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At least 199 records · Page 11Linked to original sources

PubMed1995

Differential modulation of voltage-activated conductances by intracellular and extracellular cyclic nucleotides in leech salivary glands.

1. Two-electrode voltage clamp was used to study the effects of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) on voltage-dependent ion channels in salivary gland cells of the leech, Haementeria ghilianii. 2. Intracellular cyclic AMP specifically blocked delayed rectifier K+ channels. This was shown by use of 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor), forskolin (an activator of adenylyl cyclase) and intracellular injection of cyclic AMP and its dibutyryl and 8-bromo analogues. Cyclic AMP appeared to be the second messenger for the putative neuroglandular transmitter, 5-hydroxytryptamine. 3. Intracellular injection of cyclic GMP specifically potentiated high-voltage-activated (HVA) Ca2+ current and the effect was mimicked by zaprinast, an inhibitor of cyclic GMP-dependent phosphodiesterase. 4. Extracellularly, cyclic GMP and cyclic AMP specifically decreased the amplitude and increased the rate of inactivation of HVA Ca2+ current. These effects of the cyclic nucleotides are identical to those known for extracellular ATP, which activates a presumed purinoceptor. The pyrimidine nucleotide, UTP, was almost equipotent to ATP (threshold dose < 10(-6) M), indicative of a vertebrate-type nucleotide receptor. However, suramin (5 x 10(-5) M), a non-specific P2-receptor antagonist, failed to block the effects of 5 x 10(-6) M ATP (higher suramin doses could not be reliably tested because of the depolarization and increase in membrane conductance produced by the drug). 5. Activation of the putative purinoceptor by ATP did not affect inward rectifier Na+/K+ current which is known to be potentiated by intracellular cyclic AMP and reduced by intracellular cyclic GMP. 6. The preparation may provide a useful model for study of nucleotide actions, and interactions, in channel modulation. It has technical advantages such as large cells (1200 microns in diameter) which lack intercellular coupling and may be individually dissected for biochemical studies.

Action Potentials↗

PubMed2004

Modulation of Xenopus laevis Ca-activated Cl currents by protein kinase C and protein phosphatases: implications for studies of anesthetic mechanisms.

Ca-activated Cl currents (I(Cl(Ca))) are used frequently as reporters in functional studies of anesthetic effects on G protein-coupled receptors using Xenopus laevis oocytes. However, because anesthetics affect protein kinase C (PKC), they could indirectly affect I(Cl(Ca)) if this current is regulated by phosphorylation. We therefore studied the effect of modulation of either PKC or protein phosphatases PP1alpha and PP2A on I(Cl(Ca)) stimulated either by lysophosphatidate (LPA) signaling or by microinjection of Ca. X. laevis oocytes were studied under voltage clamp. Rat PP1alpha and PP2A were overexpressed in oocytes. PP, inositoltrisphosphate (IP(3)), the PP inhibitor okadaic acid (OA), the PKC inhibitor chelerythrine, or CaCl(2) were directly injected into the oocyte. Responses to agonists (LPA 10(-6) M, IP(3) 10(-4) M, CaCl(2) 0.5 M) were measured at a holding potential of -70 mV in the presence or absence of the PP inhibitors cantharidin or OA. PP1 alpha and PP2A inhibited I(Cl(Ca)) from 7.6 +/- 0.9 microC to 2.5 +/- 0.9 microC and 3.2 +/- 1.4 microC, respectively. PP inhibition enhanced I(Cl(Ca)) in control oocytes and reversed the inhibitory effect in oocytes expressing PP1 alpha or PP2A. PKC inhibition by chelerythrine enhanced both LPA- and CaCl(2)-induced I(Cl(Ca)). Our data indicate that the Xenopus I(Cl(Ca)) is modulated by phosphorylation. This may complicate design and interpretation of studies of G protein-coupled receptors using this model.

Algorithms↗

PubMed2006

Hydrogen-bonding dynamics between adjacent blades in G-protein beta-subunit regulates GIRK channel activation.

