Monoexponential Time Course Research Articles

Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models.

TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 μM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o <30mM TMEM16A current activates with a monoexponential time course, but above 30mM, [Cl(-)]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl(-) does not alter the apparent Ca(2+) affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl(-) acts by stabilizing the open configuration induced by Ca(2+) and by contributing to the Vm dependence of activation.

Slower Piezo1 Inactivation in Dehydrated Hereditary Stomatocytosis (Xerocytosis)

Slower Piezo1 Inactivation in Dehydrated Hereditary Stomatocytosis (Xerocytosis)

Acute desensitization of acetylcholine and endothelin-1 activated inward rectifier K+ current in myocytes from the cardiac atrioventricular node

The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits ‘fade’ or desensitization in the AVN, as established for the heart’s primary pacemaker – the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37°C. 0.1–1μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0s and τs 76.9s at −120mV; 1μM ACh). 10nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6s at −120mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization.

Voltage-Dependent Modulation of α2A Adrenergic Receptor Conformations

G protein-coupled receptors (GPCRs) are proteins that span the cell membrane seven times. They are stimulated by extracellular agonists and activate heterotrimeric G proteins to elicit intracellular responses. Recent evidence suggests that GPCRs are voltage-sensitive: They exhibit gating currents similar to ion channels and respond to depolarization of the plasma membrane with changes in agonist affinity. We used a FRET-based biosensor of the α2A adrenergic receptor to analyze voltage dependence of receptor activation in HEK 293 cells by means of voltage-clamp recording. The biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (NE) and receptor activation was measured as decrease in FRET ratio. Receptor stimulation by NE was reduced by membrane depolarization and enhanced by hyperpolarization. This effect was present in wilde-type receptors and transduced to the level of G protein activation, which we determined in a FRET assay that directly detects Gαi protein activation. Depolarization-mediated inhibition of NE activated receptors was strong at low concentrations (500 nM: 60 % inhibition) but almost undetectable at saturating agonist concentrations (100 μM: 9 % inhibition). Both agonist-induced and hyperpolarization-induced receptor activation exhibited a similar monoexponential time course. For both activation modes, speed of activation was primarily dependent on agonist concentration, indicating that depolarization lowers the apparent affinity of the NE receptor interaction and thus causes receptor deactivation by means of NE release. Application of clonidine (1 μM, VM=-90 mV) resulted in a FRET response that was inhibited by 40 % at +60 mV. In contrast to NE, strong receptor-inhibition at +60 mV was present even at super-saturating concentrations of clonidine (100 μM). Therefore we conclude that negative membrane potentials promote active conformations of the α2A adrenergic receptor, increase affinity of full agonists and enhance G protein signaling.

Effects Of Old Age And Physical Activity On Oxidative Capacity In Human Skeletal Muscle

Studies of the effects of old age on muscle oxidative capacity have led to contradictory results. These discrepancies may be due in part to muscle-specific differences in the effects of altered physical activity patterns in the elderly. PURPOSE: To determine if training status has differential effects on oxidative capacity in vivo in two lower limb muscles, tibialis anterior (TA) and vastus lateralis (VL), in young and older men. METHODS: We used phosphorus magnetic resonance spectroscopy (MRS) to measure muscle oxidative capacity non-invasively in young (n=8, 25±4 yrs, mean±SD) and older (n=8, 69±4) healthy, untrained men; and young (n=7, 28±3) and older (n=3, 71±8) male runners. In separate trials, subjects performed a 16-s and a 24-s maximal voluntary isometric contraction of the TA and VL, respectively, while phosphocreatine (PCr) was measured using a 4 tesla MRS system. Resynthesis of [PCr] followed a mono-exponential time course with a rate constant, kPCr, that is attributable to ATP synthesis from oxidative phosphorylation. A three-way (group, muscle, age) ANOVA was applied. RESULTS: There was a main effect of group such that kPCr was higher in trained than untrained men (p<0.001). There was also an age-by-muscle interaction such that kPCr was greater in young compared to older men in VL (p=0.009), but not in TA, regardless of training status.TableCONCLUSION: The decrease in oxidative capacity observed in the VL but not the TA of both older groups provides evidence of a muscle-specific effect of aging that is independent of training status. These results also confirm greater muscle oxidative capacity in vivo in two muscles of the lower limb in trained compared to untrained men. NIH/NIA R01 AG21094, K02 023582; ACSM Student Grant (RGL)

