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PubMed2006

Ranolazine improves abnormal repolarization and contraction in left ventricular myocytes of dogs with heart failure by inhibiting late sodium current.

BACKGROUND: Ventricular repolarization and contractile function are frequently abnormal in ventricular myocytes from human failing hearts as well as canine hearts with experimentally induced heart failure (HF). These abnormalities have been attributed to dysfunction involving various steps of the excitation-contraction coupling process, leading to impaired intracellular sodium and calcium homeostasis. We previously reported that the slow inactivating component of the Na(+) current (late I(Na)) is augmented in myocytes from failing hearts, and this appears to play a significant role in abnormal ventricular myocytes repolarization and function. We tested the effect of ranolazine, a novel drug being developed to treat angina, on (1) action potential duration (APD), (2) peak transient and late I(Na) (I(NaT) and I(NaL), respectively), (3) early afterdepolarizations (EADs), and (4) twitch contraction (TC), including after contractions and contracture. METHODS: Myocytes were isolated from the left ventricle of normal dogs and of dogs with chronic HF caused by multiple sequential intracoronary micro-embolizations. I(NaT) and I(NaL) were recorded using conventional whole-cell patch-clamp techniques. APs were recorded using the beta-escin perforated patch-clamp configuration at frequencies of 0.25 and 0.5 Hz. TCs were recorded using an edge movement detector at stimulation frequencies ranging from 0.5 to 2.0 Hz. RESULTS: Ranolazine significantly (P<0.05) and reversibly shortened the APD of myocytes stimulated at either 0.5 or 0.25 Hz in a concentration-dependent manner. At a stimulation frequency of 0.5 Hz, 5, 10, and 20 microM ranolazine shortened the APD(90) (APD measured at 90% repolarization) from 516+/-51 to 304+/-22, 212+/-34 and 160+/-11 ms, respectively, and markedly decreased beat-to-beat variability of APD(90), EADs, and dispersion of APDs. Ranolazine preferentially blocked I(NaL) relative to I(NaT) in a state-dependent manner, with a approximately 38-fold greater potency against I(NaL) to produce tonic block (IC(50)=6.5 microM) than I(NaT) (IC(50)=294 microM). When we evaluated inactivated state blockade of I(NaL) from the steady-state inactivation mid-potential shift using a theoretical model, ranolazine was found to bind more tightly to the inactivated state than the resting state of the sodium channel underlying I(NaL), with apparent dissociation constants K(dr)=7.47 microM and K(di)=1.71 microM, respectively. TCs of myocytes stimulated at 0.5 Hz were characterized by an initial spike followed by a dome-like after contraction, which was observed in 75% of myocytes from failing hearts and coincided with the long AP plateau and EADs. Ranolazine at 5 and 10 microM reversibly shortened the duration of TCs and abolished the after contraction. When the rate of myocyte stimulation was increased from 1.0 to 2.0 Hz, there was a progressive increase in diastolic "tension," that is, contracture. Ranolazine at 5 and 10 microM reversibly prevented this frequency-dependent contracture.

Acetanilides↗

PubMed1995

Quantitative analysis of electrotonic structure and membrane properties of NMDA-activated lamprey spinal neurons.

Parameter optimization methods were used to quantitatively analyze frequency-domain-voltage-clamp data of NMDA-activated lamprey spinal neurons simultaneously over a wide range of membrane potentials. A neuronal cable model was used to explicitly take into account receptors located on the dendritic trees. The driving point membrane admittance was measured from the cell soma in response to a Fourier synthesized point voltage clamp stimulus. The data were fitted to an equivalent cable model consisting of a single lumped soma compartment coupled resistively to a series of equal dendritic compartments. The model contains voltage-dependent NMDA sensitive (INMDA), slow potassium (IK), and leakage (IL) currents. Both the passive cable properties and the voltage dependence of ion channel kinetics were estimated, including the electronic structure of the cell, the steady-state gating characteristics, and the time constants for particular voltage- and time-dependent ionic conductances. An alternate kinetic formulation was developed that consisted of steady-state values for the gating parameters and their time constants at half-activation values as well as slopes of these parameters at half-activation. This procedure allowed independent restrictions on the magnitude and slope of both the steady-state gating variable and its associated time constant. Quantitative estimates of the voltage-dependent membrane ion conductances and their kinetic parameters were used to solve the nonlinear equations describing dynamic responses. The model accurately predicts current clamp responses and is consistent with experimentally measured TTX-resistant NMDA-induced patterned activity. In summary, an analysis method is developed that provides a pragmatic approach to quantitatively describe a nonlinear neuronal system.