Functionally critical domains in the betagamma-subunits of the G-protein (Gbetagamma) do not undergo large structural rearrangements upon binding to other proteins. Here we show that a region containing Ser(67) and Asp(323) of Gbetagamma is a critical determinant of G-protein-gated inwardly rectifying K(+) (GIRK) channel activation and undergoes only small structural changes upon mutation of these residues. Using an interactive experimental and computational approach, we show that mutants that form a hydrogen-bond between positions 67 and 323 do not activate a GIRK channel. We also show that in the absence of hydrogen-bonding between these positions, other factors, such as the displacement of the crucial Ggamma residues Pro(60) and Phe(61), can impair Gbetagamma-mediated GIRK channel activation. Our results imply that the dynamic nature of the hydrogen-bonding pattern in the wild-type serves an important functional role that regulates GIRK channel activation by Gbetagamma and that subtle changes in the flexibility of critical domains could have substantial functional consequences. Our results further strengthen the notion that the dynamic regulation of multiple interactions between Gbetagamma and effectors provides for a complex regulatory process in cellular functions.

Animals↗

PubMed2001

Intercellular electrical communication among smooth muscle and endothelial cells in guinea-pig mesenteric arterioles.

1. Current clamp studies using two patch electrodes and morphological observations have been performed in guinea-pig mesenteric arterioles to evaluate intercellular electrical couplings. 2. In electron micrographs, preparations were found to have a single layer of smooth muscle cells. Typical gap junctions were readily observed between endothelial cells only. 3. While immunoreactivity to connexin 40 was strongly expressed on the membranes of endothelial cells only, that to connexin 43 was expressed on both smooth muscle and endothelial cell membranes. 4. Neurobiotin injected into a smooth muscle cell diffused into several neighbouring smooth muscle cells while that injected into an endothelial cell diffused into many endothelial cells. 5. Acetylcholine-induced hyperpolarizations were conducted from endothelial cells to smooth muscle cells with a relative amplitude of 80.1 %. Ba(2+)-induced action potentials were conducted in the opposite direction with a relative amplitude of 92.4 %. 6. An electrotonic potential produced in a smooth muscle cell by current injection diminished steeply with distance as it spread along the muscle layer, plateauing at distances beyond 25 microm. An electrotonic potential produced in an endothelial cell spread within the intima with virtually no reduction. Electrotonic potentials could conduct through myoendothelial couplings, which seemed to behave as ohmic resistors without rectification. 7. The coupling resistance between adjacent smooth muscle cells was estimated to be at least 90 MOhms and that between a smooth muscle cell and the whole endothelial layer to be 0.9 GOhms. 8. The results indicate that although the resistance of myoendothelial couplings is appreciable, the endothelium may be important as a low resistance path connecting many smooth muscle cells.

Animals↗

PubMed2004

3-D anatomically based dynamic modeling of the human knee to include tibio-femoral and patello-femoral joints.

An anatomical dynamic model consisting of three body segments, femur, tibia and patella, has been developed in order to determine the three-dimensional dynamic response of the human knee. Deformable contact was allowed at all articular surfaces, which were mathematically represented using Coons' bicubic surface patches. Nonlinear elastic springs were used to model all ligamentous structures. Two joint coordinate systems were employed to describe the six-degrees-of-freedom tibio-femoral (TF) and patello-femoral (PF) joint motions using twelve kinematic parameters. Two versions of the model were developed to account for wrapping and nonwrapping of the quadriceps tendon around the femur. Model equations consist of twelve nonlinear second-order ordinary differential equations coupled with nonlinear algebraic constraint equations resulting in a Differential-Algebraic Equations (DAE) system that was solved using the Differential/Algebraic System Solver (DASSL) developed at Lawrence Livermore National Laboratory. Model calculations were performed to simulate the knee extension exercise by applying non-linear forcing functions to the quadriceps tendon. Under the conditions tested, both "screw home mechanism" and patellar flexion lagging were predicted. Throughout the entire range of motion, the medial component of the TF contact force was found to be larger than the lateral one while the lateral component of the PF contact force was found to be larger than the medial one. The anterior and posterior fibers of both anterior and posterior cruciate ligaments, ACL and PCL, respectively, had opposite force patterns: the posterior fibers were most taut at full extension while the anterior fibers were most taut near 90 degrees of flexion. The ACL was found to carry a larger total force than the PCL at full extension, while the PCL carried a larger total force than the ACL in the range of 75 degrees to 90 degrees of flexion.