External Ba 2+ Block of Human Kv1.5 at Neutral and Acidic pH: Evidence for H o+-Induced Constriction of the Outer Pore Mouth at Rest

Previous studies have shown that low pH o accelerates depolarization-induced inactivation and decreases the macroscopic conductance by reducing channel availability. To test the hypothesis that outer pore constriction underlies the decreased conductance at low pH o, external Ba 2+ was used to examine the accessibility of the channel pore at rest under neutral and acidic conditions. At pH o 7.4, Ba 2+ block of closed channels follows a monoexponential time course and involves a low-affinity superficial site ( K D ≅ 1 mM, −80 mV, 0 mM K o + ) and a high-affinity site ( K D ≅ 4 μM) deeper in the pore. Depolarization promotes Ba 2+ dissociation and an analytical model incorporating state-dependent changes of Ba 2+ affinity is presented that replicates the frequency dependence of the time course and the extent of block. Open-channel block by Ba 2+ is weak. At pH o 5.5, both the access to and exit from the deep site is inhibited. These results are consistent with a low-pH o-induced conformational change in the outer pore that prevents Ba 2+ binding at rest or unbinding during depolarization. If a pore constriction is involved, similar to that proposed to occur during P/C-type inactivation, this would imply that closed-state inactivation in Kv1.5 occurs under acidic conditions.

Adaptive response of pulmonary arterial smooth muscle to length change

Hypervasoconstriction is associated with pulmonary hypertension and dysfunction of the pulmonary arterial smooth muscle (PASM) is implicated. However, relatively little is known about the mechanical properties of PASM. Recent advances in our understanding of plastic adaptation in smooth muscle may shed light on the disease mechanism. In this study, we determined whether PASM is capable of adapting to length changes (especially shortening) and regain its contractile force. We examined the time course of length adaptation in PASM in response to step changes in length and to length oscillations mimicking the periodic stretches due to pulsatile arterial pressure. Rings from sheep pulmonary artery were mounted on myograph and stimulated using electrical field stimulation (12-16 s, 20 V, 60 Hz). The length-force relationship was determined at L(ref) to 0.6 L(ref), where L(ref) was a reference length close to the in situ length of PASM. The response to length oscillations was determined at L(ref), after the muscle was subjected to length oscillation of various amplitudes for 200 s at 1.5 Hz. Release (or stretch) of resting PASM from L(ref) to 0.6 (and vice versa) was followed by a significant force recovery (73 and 63%, respectively), characteristic of length adaptation. All recoveries of force followed a monoexponential time course. Length oscillations with amplitudes ranging from 5 to 20% L(ref) caused no significant change in force generation in subsequent contractions. It is concluded that, like many smooth muscles, PASM possesses substantial capability to adapt to changes in length. Under pathological conditions, this could contribute to hypervasoconstriction in pulmonary hypertension.

A fast transient outward current in layer II/III neurons of rat perirhinal cortex

The perirhinal cortex (PRC) is a supra-modal cortical area that collects and integrates information originating from uni- and multi-modal neocortical regions, transmits it to the hippocampus, and receives a feedback from the hippocampus itself. The elucidation of the mechanisms that underlie the specific excitable properties of the different PRC neuronal types appears as an important step toward the understanding of the integrative functions of PRC. In this study, we investigated the biophysical properties of the transient, I (A)-type K(+) current recorded in pyramidal neurons acutely dissociated from layers II/III of PRC of the rat (P8-P16). The current activated at about -50 mV and showed a fast monoexponential decay (tau(h) >> 14 ms at -30 to +10 mV). I (A) recovery from inactivation also had a monoexponential time course. No significant differences in the biophysical properties or current density of I (A) were found in pyramidal neurons from rats of different ages. Application of 4-AP (1-5 mM) reversibly and selectively blocked I (A), and in current clamp conditions it increased spike duration and shortened the delay of the first spike during repetitive firing evoked by sustained depolarizing current injection. These properties are similar to those of the I (A) found in thalamic neurons and other cortical pyramidal neurons. Our results suggest that I (A) contributes to spike repolarization and to regulate both spike onset timing and firing frequency in PRC neurons.

