Dual stretch responses of mHCN2 pacemaker channels: accelerated activation, accelerated deactivation

Mechanoelectric feedback in heart and smooth muscle is thought to depend on diverse channels that afford myocytes a mechanosensitive cation conductance. Voltage-gated channels (e.g., Kv1) are stretch sensitive, but the only voltage-gated channels that are cation permeant, the pacemaker or HCN (hyperpolarization-activated cyclic nucleotide-gated) channels, have not been tested. To assess if HCN channels could contribute to a mechanosensitive cation conductance, we recorded I(HCN) in cell-attached oocyte patches before, during, and after stretch for a range of voltage protocols. I(mHCN2) has voltage-dependent and instantaneous components; only the former was stretch sensitive. Stretch reversibly accelerated hyperpolarization-induced I(mHCN2) activation (likewise for I(spHCN)) and depolarization-induced deactivation. HCN channels (like Kv1 channels) undergo mode-switch transitions that render their activation midpoints voltage history dependent. The result, as seen from sawtooth clamp, is a pronounced hysteresis. During sawtooth clamp, stretch increased current magnitudes and altered the hysteresis pattern consistent with stretch-accelerated activation and deactivation. I(mHCN2) responses to step protocols indicated that at least two transitions were mechanosensitive: an unspecified rate-limiting transition along the hyperpolarization-driven path, mode I(closed)-->mode II(open), and depolarization-induced deactivation (from mode I(open) and/or from mode II(open)). How might this affect cardiac rhythmicity? Since hysteresis patterns and "on" and "off" I(HCN) responses all changed with stretch, predictions are difficult. For an empirical overview, we therefore clamped patches to cyclic action potential waveforms. During the diastolic potential of sinoatrial node cell and Purkinje fiber waveforms, net stretch effects were frequency dependent. Stretch-inhibited (SI) I(mHCN2) dominated at low frequencies and stretch-augmented (SA) I(mHCN2) was progressively more important as frequency increased. HCN channels might therefore contribute to either SI or SA cation conductances that in turn contribute to stretch arrhythmias and other mechanoelectric feedback phenomena.     

Deadlift muscle force and activation under stable and unstable conditions

The objective of this study was to compare the production of force and paraspinal muscle activity between deadlifts carried out in a standard way and with different instability devices (Bosu and T-Bow). Deadlifts involve the performance of muscle activities with dynamic and isometric characteristics. Thirty-one subjects participated voluntarily in the study. Initially, they performed an isometric test for 5 seconds in each condition. After that, they performed a set of 5 repetitions with 70% of the maximum isometric force obtained in each one of the previously evaluated conditions. During the isometric tests, records of electromyographic activity and force production were obtained, whereas during the dynamic tests, only the electromyographic activity was registered. The subjects produced more force and muscle activity on the stable surface than under the other conditions during the isometric test (p < 0.05), and the same differences in muscle activity were observed during the dynamic test (p < 0.05). These data show that the performance of deadlifts under stable conditions favors a higher production of maximum strength and muscle activity. Therefore, we conclude that the use of instability devices in deadlift training does not increase performance, nor does it provide greater activation of the paraspinal muscles, leading us to question their value in the performance of other types of exercises.     

Different pathways for activation and deactivation in CaV1.2: a minimal gating model

Point mutations in pore-lining S6 segments of Ca_V1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants α and β, and γ and δ, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 Ca_V1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the “pore-releasing” sensor movement ((x(V)) was much weaker than the voltage dependence of “pore-locking” sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening Ca_V1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.     

Graphical determination of mean activation energy and standard deviation in a microheterogeneity model of enzyme deactivation