Computer Simulation↗

PubMed2004

Domains formed within the N-terminal region of the quorum-sensing activator TraR are required for transcriptional activation and direct interaction with RpoA from agrobacterium.

TraR, a quorum-sensing activator, induces transcription from its binding site, the tra-box, located upstream of Ti plasmid target promoters. TraR activated expression of a lacZ reporter in Escherichia coli only when RpoAAt from Agrobacterium tumefaciens was co-expressed. As assessed by gel retardation assays RpoAAt, but not RpoAEc, formed a ternary complex with TraR and a tra-box probe in vitro. TraR formed similar ternary complexes with alphaCTDAt but not with NTDAt, the C- and N-terminal segments of RpoAAt. As measured by surface plasmon resonance refractometry, TraR interacted directly with RpoAAt with an affinity about five times greater than that observed for its interaction with RpoAEc. The activator interacted with alphaCTDAt with kinetics and affinities similar to those of the full-sized -subunit. Positive control (PC) mutations at Asp-10 and Gly-123 of TraR did not affect DNA binding but greatly decreased the TraR-RpoAAt interaction. These two residues combine to form two patches on the activator, one of which may be involved in interaction with RpoA. When co-expressed, mutants of TraR with substitutions at Asp-10 complementing mutants with substitutions at Gly-123 for gene activation in an allele-specific manner. Co-expression studies with TraR and its PC mutants, and also with complementary PC alleles of TraR, coupled with three-dimensional structure are consistent with a hypothesis that both Asp-10/Gly-123 patches are required for activator function.

Agrobacterium tumefaciens↗

PubMed2004

Peptaibol zervamicin IIb structure and dynamics refinement from transhydrogen bond J couplings.

Zervamicin IIB (Zrv-IIB) is a channel-forming peptaibol antibiotic of fungal origin. The measured transhydrogen bond (3h)J(NC') couplings in methanol solution heaving average value of -0.41 Hz indicate that the stability of the Zrv-IIB helix in this milieu is comparable to the stability of helices in globular proteins. The N-terminus of the peptide forms an alpha-helix, whereas 3(10)-helical hydrogen bonds stabilize the C-terminus. However, two weak transhydrogen bond peaks are observed in a long-range HNCO spectrum for HN Aib(12). Energy calculations using the Empirical Conformation Energy Program for Peptides (ECEPP)/2 force field and the implicit solvent model show that the middle of the peptide helix accommodates a bifurcated hydrogen bond that is simultaneously formed between HN Aib(12) and CO Leu(8) and CO Aib(9). Several lowered (3h)J(NC') on a polar face of the helix correlate with the conformational exchange process observed earlier and imply dynamic distortions of a hydrogen bond pattern with the predominant population of a properly folded helical structure. The refined structure of Zrv-IIB on the basis of the observed hydrogen bond pattern has a small ( approximately 20 degrees ) angle of helix bending that is virtually identical to the angle of bending in dodecylphosphocholine (DPC) micelles, indicating the stability of a hinge region in different environments. NMR parameters ((1)HN chemical shifts and transpeptide bond (1)J(NC') couplings) sensitive to hydrogen bonding along with the solvent accessible surface area of carbonyl oxygens indicate a large polar patch on the convex side of the helix formed by three exposed backbone carbonyls of Aib(7), Aib(9), and Hyp(10) and polar side chains of Hyp(10), Gln(11), and Hyp(13). The unique structural features, high helix stability and the enhanced polar patch, set apart Zrv-IIB from other peptaibols (for example, alamethicin) and possibly underlie its biological and physiological properties.