Kinetic properties of human AMPA-type glutamate receptors expressed in HEK293 cells.

AMPA-type glutamate receptors (AMPAR) display a high variability in functional properties, which determine the time course of excitatory postsynaptic potentials. They are assembled as tetramers of GluR subunits 1-4 of different splice variants and nuclear edited isoforms. Presently, the kinetics of activation, desensitization and recovery from desensitization of human AMPARs (GluR1, 3 and 4 flip and flop, and GluR2 flip and flop in R and G edited forms, respectively) transiently expressed in HEK293 cells were studied with patch-clamp techniques and ultra fast agonist application. Activation time constants were identical for all receptors (0.13 ms). The GluR2 flip G variant showed the slowest desensitization (10.8 ms), GluR4 flip the fastest (1.6 ms). Recovery from desensitization varied between 3.1 ms (GluR4 flip) and 178 ms (GluR1 flip). To determine functional interactions between subunits in heteromeric receptors the GluR1 flip and the GluR2 flip R were coexpressed. The time constant of desensitization increased linearly from 2.5 ms (GluR1 flip homomers) to 6.8 ms (GluR2 flip R homomers) with the amount of GluR2 flip R cDNA transfected. Recovery followed a monoexponential time course and had a time constant of 178 ms in GluR1 flip homomeric expression. In all GluR1 flip/GluR2 flip heteromers and in GluR2 flip R homomers desensitization recovered with a time constant of approximately 50 ms. Thus, subunit interaction seems likely during recovery but not desensitization. Both parameters might influence the ability of AMPA receptors to mediate glutamate induced chronic excitotoxicity in neurodegenerative diseases.

Relationships between mechanical power, O(2) consumption, O(2) deficit and high-energy phosphates during calf exercise in humans.

Whole-body O(2) uptake ( VO(2)), O(2) deficit and the concentration of high-energy phosphates (determined by (31)P spectroscopy) in human calf muscle were measured during moderate aerobic square-wave exercise of increasing intensity in ten volunteers. Net VO(2) (above resting) increased linearly with mechanical power, yielding a delta efficiency of 13.1%. "Gross" O(2) deficit increased linearly with net VO(2). The fraction of phosphocreatine (PC) split at steady state increased linearly with the mechanical power and with the O(2) deficit. If the [PC] in resting muscle is known, the slope of the regression between PC split and O(2) deficit (in millimoles) yields the P/O(2) ratio. To calculate this, the O(2) deficit was corrected for the amount of O(2) derived from the body stores, as obtained from literature data. The value so obtained, for a resting [PC] of 30 mM was 5.9, consistent with canonical textbook values. Furthermore, the ratio of "true" O(2) deficit to steady-state VO(2) is a measure of the time constant of VO(2) kinetics at work onset at the muscle level: assuming a monoexponential time course without time delays it amounted to about 17 s, close to the value that can be expected in mammalian muscle at 37 degrees C.

Time course of functional recovery after coronary artery bypass graft surgery in patients with chronic left ventricular ischemic dysfunction