Traditionally, enzyme populations have been treated as if they were either homogenous, or heterogeneous with distinct and separable subpopulations. The microheterogeneity model, however, assumes that there is a continuous distribution of properties in the population. In the area of enzyme deactivation kinetics, this model describes the heterogeneous population as having a continuous distribution of activation energy of deactivation. This distribution is characterized by mean activation energy, and a standard deviation of activation energy. The microheterogeneity model contains two parameters, (0) and sigma. Parameter (0) is the mean value of for a heterogeneous enzyme population; is the activation energy divided by absolute temperature and the ideal gas constant. Parameter sigma is the standard deviation of the Gaussian distribution of values in the population. If the population is homogeneous, then = (0) for all enzyme molecules and sigma = 0. There are certain ratios which are independent of (0) and dependent upon sigma. Two important ratios are t(1/4)/t(1/2) and t(1/2)/t(1/2) ('), where t(1/2) (') represents t(1/2) for a homogeneous enzyme population with the same mean ((0)), as the heterogeneous population. If there is experimental deactivation data for the heterogeneous population which is well behaved, the first ratio, t(1/4)/t(1/2), can be determined by estimating the time in minutes at which the enzyme has lost 25% of its activity (t(1/4)), and the time in minutes at which the enzyme has lost 50% of its activity (t(1/2)), and then taking the ratio t(1/4)/t(1/2). The corresponding value of sigma can be estimated from a graph. The ratio t(1/2)/t(1/2) (') can be found directly as a function of t(1/4)/t(1/2), and can be estimated from another graph. The value of (0) can then be calculated from the formulasgiven in the article.     

A model for steady isometric muscle activation

The present model of the motoneuronal (MN) pool – muscle complex (MNPMC) is deterministic and designed for steady isometric muscle activation. Time-dependent quantities are treated as time-averages. The character of the model is continuous in the sense that the motor unit (MU) population is described by a continuous density function. In contrast to most already published models, the wiring (synaptic weight) between the input fibers to the MNPMC and the MNs (about which no detailed data are known) is deduced, whereas the input–force relation is given. As suggested by experimental data, this relation is assumed to be linear during MU recruitment, but the model allows other, nonlinear relations. The input to the MN pool is defined as the number of action potentials per second in all input fibers, and the excitatory postsynaptic potential (EPSP) conductance in MNs evoked by the input is assumed to be proportional to the input. A single compartment model with a homogeneous membrane is used for a MN. The MNs start firing after passing a constant voltage threshold. The synaptic current–frequency relation is described by a linear function and the frequency–force transformation of a MU by an exponential function. The sum of the MU contraction forces is the muscle force, and the activation of the MUs obeys the size principle. The model parameters were determined a priori, i.e., the model was not used for their estimation. The analysis of the model reveals special features of the activation curve which we define as the relation between the input normalized by the threshold input of the MN pool and the force normalized by the maximal muscle force. This curve for any muscle turned out to be completely determined by the activation factor, the slope of the linear part of the activation curve (during MU recruitment). This factor determines quantitatively the relation between MU recruitment and rate modulation. This property of the model (the only known model with this property) allows a quantification of the recruitment gain (Kernell and Hultborn 1990). The interest of the activation factor is illustrated using two human muscles, namely the first dorsal interosseus muscle, a small muscle with a relatively small force at the end of recruitment, and the medial gastrocnemius muscle, a strong muscle with a relatively large force at the end of recruitment. It is concluded that the present model allows us to reproduce the main features of muscle activation in the steady state. Its analytical character facilitates a deeper understanding of these features. Received: 24 November 1997 / Accepted in revised form: 30 November 1998     

Structural basis of blocking integrin activation and deactivation for anti-inflammation

Integrins mediate leukocyte accumulation to the sites of inflammation, thereby enhancing their potential as an important therapeutic target for inflammatory disorders. Integrin activation triggered by inflammatory mediators or signaling pathway is a key step to initiate leukocyte migration to inflamed tissues; however, an appropriately regulated integrin deactivation is indispensable for maintaining productive leukocyte migration. While typical integrin antagonists that block integrin activation target the initiation of leukocyte migration, a novel class of experimental compounds has been designed to block integrin deactivation, thereby perturbing the progression of cell migration. Current review discusses the mechanisms by which integrin is activated and subsequently deactivated by focusing on its structure-function relationship.     

Structural aspects of AMPA receptor activation, desensitization and deactivation

Glutamate mediates most of the excitatory neurotransmission in the mammalian central nervous system by activating ionotropic glutamate receptors. Structural and functional studies of ionotropic glutamate receptors have offered detailed insight into the mechanism by which these integral membrane proteins function. In particular, advances in our understanding of the atomic structure of the agonist-binding domain have provided new opportunities to consider the conformational changes that take place in a functioning ligand-gated ion channel. Several recent studies have turned up important new ideas about the structural determinants of channel activation, deactivation and desensitization of AMPA receptors. Working hypotheses derived from this structural insight offer a rare opportunity to enrich and guide functional studies.     

