION OF ITO CELLS (FAT-STORING CELLS) IN ACUTE CARBON TETRACHLORIDE LIVER INJURY: A Light and Electron Microscopic Autoradiographic Study

The proliferative activity of Ito cells in acute liver injury induced by carbon tetrachloride (CCl₄) was studied by light and electron microscopic autoradio-graphy. At 48 hours after a single intraperitoneal injection of CCl₄, the livers of the mice given vitamin A per os for preceding 9 days and those of the mice without vitamin A-pretreatment were removed. Small tissue blocks of each group were respectively incubated at 37°CC for 1 hour in culture medium containing ³H-thymidine. After CC1₄, injection, perisinusoidal and sinusoidal cells adjacent to centrilobular necrotic liver cells increased in number and size. Some of them were labelled by ³H-thymidine. On the other hand, the perisinusoidal and sinusoidal cells in the peripheral zone in which liver cells are not markedly degenerated nor necrotic showed no noticeable increase in number. They contained very few or no silver grains after ³H-thymidine. In control mice the labelling of perisinusoidal cells was hardly observed. Electron microscopic autoradiography revealed that most of the labelled perisinusoidal cells in the centrilobular zone possess characteristics of Ito cells in their location and in the fine structures such as the presence of small fat droplets, well-developed rough endoplasmic reticulum, and Golgi complex in the cytoplasm. These findings indicate that Ito cells incorporate ³H-thymidine in DNA synthesis after hepatocellular necrosis resulting in cell proliferation. ACTA PATHOL. JPN. 35: 1301–1308, 1985.     

Actomyosin energy turnover declines while force remains constant during isometric muscle contraction

Energy turnover was measured during isometric contractions of intact and Triton-permeabilized white fibres from dogfish ( Scyliorhinus canicula ) at 12°C. Heat + work from actomyosin in intact fibres was determined from the dependence of heat + work output on filament overlap. Inorganic phosphate (P_i) release by permeabilized fibres was recorded using the fluorescent protein MDCC-PBP, N-(2-[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3 carboxamide phosphate binding protein. The steady-state ADP release rate was measured using a linked enzyme assay. The rates decreased five-fold during contraction in both intact and permeabilized fibres. In intact fibres the rate of heat + work output by actomyosin decreased from 134 ± s.e.m. 28 μW mg⁻¹ ( n = 17) at 0.055 s to 42% of this value at 0.25 s, and to 20% at 3.5 s. The force remained constant between 0.25 and 3.5 s. Similarly in permeabilized fibres the P_i release rate decreased from 5.00 ± 0.39 mmol l⁻¹ s⁻¹ at 0.055 s to 39% of this value at 0.25 s and to 19% at 0.5 s. The steady-state ADP release rate at 15 s was 21% of the P_i rate at 0.055 s. Using a single set of rate constants, the time courses of force, heat + work and P_i release were described by an actomyosin model that took account of the transition from the initial state (rest or rigor) to the contracting state, shortening and the consequent work against series elasticity, and reaction heats. The model suggests that increasing P_i concentration slows the cycle in intact fibres, and that changes in ATP and ADP slow the cycle in permeabilized fibres.     

Delayed Elimination of the LCM Virus from Acid Sphingomyelinase-Deficient Mice due to Reduced Expansion of Virus-Specific CD8+ T Lymphocytes

The phagolysosomally localized acid sphingomyelinase (ASMase) activated by proinflammatory cytokines such as TNF and IFN-γ generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin D. These characteristics of ASMase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. We show here that ASMase^–/– mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (LCM) virus as rapidly as littermate wildtype mice. Investigation of the immune response revealed a reduced expansion of CD8⁺ T cells. The secretion of IFN-γ in response to contact with target cells as well as the cytolytic activity of virus-specific CD8⁺ T cells was severely impaired. Additionally, both phases of the LCM virus-specific DTH response, mediated by CD8⁺ and CD4⁺ T cells consecutively, were diminished in ASMase^–/– mice. However, the secondary memory response of virus-specific CTL was not altered, and the virus was effectively controlled for at least 3 months by ASMase^–/– mice. In conclusion, the results of this study suggest an involvement of the ASMase in the activation, expansion or maturation of virus-specific CD8⁺ T cells during the acute infection of mice with the LCM virus.     