Chronic left ventricular (LV) ischemic dysfunction, a condition often referred to as myocardial hibernation, is associated in humans with ultrastructural alterations of the myocytes, including the loss of myofilaments and the accumulation of glycogen. Given the severity of these structural changes, contractile function is unlikely to resume immediately upon revascularization. Therefore, the aim of the present study was to assess the time course of functional improvement after successful revascularization as well as its potential structural correlates. We studied 32 patients with coronary disease and chronic LV ischemic dysfunction who underwent bypass surgery. Dynamic positron emission tomography with N-13 ammonia and F-18 deoxyglucose to assess myocardial perfusion and glucose metabolism was performed in 29 patients. In all patients, a transmural biopsy was harvested from the center of the dysfunctional area, to quantify the increase in extracellular matrix and the presence of structurally altered cardiomyocytes. LV function was serially measured by digitized 2-dimensional echocardiography before and at 10 days, 2 months, and 6 months after revascularization. The time course of recovery of regional function was estimated from the monoexponential decrease in dysfunctional wall motion score. At follow-up, 19 patients had improved LV function, whereas 13 patients showed persistent dysfunction. Before revascularization, reversibly dysfunctional segments had higher myocardial blood flow (82 ± 29 vs 53 ± 21 ml · (min · 100 g)−1, p = 0.044), higher glucose uptake (40 ± 16 vs 21 ± 9 μmol · (min · 100 g)−1, p = 0.001), and less increase in extracellular matrix (25 ± 15% vs 46 ± 17%, p = 0.0008) than segments with persistent dysfunction. The extent to which function recovered was positively correlated with myocardial blood flow and negatively correlated with the increase in the extracellular matrix. In patients with reversible dysfunction, the return of segmental function was progressive and followed a monoexponential time course with a median time constant of 23 days (range 6 to 78). The rate of recovery correlated best with the proportion of altered cardiomyocytes in the biopsy. The present study thus indicates that the recovery of regional and global LV function after successful revascularization is progressive and follows a monoexponential time course that is influenced by the extent of the structural changes affecting cardiomyocytes.

Effects of cationic substitutions on delayed rectifier current in type I vestibular hair cells.

The resting potassium current (I(KI)) in gerbil dissociated type I vestibular hair cells has been characterized under various ionic conditions in whole cell voltage-clamp. When all K(+) in the patch electrode solution was replaced with Na(+), (Na(+))(in) or Cs(+), (Cs(+))(in), large inward currents were evoked in response to voltage steps between -90 and -50 mV. Activation of these currents could be described by a Hodgkin-Huxley-type kinetic scheme, the order of best fit increasing with depolarization. Above approximately -40 mV currents became outward and inactivated with a monoexponential time course. Membrane resistance was inversely correlated with external K(+) concentration. With (Na(+))(in), currents were eliminated when K(+) was removed from the external solution or following extracellular perfusion of 4-aminopyridine, indicating that currents flowed through I(KI) channels. Also, reduction of K(+) entry through manipulation of membrane potential reduced the magnitude of the outward current. Under symmetrical Cs(+), 0 K(+) conditions I(KI) is highly permeable to Cs(+). However, inward currents were reduced when small amounts of external K(+) were added. Higher concentrations of K(+) resulted in larger currents indicating an anomalous mole fraction effect in mixtures of external Cs(+) and K(+).

Enhancement of retention and cytotoxicity of 2-chlorodeoxyadenosine in cultured human leukemic lymphoblasts by nitrobenzylthioinosine, an inhibitor of equilibrative nucleoside transport.

In leukemic cells exposed to 2-chlorodeoxyadenosine (2-CdA), levels of the nucleoside drug and its phosphate metabolites decay with time in the absence of external 2-CdA; an intrinsic part of this process is the efflux of 2-CdA. The effects of nitrobenzylthioinosine (NBMPR) and of dipyridamole (DPM), both potent inhibitors of es (e, equilibrative; s, sensitive to NBMPR) nucleoside transport processes, were studied in four lines of cultured leukemic lymphoblasts. Suspensions of 2-CdA-loaded cells were diluted 10-fold with 2-CdA-free medium to initiate the cellular 2-CdA decay processes, which followed a biexponential time course. When diluting media contained NBMPR or DPM, intracellular levels of 2-CdA and its metabolites were substantially increased (P < 0.001) compared with cells in media lacking the transport inhibitors, and 2-CdA loss followed a monoexponential time course. As a consequence, the AUCs (area under time-course plots of intracellular 2-CdA and its metabolites) were significantly (P < 0.001) lower in untreated control cells compared to inhibitor-treated cells. These results suggest that nucleoside transport processes contribute to the efflux of 2-CdA from the cultured lymphoblasts. The cytotoxicity of 1-h exposure to 2-CdA of Reh-A2 and CCRF-CEM cells was enhanced three-fold by subsequent exposure to 0.5 microM NBMPR relative to that of control cells subjected to the same manipulations without NBMPR exposure. However, before such a strategy may be considered to have a therapeutic application, careful examination of effects in normal lymphocytes and ex vivo leukemic lymphoblasts must first be undertaken. Leukemia (2000) 14, 52-60.