Theme and variations on kinetics of GPCR activation/deactivation

G protein-coupled receptors (GPCRs) initiate intracellular signaling pathways in response to physiologically and medically important extracellular ligands such as peptide and large glycoprotein hormones, neurotransmitters, sensory stimuli (odorant and taste molecules, light), calcium, l-amino acids, and are the target of many clinical drugs. The conversion of these extracellular stimuli into intracellular signals involves sequential and reversible reactions that initially take place at the plasma membrane. These reactions are mediated not only by dynamic interactions between ligands, receptors and heterotrimeric G proteins, but also by conformational changes associated with the activation/deactivation process of each protein. This review discusses the kinetic characteristics and rate-limiting reactions engaged in signal propagation that are involved in systems as diverse as neurotransmitter and hormonal signaling, and that have been recorded in live cells by Förster resonance energy transfer (FRET) approaches.     

Effect of activation energy microheterogeneity on first-order enzyme deactivation

The influence of microheterogeneity on enzyme inactivation kinetics is examined. A continuous normal distribution of the thermal activation energy is assumed, and using this, a simple mathematical model is developed to find the activity-time trajectories for a microheterogeneous enzyme. Using an example, the model is used to show the quantitative effects of microheterogeneity such as increased order and stability observed during an enzyme inactivation. Experimental measurement of the extent of microheterogeneity in an enzyme sample is also discussed.     

A dynamical model of muscle activation, fatigue, and recovery

A dynamical model is presented as a framework for muscle activation, fatigue, and recovery. By describing the effects of muscle fatigue and recovery in terms of two phenomenological parameters (F, R), we develop a set of dynamical equations to describe the behavior of muscles as a group of motor units activated by voluntary effort. This model provides a macroscopic view for understanding biophysical mechanisms of voluntary drive, fatigue effect, and recovery in stimulating, limiting, and modulating the force output from muscles. The model is investigated under the condition in which brain effort is assumed to be constant. Experimental validation of the model is performed by fitting force data measured from healthy human subjects during a 3-min sustained maximal voluntary handgrip contraction. The experimental results confirm a theoretical inference from the model regarding the possibility of maximal muscle force production, and suggest that only 97% of the true maximal force can be reached under maximal voluntary effort, assuming that all motor units can be recruited voluntarily. The effects of different motor unit types, time-dependent brain effort, sources of artifacts, and other factors that could affect the model are discussed. The applications of the model are also discussed.     

Transient model of thermal deactivation of enzymes

The kinetics of enzyme deactivation provide useful insights on processes that determine the level of biological function of any enzyme. Photinus pyralis (firefly) luciferase is a convenient enzyme system for studying mechanisms and kinetics of enzyme deactivation, refolding, and denaturation caused by various external factors, physical or chemical by nature. In this report we present a study of luciferase deactivation caused by increased temperature (i.e., thermal deactivation). We found that deactivation occurs through a reversible intermediate state and can be described by a Transient model that includes active and reversibly inactive states. The model can be used as a general framework for analysis of complex, multiexponential transient kinetics that can be observed for some enzymes by reaction progression assays. In this study the Transient model has been used to develop an analytical model for studying a time course of luciferase deactivation. The model might be applicable toward enzymes in general and can be used to determine if the enzyme exposed to external factors, physical or chemical by nature, undergoes structural transformation consistent with thermal mechanisms of deactivation.     

Calcium-dependent activation and deactivation of rod outer segment phosphodiesterase is calmodulin-independent

ATP-dependent activation and deactivation of retinal rod outer segment phosphodiesterase is affected by calcium [Kawamura, S. and Bownds, M. D., J. Gen. Physiol. 77:571-591(1981)]. Our data demonstrate that although calmodulin has been found in rod outer segments [Liu, Y. P. and Schwartz, H., Biochim. Biophys. Acta 526:186-193(1978); Kohnken, R. E. et al, J. Biol. Chem. 256:12517-12522(1981)], this protein is not involved in calcium-dependent phosphodiesterase activation at light levels at which calcium clearly affects this enzyme's activity. Furthermore, calmodulin does not mediate the calcium-dependent deactivation of phosphodiesterase.     