Animals↗

PubMed2004

Testosterone induces cytoprotection by activating ATP-sensitive K+ channels in the cardiac mitochondrial inner membrane.

BACKGROUND: Whereas in the past, androgens were mainly believed to exert adverse effects on the cardiovascular system, recent experimental data postulate a benefit of testosterone for recovery of myocardial function after ischemia/reperfusion injury. Thus, we examined whether testosterone might improve myocardial tolerance to ischemia due to activation of mitochondrial (mitoK(ATP)) and/or sarcoplasmatic (sarcK(ATP)) K(ATP) channels. METHODS AND RESULTS: In a cellular model of ischemia, testosterone significantly decreased the rate of ischemia-induced death of cardiomyocytes that could be prevented by 5-hydroxydecainoic acid but was unaffected by the sarcK(ATP) blocker HMR1098 and the testosterone receptor antagonist flutamide. To index mitoK(ATP), mitochondrial flavoprotein fluorescence was measured. Testosterone induced a highly significant increase in mitochondrial flavoprotein fluorescence in intact myocytes and isolated mitoplasts that could be abolished by 5-hydroxydecainoic acid. Testosterone-mediated flavoprotein oxidation of mitoplasts was K+ dependent and ATP sensitive. In mitoplast-attached single-channel recordings, testosterone directly activated an ATP-sensitive K+ channel of the inner mitochondrial membrane. Addition of the K(ATP) channel opener diazoxide and pinacidil to the cytosolic solution activated the ATP-sensitive K+ current comparable to testosterone, whereas 5-hydroxydecainoic acid and glibenclamide inhibited the testosterone-induced current. Patch-clamp experiments of intact myocytes in whole-cell configuration did not demonstrate any effect of testosterone on sarcK(ATP) channels. CONCLUSIONS: Our results provide direct evidence for the existence of cardiac mitoK(ATP) and a link between testosterone-induced cytoprotection and activation of mitoK(ATP). Endogenous testosterone might play a more important role in recovery after myocardial infarction than is currently assumed.

Adenosine Triphosphate↗

PubMed2001

Murine ventricular L-type Ca(2+) current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor.

1. The functional coupling of beta(2)-adrenoceptors (beta(2)-ARs) to murine L-type Ca(2+) current (I(Ca(L))) was investigated with two different approaches. The beta(2)-AR signalling cascade was activated either with the beta(2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta(2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta(2)-ARs). Ca(2+) and Ba(2+) currents were recorded in the whole-cell and cell-attached configuration of the patch-clamp technique, respectively. 2. Zinterol (10 microM) significantly increased I(Ca(L)) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta(1)-AR subtype, since it was blocked by the beta(1)-AR selective antagonist CGP 20712A (300 nM). The beta(2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I(Ca(L)) to zinterol. 3. In myocytes with beta(2)-AR overexpression I(Ca(L)) was not stimulated by the activated signalling cascade. On the contrary, I(Ca(L)) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I(Ca(L)). The beta(2)-AR inverse agonist ICI 118,551 did not further decrease I(Ca(L)). PTX-treatment increased current amplitude to values found in control myocytes. 4. In conclusion, there is no evidence for beta(2)-AR mediated increases of I(Ca(L)) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta(2)-AR responses to zinterol, but augments beta(1)-AR mediated increases of I(Ca(L)). In the mouse model of beta(2)-AR overexpression I(Ca(L)) is reduced due to tonic activation of Gi-proteins.

Adrenergic beta-1 Receptor Agonists↗

PubMed1993

Three-dimensional structure in solution of the calcium channel blocker omega-conotoxin.