Identification by microscopically controlled comet assay of peritoneal macrophages in a mixture of peritoneal exudate for DNA strand break analysis

A simple modification of the alkaline comet assay allows the study of DNA damage in a specific cell type in a mixture of primary cells. Peritoneal macrophages from mice are selected from other peritoneal exudate cells without complex preparation and separation steps by their size and shape of the nuclei and their comets. The DNA damage can be well characterised by the manually monitored parameter `tail length'. Complex measurement of the `tail moment', often used for characterising DNA damage is not required, a fact which further simplifies the protocol. The distribution of tail length within one sample is symmetric and can be described by a Gaussian distribution and the mean tail length. As a first application, UV-A sensitivity of resident and stimulated macrophages was studied. The resident macrophages were more sensitive to UV-A than the stimulated ones. DNA damage repair follows the same simple monoexponential time course for both cell types. The simplicity of results, i.e., applicability of tail lengths and Gaussian statistics as well as monoexponential kinetics, suggest that microscopically controlled comet assay is well suited to study elementary processes of DNA damage induction and repair.

Effects of local anesthetics on Na+ channels containing the equine hyperkalemic periodic paralysis mutation.

We examined the ability of local anesthetics to correct altered inactivation properties of rat skeletal muscle Na+ channels containing the equine hyperkalemic periodic paralysis (eqHPP) mutation when expressed in Xenopus oocytes. Increased time constants of current decay in eqHPP channels compared with wild-type channels were restored by 1 mM benzocaine but were not altered by lidocaine or mexiletine. Inactivation curves, which were determined by measuring the dependence of the relative peak current amplitude after depolarization to -10 mV on conditioning prepulse voltages, could be shifted in eqHPP channels back toward that observed for wild-type (WT) channels using selected concentrations of benzocaine, lidocaine, and mexiletine. Recovery from inactivation at -80 mV (50-ms conditioning pulse) in eqHPP channels followed a monoexponential time course and was markedly accelerated compared with wild-type channels (tauWT = 10.8 +/- 0.9 ms; taueqHPP = 2.9 +/- 0.4 ms). Benzocaine slowed the time course of recovery (taueqHPP,ben = 9.6 +/- 0.4 ms at 1 mM) in a concentration-dependent manner. In contrast, the recovery from inactivation with lidocaine and mexiletine had a fast component (taufast,lid = 3.2 +/- 0.2 ms; taufast,mex = 3.1 +/- 0.2 ms), which was identical to the recovery in eqHPP channels without drug, and a slow component (tauslow,lid = 1,688 +/- 180 ms; tauslow,mex = 2,323 +/- 328 ms). The time constant of the slow component of the recovery from inactivation was independent of the drug concentration, whereas the fraction of current recovering slowly depended on drug concentrations and conditioning pulse durations. Our results show that local anesthetics are generally incapable of fully restoring normal WT behavior in inactivation-deficient eqHPP channels.

Pre-steady State of Reaction of Nucleoside Diphosphate Kinase with Anti-HIV Nucleotides

The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addition according to a monoexponential time course. The pseudo-first order rate constant was determined for different concentrations of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast association of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate constants and dissociation equilibrium constants determined for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymatic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s-1 and a KS of 1-5 mM. The equilibrium dissociation constants for ADP, GDP, ddADP, and ddGDP were also determined by fluorescence titration of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3'-OH in ddNTP causes a 10-fold increase in KD. Contrary to "natural" NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in kcat values.