Hydrogen Bonding↗

PubMed2006

Role of pairwise interactions between M1 and M2 domains of the nicotinic receptor in channel gating.

The adult form of the nicotinic acetylcholine receptor (AChR) consists of five subunits (alpha(2)betaepsilondelta), each having four transmembrane domains (M1-M4). The atomic model of the nicotinic acetylcholine receptor shows that the pore-lining M2 domains make no extensive contacts with the rest of the transmembrane domains. However, there are several sites where close appositions between segments occur. It has been suggested that the pair alphaM1-F15' and alphaM2-L11' is one of the potential interactions between segments. To determine experimentally if these residues are interacting and to explore if this interhelical interaction is essential for channel gating, we combined mutagenesis with single-channel kinetic analysis. Mutations in alphaM1-F15' lead to profound changes in the opening rate and slighter changes in the closing rate. Channel gating is impaired as the volume of the residue increases. Rate-equilibrium linear free-energy relationship analysis reveals an approximately 70% open-state-like environment for alphaM1-F15' at the transition state of the gating reaction, suggesting that it moves early during the gating process. Replacing the residue at alphaM1-15' by that at alphaM2-11' and vice versa profoundly alters gating, but the combination of the two mutations restores gating to near normal, indicating that alphaM1-F15' and alphaM2-L11' are interchangeable. Double-mutant cycle analysis shows that these residues are energetically coupled. Thus, the interaction between M1 and M2 plays a key role in channel gating.

Acetylcholine↗

PubMed1995

The control of calcium release in heart muscle.

The control of calcium release from intracellular stores (the sarcoplasmic reticulum) in cardiac muscle was examined with the use of a confocal microscope and voltage clamp techniques. Depolarization evoked graded calcium release by altering the extent of spatial and temporal summation of elementary calcium release events called "calcium sparks." These evoked sparks were triggered by local L-type calcium channel currents in a stochastic manner, were similar at different potentials, and resembled spontaneous calcium sparks. Once triggered, the calcium release from the sarcoplasmic reticulum during a calcium spark was independent of the duration of the triggering calcium influx. These results were used to develop a unifying model for cardiac excitation-contraction coupling that explains the large (but paradoxically stable) amplification of the trigger calcium influx by a combination of digital and analog behavior.

Animals↗

PubMed2005

Ca(2+)- and volume-sensitive chloride currents are differentially regulated by agonists and store-operated Ca2+ entry.

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca(2+)-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca(2+)-activated (I(Cl(Ca))) and volume-regulated (I(Cl, swell)) chloride currents. NPPB and DIDS more efficiently inhibited I(Cl(Ca)) and I(Cl, swell), respectively. Cell swelling caused by hypotonic solution invariably activated I(Cl, swell) while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling-induced I(Cl, swell), while its inactive analogue U-73343 had no effect. I(Cl(Ca)) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, I(Cl, swell) could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced I(Cl, swell) in a nonadditive manner, suggesting their convergence on a common pathway. I(Cl, swell) and I(Cl(Ca)) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated I(Cl(Ca)), but abolished I(Cl, swell), thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that I(Cl, swell) can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a "selection" switch between closely localized channels.

Adenosine Triphosphate↗

PubMed2004

Cultured neurons coupled to microelectrode arrays: circuit models, simulations and experimental data.

The purpose of this paper is to characterize the neuron-microelectrode junction, based on the equivalent electric-circuit approach. As a result, recording of action potentials can be simulated with a general-purpose circuit simulation program such as HSPICE. The response of the microelectrode was analyzed as a function of parameters such as sealing resistance and adhesion conditions. The models of the neuron and microelectrode implemented in HSPICE were first described. These models were used to simulate the behavior of the junction between a patch of neuronal membrane (described by the compartmental model) and a microelectrode.

Action Potentials↗

PubMed2005

Estrogen modulation of hypothalamic neurons: activation of multiple signaling pathways and gene expression changes.