The 27 amino acid residue polypeptide omega-conotoxin GVIA, from venom of the cone shell Conus geographus, blocks neuronal voltage-activated calcium channels at picomolar concentrations. The three-dimensional structure in aqueous solution of synthetic omega-conotoxin has been determined from two-dimensional 1H n.m.r. data recorded at 600 MHz. Structural constraints consisting of interproton distances inferred from NOEs and dihedral angles from spin-spin coupling constants were used as input for distance geometry calculations with the program DSPACE. The structures were then refined using back-calculation of NOESY spectra. The family of structures obtained in this way is well defined by the n.m.r. data, the best 12 structures having pairwise root-mean-square differences of 0.68 (+/- 0.15) A over the backbone heavy atoms (N, C alpha and C) and 1.15 (+/- 0.17) A over all heavy-atoms. The molecule adopts a compact structure consisting of a small, triple-stranded, anti-parallel beta-sheet and several reverse turns. All three tyrosine residues are located on the molecular surface, which is noteworthy for its abundance of side-chain hydroxyl groups. There is no negatively charged group in conotoxin, but the five positively charged groups are distributed in three small patches on the surface, one of which, made up of the ammonium moieties of the N terminus and Lys2, may contribute to the receptor-binding surface of the molecule. An isomer of conotoxin with the same amino acid sequence, but different disulfide pairings, has also been investigated. Its structure is less well ordered than that of native conotoxin and it shows significant heterogeneity, probably as a result of cis-trans isomerism preceding hydroxyproline residues.

Amino Acid Sequence↗

PubMed2002

Fluid dynamics, wall mechanics, and oxygen transfer in peripheral bypass anastomoses.

Intimal hyperplasia at vascular anastomoses seems to be promoted by altered flow conditions and stress distributions within the anastomotic region. In order to gain deeper insight into postoperative disease processes, and subsequently, to contribute to the development of improved vascular reconstructions, detailed studies, also on local flow dynamics and related mass transport and wall mechanical effects, are required. In context with in vivo studies, computer simulation based on casts of femoro-popliteal bypasses implanted into sheep were performed to analyze the flow dynamics, the oxygen transport, and the wall and suture mechanics in anatomically correct bypass configurations related to three established surgical techniques and resulting geometries (conventional type anastomosis, Taylor-patch and Miller-cuff anastomoses with venous interposition grafts of different modifications). The influence of geometry, compliance of the graft, the interponated vein patch and vein cuff, and of the artery was included. Time-dependent, three-dimensional Navier-Stokes equations describing the flow field, and a nonlinear shell structure for the vessel walls were coupled using finite element methods. The numerical results demonstrate nonphysiological flow patterns in the anastomotic region. Strongly skewed axial velocity profiles and secondary velocities occur in the junction region. In the Miller-cuff a vortex may induce a wash-out effect which protects the host artery. On the artery floor opposite the junction flow separation and zones of recirculation were found. The analysis of oxygen transport illustrates a correlation between zones of low wall shear stress and reduced oxygen flux into the wall. Wall mechanics show that increased compliance mismatch leads to increased and discontinuous intramural stresses. Comparison to histomorphological findings on intimal hyperplasia shows certain correlations, particularly increased compliance mismatch has a proliferate influence on suture line hyperplasia. The reduction of compliance mismatch using vein interposition results in decreased generation of intimal hyperplasia, and therefore, contributes to improvement of patency rates, while the geometrical modification and the resulting change of the flow pattern seems to be less important for the growth of anastomotic intimal hyperplasia.

Anastomosis, Surgical↗

PubMed2002

Dynamic docking and electron transfer between Zn-myoglobin and cytochrome b(5).