Dynamics of viscoelastic properties of rat cardiac sarcomeres during the diastolic interval: involvement of Ca2+.

1. Cardiac sarcomere stiffness was investigated during diastole in eighteen trabeculae dissected from the right ventricle of rat heart. The trabeculae were stimulated at 0.5 Hz, in a modified Krebs-Henseleit solution (pH, 7.4; 25 degrees C). Sarcomere length (SL) was measured using high resolution (+/-2 nm) laser diffraction techniques. Force (F) was measured with a silicon strain gauge. 2. SL increased exponentially (amplitude, 25 +/- 9 nm; n = 15) throughout diastole. This increase occurred even at slack SL, showing that this phenomenon was due to an internal expansion. The majority of the muscles showed discrete spontaneous fluctuations of SL (amplitude < 20 nm) starting approximately 1 s after the end of the twitch. 3. The intracellular free Ca2+ concentration ([Ca2+]i) was measured from the fluorescence of microinjected fura-2 salt in seven trabeculae under the same experimental conditions. [Ca2+]i continuously declined (from 240 to 90 nM) during diastole following a monoexponential time course (time constant, 210-325 ms). 4. The stiffness of the sarcomere was evaluated at 10, 30, 50, 70 and 90% of diastole using bursts (30 ms) of 500 Hz sinusoidal perturbations of muscle length (amplitude of SL oscillations < 30 nm). At 1 nM external Ca2+ concentration ([Ca2+]o), the average stiffness modulus (Mod) increased from 9.3 +/- 0.6 to 12 +/- 0.6 nN mm-2 micron-1 (n = 18; P < 0.05), while the average phase shift (phi) between F and SL signals decreased from 84 +/- 3 to 73 +/- 4 deg (n = 18; P < 0.05) between 10 and 90% during diastole. The increase in Mod and the decrease in phi reversed when spontaneous activity occurred. When [Ca2+]o was raised to 2 mM, the stiffness time course reversed approximately 450 ms earlier, simultaneously with the occurrence of spontaneous activity. 5. Our results show that diastole is only an apparent steady state and suggest that the structural system responsible for the viscoelastic properties of the sarcomere is regulated by [Ca2+]i in the submicromolar range. Different possible origins of the dynamic changes in viscoelasticity during diastole are discussed.

Acetylcholine-induced K+ currents in smooth muscle cells of intact rat small arteries.

1. The mechanism of the sustained acetylcholine-induced endothelium-dependent hyperpolarization (EDH) in intact rat small mesenteric arteries prestimulated with noradrenaline (10(-6) M) was investigated by means of the single microelectrode voltage-clamp method. 2. The vascular smooth muscle cells (VSMCs) in this preparation are poorly or even not coupled for the reasons that: (1) the mean input resistance Rlnp of the clamped vascular smooth muscle increases from 120 M omega under control conditions to 440 M omega after application of K+ channel blocking drugs, (2) the voltage relaxation after injection of hyperpolarizing currents has a monoexponential time course and is linearly dependent on Rlnp, and (3) voltage steps induced by current-clamp steps are not transferred to locations in the vascular musculature 120 microns apart from the current injecting microelectrode. 3. Sustained (> 5 min) application of ACh (10(-5) M) hyperpolarized the VSMCs by induction of a hyperpolarizing current. This effect was completely blocked by the inhibitor of the nitric oxide (NO) synthase L-NAME (10(-3) M) but not by the inhibitor of the soluble guanylate cyclase (sGCl) Methylene Blue (MB, 10(-4) M). 4. Application of the NO donor sodium nitroprusside (SNP, 10(-6) M) for more than 5 min mimicked the induction of the endothelium-dependent hyperpolarizing current in vessels with destroyed endothelium. The reversal potential of this current is dependent on the extracellular K+ concentration. The effect of SNP could also not be blocked by MB. 5. The blockers of ATP-dependent and Ca(2+)-dependent K+ channels, glibenclamide (Glb, 10(-5) M) and charybdotoxin (CTX, 5 x 10(-8) M), respectively, blocked a hyperpolarizing current in the VSMCs similar to the ACh- or SNP-induced current. 6. The isolated application of either Glb or CTX did not block the activation of the hyperpolarizing current by SNP. Only the combined administration of Glb and CTX blocked the SNP-induced current completely. 7. Our results suggest that in rat small mesenteric artery, ACh hyperpolarizes the VSMCs tonically by activating both ATP- and Ca(2+)-dependent K+ currents, only via release of NO from the endothelium without need for activation of the sGCl.