Hypothalamic target neurons of estrogen include neurosecretory neurons such as gonadotropin-releasing hormone (GnRH) and dopamine neurons, and local circuitry neurons such as proopiomelanocortin (POMC) and gamma-aminobutyric acid (GABA) neurons. These and other hypothalamic neurons are involved in regulating numerous homeostatic functions including reproduction, thermoregulation, stress responses, feeding and motivated behaviors. Using a combination of techniques to examine the molecular mechanisms leading to physiological changes induced by estrogen, we find that both rapid effects and transcriptional changes alter excitability of hypothalamic neurons. We have identified membrane-initiated, rapid signaling pathways through which 17beta-estradiol (E2) alters synaptic responses in these neurons using whole-cell patch recording in hypothalamic slices from ovariectomized female guinea pigs. E2 rapidly uncouples mu-opioid and GABA(B) receptors from G protein-gated inwardly rectifying K+ (GIRK) channels in POMC and dopamine neurons as manifested by a reduction in the potency of mu-opioid and GABA(B) receptor agonists to activate these channels. Inhibitors of phospholipase C, protein kinase C and protein kinase A block the actions of E2, indicative that the E2 receptor is G protein-coupled to activation of this cascade. Taking advantage of an animal model we developed to investigate estrogen's feedback actions on secretion of gonadotropin-releasing hormone (GnRH), we studied the transcriptional changes induced by estrogen using suppression subtractive hybridization (SSH) and microarray analysis. Many of the observed mRNA expression changes include transcripts encoding proteins critical for neurotransmitter release and receptor dynamics. Some of these include gec-1, PI3-kinase p55gamma, rab11a GTPase, synaptobrevin2, synaptogyrin, taxilin, Ca2+-dependent activator protein for secretion (CAPS) and a number of proteins containing pleckstrin homology domains-domains that are involved in plasma membrane targeting of their host protein. In situ hybridization and quantitative film autoradiography analysis on selected transcripts show differential distribution and expression in hypothalamic nuclei. Furthermore, single-cell PCR analysis reveals these genes to be expressed in neurons such as POMC (and GnRH). Whether these expression changes are mediated by the classical or membrane estrogen receptors has yet to be delineated. More detailed investigations of transcript spatial localization within neurons and their temporal expression, i.e., within minutes or hours, will provide more insight regarding how estrogen alters neuronal excitability and synaptic efficacy that ultimately lead to changes in complex behavior.

Animals↗

PubMed1998

Functional interaction between InsP3 receptors and store-operated Htrp3 channels.

Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (I(crac)). A critical question in cell signalling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).

Animals↗

PubMed1995

Thermodynamic determination of the Na+: glucose coupling ratio for the human SGLT1 cotransporter.

Phlorizin-sensitive currents mediated by a Na-glucose cotransporter were measured using intact or internally perfused Xenopus laevis oocytes expressing human SGLT1 cDNA. Using a two-microelectrode voltage clamp technique, measured reversal potentials (Vr) at high external alpha-methylglucose (alpha MG) concentrations were linearly related to In[alpha MG]o, and the observed slope of 26.1 +/- 0.8 mV/decade indicated a coupling ratio of 2.25 +/- 0.07 Na ions per alpha MG molecule. As [alpha MG]o decreased below 0.1 mM, Vr was no longer a linear function of In[alpha MG]o, in accordance with the suggested capacity of SGLT1 to carry Na in the absence of sugar (the "Na leak"). A generalized kinetic model for SGLT1 transport introduces a new parameter, Kc, which corresponds to the [alpha MG]o at which the Na leak is equal in magnitude to the coupled Na-alpha MG flux. Using this kinetic model, the curve of Vr as a function of In[alpha MG]o could be fitted over the entire range of [alpha MG]o if Kc is adjusted to 40 +/- 12 microM. Experiments using internally perfused oocytes revealed a number of previously unknown facets of SGLT1 transport. In the bilateral absence of alpha MG, the phlorizin-sensitive Na leak demonstrated a strong inward rectification. The affinity of alpha MG for its internal site was low; the Km was estimated to be between 25 and 50 mM, an order of magnitude higher than that found for the extracellular site. Furthermore, Vr determinations at varying alpha MG concentrations indicate a transport stoichiometry of 2 Na ions per alpha MG molecule: the slope of Vr versus In[alpha MG]o averaged 30.0 +/- 0.7 mV/decade (corresponding to a stoichiometry of 1.96 +/- 0.04 Na ions per alpha MG molecule) whenever [alpha MG]o was higher than 0.1 mM. These direct observations firmly establish that Na ions can utilize the SGLT1 protein to cross the membrane either alone or in a coupled manner with a stoichiometry of 2 Na ions per sugar, molecule.