We present a broad study of the effect of neutralizing the two negative charges of the Mb propionates on the interaction and electron transfer (ET) between horse Mb and bovine cyt b(5), through use of Zn-substituted Mb (ZnMb, 1) to study the photoinitiated reaction, ((3)ZnP)Mb + Fe(3+)cyt b(5) --> (ZnP)(+)Mb + Fe(2+)cyt b(5). The charge neutralization has been carried out both by replacing the Mb heme with zinc-deuteroporphyrin dimethylester (ZnMb(dme), 2), which replaces the charges by small neutral hydrophobic patches, and also by replacement with the newly prepared zinc-deuteroporphyrin diamide (ZnMb(diamide), 3), which converts the charged groups to neutral, hydrophilic ones. The effect of propionate neutralization on the conformation of the zinc-porphyrin in the Mb heme pocket has been studied by multinuclear NMR with an (15)N labeled zinc porphyrin derivative (ZnMb((15)N-diamide), 4). The rates of photoinitiated ET between the Mb's (1-3) and cyt b(5) have been measured over a range of pH values and ionic strengths. Isothermal titration calorimetry (ITC) and NMR methods have been used to independently investigate the effect of charge neutralization on Mb/b(5) binding. The neutralization of the two heme propionates of ZnMb by formation of the heme diester or, for the first time, the diamide increases the second-order rate constant of the ET reaction between ZnMb and cyt b(5) by as much as several 100-fold, depending on pH and ionic strength, while causing negligible changes in binding affinity. Brownian dynamic (BD) simulations and ET pathway calculations provide insight into the protein docking and ET process. The results support a new "dynamic docking" paradigm for protein-protein reactions in which numerous weakly bound conformations of the docked complex contribute to the binding of cyt b(5) to Mb and Hb, but only a very small subset of these are ET active, and this subset does not include the conformations most favorable for binding; the Mb surface is a large "target" with a small "bullseye" for the cyt b(5) "arrow". This paradigm differs sharply from the more familiar, "simple" docking within a single, or narrow range of conformations, where binding strength and ET reactivity increase in parallel. Likewise, it is distinct from, although complementary to, the well-known picture of conformational control of ET through "gating", or a related picture of "conformational coupling". The new model describes situations in which tight binding does not correlate with efficient ET reactivity, and explains how it is possible to modulate reactivity without changing affinity. Such "decoupling" of reactivity from binding clearly is of physiological relevance for the reduction of met-Mb in muscle and of met-Hb in a red cell, where tight binding of cyt b(5) to the high concentration of ferrous-Mb/Hb would prevent the cytochrome from finding and reducing the oxidized proteins; it likely is of physiological relevance in other situations, as well.

Animals↗

PubMed1998

Activating Smoothened mutations in sporadic basal-cell carcinoma.

Basal-cell carcinomas (BCCs) are the commonest human cancer. Insight into their genesis came from identification of mutations in the PATCHED gene (PTCH) in patients with the basal-cell nevus syndrome, a hereditary disease characterized by multiple BCCs and by developmental abnormalities. The binding of Sonic hedgehog (SHH) to its receptor, PTCH, is thought to prevent normal inhibition by PTCH of Smoothened (SMO), a seven-span transmembrane protein. According to this model, the inhibition of SMO signalling is relieved following mutational inactivation of PTCH in basal-cell nevus syndrome. We report here the identification of activating somatic missense mutations in the SMO gene itself in sporadic BCCs from three patients. Mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblast cells in culture. Furthermore, skin abnormalities similar to BCCs developed in transgenic murine skin overexpressing mutant SMO. These findings support the role of SMO as a signalling component of the SHH-receptor complex and provide direct evidence that mutated SMO can function as an oncogene in BCCs.

Adenovirus E1A Proteins↗

PubMed2001

Confocal microscopic study of glial-vascular relationships in the retinas of pigmented rats.

Astroglia are interposed between the cerebral vasculature and neurons, where they may mediate the transfer of substances from the circulation to neurons and couple changes in neuronal activity to changes in cerebral blood flow. The retina is a particularly advantageous model system for studying glial-vascular interactions in situ. Confocal microscopy and three-dimensional image reconstruction were used to study the anatomical relationships between glia and the surface vasculature in retinas acutely isolated from adult pigmented rats. Retinas were immunostained using antibodies directed against the basal lamina surrounding the vasculature as well as antibodies directed against glial fibrillary acidic protein. Surface vessels of all calibers were contacted by the processes of astrocytes. The vitreal surfaces of the large retinal vessels were covered by a meshwork of immunoreactive astrocyte processes of a variety of shapes, whereas the scleral surfaces of the vessels were supported by thick bundles of astrocyte processes. In addition, glial cells were filled intracellularly with the gap junction-permeable tracers Lucifer yellow and Neurobiotin. Intracellular fills clearly demonstrated the presence of astrocytes with somata that were closely apposed to the large retinal vessels. Tracer-filled astrocytes displayed a variety and complexity of shapes that was not apparent in immunostained material. Gap junctional coupling was stronger between astrocytes adjacent to the same artery than between periarterial astrocytes and astrocytes located away from arteries. Significantly fewer Müller cells were labeled when Neurobiotin was injected into astrocytes associated with arteries than when Neurobiotin was injected into astrocytes that were distant from arteries.