Interaction between low voltage-activated currents in reticular thalamic neurons in a rat model of absence epilepsy.

A transient potassium (K+) outward current (IA) contributes to the distinctive patterns of low-threshold spike firing observed in various classes of thalamic neurons through a functional interaction with a calcium (Ca2+)-mediated inward current (IT). The present study was undertaken to investigate the properties of transient K+ currents and their interaction with IT in neurons of the reticular thalamic nucleus, and to compare these properties in reticular thalamic nucleus neurons from a rat model of absence epilepsy, designated the Genetic Absence Epilepsy Rat from Strasbourg (GAERS), with those from a Non-epileptic Control strain (NEC). This comparative approach appeared to be particularly important in view of the recent finding of a selective increase in IT in reticular thalamic nucleus neurons from GAERS. Neurons were acutely isolated from the reticular thalamic nucleus through enzymatic procedures, and identified by morphological and immunocytochemical criteria. Ionic currents were analysed using whole-cell patch-clamp techniques. Transient K+ currents in reticular thalamic nucleus neurons with properties indicative of IA activated at approximately -55 mV (with half-activation at -27 and -33 mV in NEC and GAERS respectively), declined rapidly with a voltage-dependent time constant (tau = 4 ms at +45 mV), were 50% steady-state-inactivated at -81 and -86 mV in the two strains of rats respectively, and rapidly recovered from inactivation with a monoexponential time course (tau = 31 and 37 ms respectively). No significant differences in IA properties or densities were found between reticular thalamic nucleus neurons from GAERS and NEC rats. Analysis of the interaction between IA and IT indicated a shift in the balance between the two opposing membrane conductances towards the generation of a low-voltage-activated inward current in reticular thalamic nucleus neurons from GAERS compared with NEC, and a lack of IA to functionally compensate for this shift, which in turn may contribute to pathological forms of low-threshold spike firing characterizing spike-and-wave discharges.

Kinetics of slow inactivation of persistent sodium current in layer V neurons of mouse neocortical slices.

1. In whole cell recordings from layer V neurons in slices of mouse somatosensory neocortex, tetrodotoxin (TTX)-sensitive persistent Na+ current (INaP) was studied by blocking K+ currents with intracellular Cs+ and Ca2+ currents with extracellular Cd2+. During slow voltage ramps, INaP began to activate at around -60 mV, and attained a peak at around -25 mV. The peak amplitude of INaP varied widely from cell to cell (range 60-3,160 pA; median 308 pA, n = 77). At potentials more positive than -35 mV, INaP in all cells was superimposed on a large, TTX-resistant outward current. 2. In hybrid clamp experiments, INaP was significantly reduced by a preceding high-frequency train of spikes. 3. INaP underwent pronounced slow inactivation, which was revealed by systematically varying the ramp speed between 233 and 2.33 mV/s, or varying the duration of a depolarizing prepulse between 0.1 and 10 s. 4. Onset of slow inactivation at +20 mV was monoexponential with tau = 2.06 s (n = 17 cells). Recovery from slow inactivation was voltage dependent. It followed a monoexponential time course with tau = 2.31 s (n = 6) at -70 mV and tau = 1.10 s (n = 4) at -90 mV. These values are not significantly different than values previously reported for slow inactivation of fast-inactivating INa. 5. Slow inactivation of neocortical INaP will influence all neuronal functions in which this current plays a role, including spike threshold determination, synaptic integration, and active propagation in dendrites. The kinetics of slow inactivation suggest that it may be a factor not only during extremely intense spiking, but also during periods of "spontaneous" activity.