Animals↗

PubMed2002

Hedgehog signalling: pulling apart patched and smoothened.

Two integral membrane proteins, Patched and Smoothened, were for a long time thought to comprise a preformed receptor complex for secreted Hedgehog signalling proteins. Recent analyses of the subcellular distribution of these proteins argue strongly against this simple model.

Animals↗

PubMed1999

Bursting in inhibitory interneuronal networks: A role for gap-junctional coupling.

Much work now emphasizes the concept that interneuronal networks play critical roles in generating synchronized, oscillatory behavior. Experimental work has shown that functional inhibitory networks alone can produce synchronized activity, and theoretical work has demonstrated how synchrony could occur in mutually inhibitory networks. Even though gap junctions are known to exist between interneurons, their role is far from clear. We present a mechanism by which synchronized bursting can be produced in a minimal network of mutually inhibitory and gap-junctionally coupled neurons. The bursting relies on the presence of persistent sodium and slowly inactivating potassium currents in the individual neurons. Both GABAA inhibitory currents and gap-junctional coupling are required for stable bursting behavior to be obtained. Typically, the role of gap-junctional coupling is focused on synchronization mechanisms. However, these results suggest that a possible role of gap-junctional coupling may lie in the generation and stabilization of bursting oscillatory behavior.

Action Potentials↗

PubMed2004

Proximal renal tubular acidosis in TASK2 K+ channel-deficient mice reveals a mechanism for stabilizing bicarbonate transport.

The acid- and volume-sensitive TASK2 K+ channel is strongly expressed in renal proximal tubules and papillary collecting ducts. This study was aimed at investigating the role of TASK2 in renal bicarbonate reabsorption by using the task2 -/- mouse as a model. After backcross to C57BL6, task2 -/- mice showed an increased perinatal mortality and, in adulthood, a reduced body weight and arterial blood pressure. Patch-clamp experiments on proximal tubular cells indicated that TASK2 was activated during HCO3- transport. In control inulin clearance measurements, task2 -/- mice showed normal NaCl and water excretion. During i.v. NaHCO3 perfusion, however, renal Na+ and water reabsorption capacity was reduced in -/- animals. In conscious task2 -/- mice, blood pH, HCO3- concentration, and systemic base excess were reduced but urinary pH and HCO3- were increased. These data suggest that task2 -/- mice exhibit metabolic acidosis caused by renal loss of HCO3-. Both in vitro and in vivo results demonstrate the specific coupling of TASK2 activity to HCO3- transport through external alkalinization. The consequences of the task2 gene inactivation in mice are reminiscent of the clinical manifestations seen in human proximal renal tubular acidosis syndrome.

Acidosis, Renal Tubular↗

PubMed1990

Coupling of K(+)-gating and permeation with Ca2+ block in the Ca2(+)-activated K+ channel in Chara australis.

The patch-clamp technique is used here to investigate the kinetics of Ca2+ block in single high-conductance Ca2(+)-activated K+ channels. These channels are detected in the membrane surrounding cytoplasmic drops from Chara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (-300 to 200 mV). Ca2+ on either side of the channel reduces its K+ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca2(+)-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca2+ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca2+ indicates that (i) external Ca2+ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca2+ or K+, and (iii) the kinetics of Ca2+ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca2+ indicates that more than one Ca2+ is involved.

Calcium↗