Freezing point and melting point of barnacle muscle fibers

The freezing point and the melting point of myoplasm were measured with two experimental models. In all samples, a supercooled stage was reached by lowering the temperature of the sample to approximately - 7 degrees C, and the freezing of the sample was mechanically induced. The freezing process was associated with a phase transition in the interstices between the contractile filaments. In intact muscle fibers, the freezing point showed a structural component (0.43 degrees C), and the melting point indicated that the intracellular and the extracellular compartments are isotonic. When the sample of myoplasm, previously inserted in a cylindrical cavity was incubated in an electrolyte solution, the freezing point showed a structural component similar to that of the intact muscle fiber, but the melting point was lower than the freezing and the melting points of the embedding solution. This was interpreted as evidence that the counterions around the contractile filaments occupied a nonnegligible fraction of the intracellular compartment.     

A deactivation model for immobilized and soluble enzymes

Summary A first-order deactivation model which includes a step that does not destroy enzyme activity, but is a compulsory precursor of the step that does, is shown to fit reasonably well the immobilized and soluble enzyme deactivation data presented. The deactivation rate coefficient increases with time, and the model may be generalized to include n such compulsory precursors.     

Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.     

Smooth muscle adherens junctions associated proteins are stable at the cell periphery during relaxation and activation

This study was performed to determine the stability of the adherens junction (AJ)-associated proteins at the smooth muscle cell (SMC) plasma membrane during relaxing and activating conditions. Dog stomach, ileum, colon, and trachea tissues were stored in Ca²⁺-free PSS or regular PSS or were activated in 10 µM carbachol in PSS before rapid freezing. The tissues were subsequently sectioned and immunoreacted using antibodies for vinculin, talin, fibronectin, and caveolin to determine their cellular distribution in these tissues under these conditions. In all four tissues and under all three conditions, the distribution of these four proteins remained localized to the periphery of the cell. In transverse tissue sections, the AJ-associated proteins formed a distinct punctate pattern around the periphery of the SMCs at the plasma membrane. These domains alternated with the caveolae (as identified by the presence of caveolin). In longitudinal tissue sections, the AJ-associated proteins formed continuous tracks or staves, while the caveolae remained punctate in this dimension as well. Caveolin is not present in the tapered ends of the SMCs, where the AJ-associated proteins appear continuous around the periphery. Densitometry of the fluorophore distribution of these proteins showed no shift in their localization from the SMC periphery when the tissues were relaxed or when they were activated before freezing. These results suggest that under physiologically relaxing and activating conditions, AJ-associated proteins remain stably localized at the plasma membrane. vinculin; talin; fibronectin; caveolin; stomach; ileum; colon; trachea     

Responses of Rubisco activation and deactivation rates to variations in growth-light conditions

Basil (Ocimum basilicum) and impatiens (Impatiens wallerana) were grown in sun, shade, or fluctuating light (15 min sun, 15 min shade) to examine the effects of growth-light conditions on the rates of light-induced Rubisco activation and deactivation. Rubisco activation and deactivation rates were determined from gas-exchange measurements of photosynthesis following a step increase in PFD. Rubisco deactivation rates were also determined from biochemical analyses of leaf extracts. There were no significant differences in Rubisco activation rate among the growth conditions or between the two species. However, there were significant differences in Rubisco deactivation rate among the growth conditions in basil and between the two species. In basil, Rubisco deactivated more slowly following a decrease in PFD in sun- and fluctuating-light grown plants than in shade grown plants. Slower rates of Rubisco deactivation during periods at low PFD resulted in higher activation states at the onset of increased PFD. Thus, the contribution of Rubisco activation to the induction process was less for basil plants grown under sun and fluctuating light than for those grown under shade. Impatiens deactivated Rubisco more rapidly than in basil, but there was no substantial effect of the three growth-light conditions on Rubisco deactivation rates in impatiens.