Animals↗

PubMed2002

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface.

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.

Binding Sites↗

PubMed2001

Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptor-deficient mice.

The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PAC1) is a G-protein-coupled receptor binding the strongly conserved neuropeptide PACAP with 1000-fold higher affinity than the related peptide vasoactive intestinal peptide. PAC1-mediated signaling has been implicated in neuronal differentiation and synaptic plasticity. To gain further insight into the biological significance of PAC1-mediated signaling in vivo, we generated two different mutant mouse strains, harboring either a complete or a forebrain-specific inactivation of PAC1. Mutants from both strains show a deficit in contextual fear conditioning, a hippocampus-dependent associative learning paradigm. In sharp contrast, amygdala-dependent cued fear conditioning remains intact. Interestingly, no deficits in other hippocampus-dependent tasks modeling declarative learning such as the Morris water maze or the social transmission of food preference are observed. At the cellular level, the deficit in hippocampus-dependent associative learning is accompanied by an impairment of mossy fiber long-term potentiation (LTP). Because the hippocampal expression of PAC1 is restricted to mossy fiber terminals, we conclude that presynaptic PAC1-mediated signaling at the mossy fiber synapse is involved in both LTP and hippocampus-dependent associative learning.

Animals↗

PubMed1998

Studying how different terminal groups change the motion of H2NSO2C6H4CONH(EG)3R when bound to the active site of human carbonic anhydrase II.

Molecular dynamics simulations have been used to explore the motions of series of ligands containing coupled benzenesulfonamide and oligoethylene glycol moieties (H2NSO2C6H4CONH(CH2-CH2OCH2CH2OCH2CH2)R+; R+ = NH3+, NHCOCH2NH3+, NHCOCH(CH2Ph)NH3+) bound at the active site of human carbonic anhydrase II (HCAII; E.C. 4.2.1.1). These complexes have been examined previously by X-ray crystallography; the locations of the terminal groups of these ligands were not defined in the crystal structures. These stimulations, carried out in the presence of water, provide dynamic information about the motion of the bound ligand that supplements the quasistatic information from crystallography. Our results suggested that the Gly and Phe groups of these ligands interacted weakly with the protein adjacent to the active site. Quantitative estimates of energies of binding did not correlate usefully with observed free energies of binding, but in the absence of information about entropies, it is not possible to tell if the lack of correlation between calculated energies and observed free energies represents inaccuracies in the energies, or a compensation between enthalpies and entropies. When the terminal Phe group was placed near a previously identified hydrophobic patch in the active site (Phe20 and Pro202) the average conformation of the ligand inferred from this simulation was inconsistent with that from the crystal structure; this result illustrates the problems of misleading local minima in these types of simulations.

Binding Sites↗

PubMed2005

Ligand-dependent linkage of the ATP site to inhibition gate closure in the KATP channel.

Major advances have been made on the inhibition gate and ATP site of the K(ir)6.2 subunit of the K(ATP) channel, but little is known about conformational coupling between the two. ATP site mutations dramatically disrupt ATP-dependent gating without effect on ligand-independent gating, observed as interconversions between active burst and inactive interburst conformations in the absence of ATP. This suggests that linkage between site and gate is conditionally dependent on ATP occupancy. We studied all substitutions at position 334 of the ATP site in K(ir)6.2deltaC26 that express in Xenopus oocytes. All substitutions disrupted ATP-dependent gating by 10-fold or more. Only positive-charged arginine or lysine at 334, however, slowed ligand-independent gating from the burst, and this was in some but not all patches. Moreover, the polycationic peptide protamine reversed the slowed gating from the burst of 334R mutant channels, and speeded the slow gating from the burst of wild-type SUR1/K(ir)6.2 in the absence of ATP. Our results support a two-step ligand-dependent linkage mechanism for K(ir)6.2 channels in which ATP-occupied sites function to electrostatically dissociate COOH-terminal domains from the membrane, then as in all K(ir) channels, free COOH-terminal domains and inner M2 helices transit to a lower energy state for gate closure.