Creatine and guanidinoacetate: diagnostic markers for inborn errors in creatine biosynthesis and transport

In this study, measurements of guanidinoacetate (GAA) and creatine (Cr) in urine, plasma, and cerebrospinal fluid (CSF) were performed using stable isotope dilution gas chromatography-mass spectrometry. Both compounds were analyzed in a single analysis. Reference values were established for GAA and Cr. These values were age dependent. No differences with gender were observed. Eight guanidinoacetate methyltransferase (GAMT) deficient patients and eight creatine transporter SLC6A8 deficient patients were investigated. In urine, plasma, and CSF of GAMT deficient patients increased levels of GAA are present. The SLC6A8 deficient patients all show increased creatine/creatinine (Cr/Crn) ratio in urine demonstrating the importance of the Cr/Crn ratio as a pathognomonic marker of the SLC6A8 deficiency.     

Polycythemia Vera treated with 32P and Myleran: Development of chronic granulocytic leukemia with chromosomal abnormalities in one patient

Chronic granulocytic leukemia developed in a 59–year-old woman who had previously received a total of 21 mCi ³²P for polycythemia Vera. She was treated with Myleran (busulphan) for her chronic granulocytic leukemia. Cytogenetic studies revealed deletion of chromosomes No. 8 and 12, and translocation between 1 and 8. The patient also developed a severe autoimmune hemolytic anemia, for which she received prednisone treatment. She died with a perforated stomach ulcer.     

Calmodulin binding to M-type K+ channels assayed by TIRF/FRET in living cells

Calmodulin (CaM) binds to KCNQ2–4 channels within their carboxy termini, where it regulates channel function. The existing data have not resolved the Ca²⁺ dependence of the interaction between the channels and CaM. We performed glutathione S-transferase (GST)-pull-down assays between purified KCNQ2–4 carboxy termini and CaM proteins to determine the Ca²⁺ dependence of the interaction in vitro . The assays showed substantial Ca²⁺ dependence of the interaction of the channels with wild-type (WT) CaM, but not with dominant-negative (DN) CaM. To demonstrate CaM–channel interactions in individual living cells, we performed fluorescence resonance energy transfer (FRET) between ECFP-tagged KCNQ2–4 channels and EYFP-tagged CaM expressed in CHO cells, performed under total internal reflection fluorescence (TIRF) microscopy, in which excitation light only penetrates several hundred nanometres into the cell, thus isolating membrane events. FRET was assayed between the channels and either WT or DN CaM, performed under conditions of normal [Ca²⁺]_i, low [Ca²⁺]_i or high [Ca²⁺]_i induced by empirically optimized bathing solutions. The FRET data suggest a strong Ca²⁺ dependence for the interaction between WT CaM and KCNQ2, but less so for KCNQ3 and KCNQ4. FRET between all KCNQ2–4 channels and DN CaM was robust, and not significantly Ca²⁺ dependent. These data show interactions between CaM and KCNQ channels in living cells, and suggest that the interactions between KCNQ2–4 channels and CaM are likely to have Ca²⁺-dependent and Ca²⁺-independent components.     

Gene targeting approach to clarification of ion channel function: studies of Kir6.x null mice

ATP-sensitive potassium (K_ATP) channels are present in many tissues, including pancreatic β-cells, heart, skeletal muscle, vascular smooth muscle and brain, in which they couple the cell metabolic state to membrane potential. K_ATP channels are hetero-octameric proteins composed of the pore-forming subunits Kir6.x (Kir6.1 or Kir6.2) of the inwardly rectifying K⁺ channel family and the regulatory subunits SURx (SUR1, SUR2A or SUR2B), the receptor of the sulphonylureas widely used in treatment of type 2 diabetes mellitus. Different combinations of Kir6.x and SURx comprise K_ATP channels with distinct electrophysiological and pharmacological properties, but their physiological functions in the various tissues are unclear. Our studies of Kir6.2 null (knockout) and Kir6.1 null mice have shown that K_ATP channels are critical metabolic sensors in protection against acute metabolic stress such as hyperglycaemia, hypoglycaemia, ischaemia and hypoxia.