Adenosine Triphosphate↗

PubMed2000

Acute regulation of the betaine/GABA transporter BGT-1 expressed in Xenopus oocytes by extracellular pH.

Besides uptake of Na(+) and Cl(-), mammalian cells counteract osmotic cell shrinkage also by Na(+)-coupled uptake of osmolytes, e. g., myo-inositol, taurine or betaine. The expression of the corresponding transporters is transcriptionally regulated by the ambient pH and osmolarity and is increased upon cell shrinkage, a process requiring hours. The present study has been performed to disclose rapid regulation by pH of osmolyte transport via BGT-1. Transport of GABA was investigated by using the two-electrode voltage-clamp technique with BGT-1 expressing Xenopus oocytes. GABA was used as a substrate, because of the low oocyte endogenous transport activity. Extracellular acidification to pH 5.5 reversibly decreased and extracellular alkalinization to pH 8.5 increased GABA-induced currents. Kinetic analysis revealed that extracellular alkalinization increases the affinity for Cl(-) as reflected by a decrease of the apparent K(m)-value for Cl(-) from >500 mM to 55.8 +/- 4.7 mM upon an increase of the pH from 7.0 to 8.5. The apparent K(m)- values for Na(+) and GABA remained unaltered in the pH range from 6.0 to 8.5. Instead, alkalinization increased the maximal current induced by saturating Na(+) and GABA concentrations. The results are compatible with a model of interference of H(+) ions with Cl(-) binding and a pH-dependent reduction of V(max) for Na(+) and GABA.

Animals↗

PubMed2006

Calcium-mediated coupling between mitochondrial substrate dehydrogenation and cardiac workload in single guinea-pig ventricular myocytes.

We measured mitochondrial NADH autofluorescence or Ca(2+) using Rhod-2, simultaneously with cell shortening in isolated guinea-pig ventricular myocytes. When both frequency and amplitude of twitch shortening (work intensity) were increased by raising stimulus frequency in incremental steps from 0.1 to 3.3 Hz, the steady level of NADH signal increased in a frequency-dependent manner. Mitochondrial Ca(2+) also increased with increasing work intensity. Applying Ru360, an inhibitor of mitochondrial Ca(2+) uniporter, largely attenuated the response of both NADH fluorescence and mitochondrial Ca(2+). The increase in mitochondrial Ca(2+) was slow with t(1/2)=~12 s and no obvious cyclic changes were observed in the NADH signal. When a step change from 0.1 to 3.3 Hz stimulation was applied, the NADH signal first decreased to 83% and then increased to 155% of the control level. Upon returning to 0.1 Hz, the NADH signal showed an overshoot before declining to the control level. The biphasic onset time course was well explained by the delayed Ca(2+) activation of the substrate dehydrogenation superimposed on the feedback control of the ATP synthesis, while the offset time course with a delayed deactivation of dehydrogenation. A computer simulation using an oxidative phosphorylation linked to the cardiac excitation contraction model well reconstructed the response of NADH. This model simulation predicts that the activation of substrate dehydrogenation provides ~23% of driving force of the ATP synthesis to meet the increased workload induced by the jump of stimulus from 0.1 to 3.3 Hz, and remaining ~77% is supplied by the feedback control.

Animals↗

PubMed2005

Classic benzodiazepines modulate the open-close equilibrium in alpha1beta2gamma2L gamma-aminobutyric acid type A receptors.

BACKGROUND: Classic benzodiazepine agonists induce their clinical effects by binding to a site on gamma-aminobutyric acid type A (GABAA) receptors and enhancing receptor activity. There are conflicting data regarding whether the benzodiazepine site is allosterically coupled to gamma-aminobutyric acid binding versus the channel open-close (gating) equilibrium. The authors tested the hypothesis that benzodiazepine site ligands modulate alpha1beta2gamma2L GABAA receptor gating both in the absence of orthosteric agonists and when the orthosteric sites are occupied. METHODS: GABAA receptors were recombinantly expressed in Xenopus oocytes and studied using two-microelectrode voltage clamp electrophysiology. To test gating effects in the absence of orthosteric agonist, the authors used spontaneously active GABAA receptors containing a leucine-to-threonine mutation at residue 264 on the alpha1 subunit. To examine effects on gating when orthosteric sites were fully occupied, they activated wild-type receptors with high concentrations of a partial agonist, piperidine-4-sulfonic acid. RESULTS: In the absence of orthosteric agonists, the channel activity of alpha1L264Tbeta2gamma2L receptors was increased by diazepam and midazolam and reduced by the inverse benzodiazepine agonist FG7142. Flumazenil displayed very weak agonism and blocked midazolam from further activating mutant channels. In wild-type receptors activated with saturating concentrations of piperidine-4-sulfonic acid, midazolam increased maximal efficacy. CONCLUSIONS: Independent of orthosteric site occupancy, classic benzodiazepines modulate the gating equilibrium in alpha1beta2gamma2L GABAA receptors and are therefore allosteric coagonists. A Monod-Wyman-Changeux coagonist gating model quantitatively predicts these effects, suggesting that benzodiazepines minimally alter orthosteric ligand binding.

Algorithms↗

PubMed2004

Measuring the modulatory effects of RGS proteins on GIRK channels.

Discovery of "regulators of G-protein signaling" (RGS) as GTPase-activating proteins for heterotrimeric G proteins has provided a highly sought "missing link," reconciling past discrepancies between the in vitro GTPase activity of purified G proteins and the kinetics of physiological responses mediated by G-protein signaling in vivo. With the number of RGS genes in the mammalian genome at more than 30, associating specific RGS proteins to specific G-protein-coupled receptor (GPCR) signaling events has become a focus of RGS investigators. The ubiquitous expression of multiple RGS proteins has complicated this effort, yet the outlook has been encouraged with the identification of RGS9 as the determinant mediating rapid recovery of the transducin-dependent photoresponse. G-protein-gated inwardly rectifying potassium (GIRK) channels that mediate inhibitory synaptic transmission via GPCR activation of pertussis toxin-sensitive G proteins are similarly accelerated by RGS proteins when reconstituted in heterologous cell expression systems and fully reproduce the gating properties of native GIRK channels in neurons and cardiomyocytes. The endogenous neuronal and cardiac RGS protein(s) that accelerate GPCR-->GIRK channel-gating kinetics are currently not known. This article describes methods used to measure the receptor-dependent GIRK channel-gating parameters reconstituted in Chinese hamster ovary (CHO-K1) cells and Xenopus oocytes, as well as rat atrial myocytes and rat cerebellar granule neurons as model cells with native GPCR-->GIRK channel signaling. Applications of these methods for structure-function-based studies of RGS proteins, G proteins, and GPCRs are discussed. We also describe single cell reverse transcriptase polymerase chain reaction methods developed to profile atrial myocyte and neuronal RGS expression to identify specific RGS proteins for targeted knockdown or knockout.

Animals↗

PubMed1999

Ionic selectivity of the coupled and uncoupled currents carried by the amino acid transporter KAAT1.

The ability of the intestinal amino acid co-transporter KAAT-1 expressed in Xenopus oocytes to transport different cations in either amino acid coupled or uncoupled manner was studied using voltage-clamp conditions. KAAT1-expressing oocytes exhibit a transporter-related current in the absence of organic substrate (uncoupled current). In the presence of various alkali cations the amplitude of this current follows the sequence: ILi > INa > IK approximately equal to IRb approximately equal to ICs. Addition of 1 mM leucine causes large increases in K+ and Na+ currents, while the Li+ current undergoes a more complex change and Rb+ and Cs+ currents are only marginally affected. Pre-steady-state currents in the absence of organic substrate are apparent when Na+, K+, or Li+ are the bathing ions; analysis of these currents in terms of charge movement reveals that Na+, K+, and Li+ interact differently with the transporter. The uncoupled current in mixtures of Na+ and Li+ fails to exhibit anomalous mole-fraction behavior. Kinetic analysis of ion binding and uncoupled permeation argues against a multi-ion single-file mechanism in the KAAT1 cotransporter.

Amino Acid Transport Systems, Neutral↗