Point mutations in IIS4 alter activation and inactivation of rat brain IIA Na channels in Xenopus oocyte macropatches

Macroscopic currents of wild-type rat brain IIA (RBIIA) and mutant Na channels were recorded in excised patches from Xenopus oocytes. A charge deletion (K859Q) and an adjacent conservative mutation (L860F) in the second domain S4 membrane-spanning region differentially altered voltage sensitivity and kinetics. Analysis of voltage dependence was confined to Na currents with fast inactivation kinetics, although RBIIA and K859Q (but not L860F) also showed proportional shifts between at least two gating modes, rendering currents with fast or slow inactivation kinetics, respectively. Compared to RBIIA, the midpoint of the activation curve was shifted in both K859Q and L860F by 22 mV to more positive potentials, yet this shift was not associated with a corresponding change in the voltage dependence of time constants for activation ( _a) or inactivation ( _h1, _h2). L860F showed faster activation time constants _a than RBIIA, while K859Q was slower for both the activation ( _a) and the inactivation components ( _h1). Similarly, the steady-state inactivation curve of L860F but not K859Q shifted by 9 mV in the hyperpolarizing direction. Thus, the fourth charge in the IIS4 transmembrane segment exerts control over voltage sensitivity and kinetics of activation and may interact with structure that influence other aspects of channel gating.     

Postsynaptic actions of ethanol and methanol in crayfish neuromuscular junctions

Actions of ethanol and methanol on excitatory postsynaptic channels activated by quisqualate were investigated in opener muscles from the first walking leg and the claw of crayfish. Both ethanol and methanol reduced the elementary currents |i| that flow through channels operated by quisqualate in a concentration-dependent manner but did not affect the apparent mean open time, _noise, of the channels estimated from power spectra. 0.26 mol/l ethanol, or 1 mol/l methanol, respectively, reduced |i|e-fold. Ethanol also markedly decreased the size and the decay time constant (sEPSCs) of spontaneous excitatory postsynaptic currents (sEPSCs). At ten fibres, on the average, 0.26 mol/l ethanol decreased (sEPSCs) by a factor 1.56±0.24 (SD). (sIPSCs) and _noise of inhibitory postsynaptic currents apparently were not affected by ethanol. Moreover the size of elementary inhibitory postsynaptic currents did not decrease in the presence of this alcohol. Thus, in crayfish opener muscles ethanol seems to selectively depress excitatory postsynaptic currents.This investigation was supported by the Deutsche Forschungsgemeinschaft, Project Fi 305/1-1/1-2     

Neuronal mechanisms for pitch analysis in the time domain

Summary Many units in the auditory midbrain nucleus (MLD) of the Guinea fowl are found to be tuned to amplitude modulated tones (AM). For a given response maximum the relationship of the period _m of the modulation frequency f_m and the period _c of the carrier frequency f_c may be given by an empirical equation: m · _m + n · _c = 1 · ₁, where m, n and 1 are small integers typical for a unit. ₁ is a time constant of 0.4 ms. The temporal pattern of the neuronal response support these findings. The averages of spike trains oscillate with periods multiple to ₁. These oscillations are elicited by stimulus onsets and zero crossings of f_m and may be coupled strongly to f_m depending on f_c. Variation of f_m or f_c shifts the mean delay of the phase coupled activity proportional to m · _m and n · _c, respectively. These effects may be explained with activity phase coupled to f_c which coincides at the level of the recorded units with oscillations coupled to f_m. This is expressed by the above given periodicity equation. Psychophysical results with AM-stimuli indicate that the mechanisms described and the periodicity equation are adequate for the explanation of the analysis of periodicity pitch in humans. Hence the period corresponding to pitch is defined by m · _m + n · _c = 1 · ₁, where n and 1 are integers and ₁ = 0.4 ms. Plots of _p as a function of _c reveal steps at 0.4 ms intervals indicating that the neuronal time constant is the same in both species.Supported by the DFG, SFB 45     

Glutathione accelerates sodium channel inactivation in excised rat axonal membrane patches

The effects of glutathione were studied on the gating behaviour of sodium channels in membrane patches of rat axons. Depolarizing pulses from –120 to –40 mV elicited sodium currents of up to 500 pA, indicating the simultaneous activation of up to 250 sodium channels. Inactivation of these channels in the excised, inside-out configuration was fitted by two time constants ( _h1=0.81 ms; _h2= 5.03 ms) and open time histograms at 0 mV revealed a biexponential distribution of channel openings ( _short=0.28 ms; _long=3.68 ms). Both, the slow time constant of inactivation and the long lasting single channel openings disappeared after addition of the reducing agent glutathione (2–5 mM) to the bathing solution. Sodium channels of excised patches with glutathione present on the cytoplasmatic face of the membrane had inactivation kinetics similar to channels recorded in the cell-attached configuration. These observations indicate that redox processes may contribute to the gating of axonal sodium channels.     

Kinetic mode switch of rat brain IIA Na channels in Xenopus oocytes excised macropatches

Na currents recorded from inside-out macropatches excised from Xenopus oocytes expressing the subunit of the rat brain Na channel IIA show at least two distinguishable components in their inactivation time course, with time constants differing about tenfold ( _h1 = approx. 150 s and _h2 = approx. 2 ms). In excised patches, the inactivation properties of Na currents changed with time, favoring the faster inactivation kinetics. Analysis of the fast and slow current kinetics shows that only the relative magnitudes of _h1 and _h2 components are altered without significant changes in the time constants of activation or inactivation. In addition, voltage dependence of both activation and steady-state inactivation of Na currents are shifted to more negative potentials in patches with predominantly fast inactivation, although reversal potentials and valences remained unaltered. We conclude that the two inactivation modes discerned in this study are conferred by two states of Na channel the interconversion of which are regulated by an as yet unknown mechanism that seems to involve cytosolic factors.     

Demonstration of A-currents in pancreatic islet cells

Voltage-activated K⁺ currents resistant to TEA but blockable by 4-AP were recorded from mouse pancreatic islet cells. These currents first become observable during depolarizations to voltages more positive than –40 mV, reaching a peak amplitude of 120±34 pA at +6 mV (n=4), display rapid turn on (=3.3±1.1 ms at +6 mV) and inactivate completely within 250 ms (=65±5 at +6 mV). The current is subject to steady-state inactivation. The midpoint (V _h) of the inactivation curve (h) was observed at –72±2 mV. The properties of this current resemble those reported for the A-current in neurons.     

Modulation of ATP-sensitive potassium channel activity by flash-photolysis of ‘caged-ATP’ in rat heart cells

We have used caged-ATP to investigate the kinetic behavior of K_ATP channels in ventricular cells from rat heart. In whole cells, loaded with caged-ATP, an increase of intracellular [ATP] following a UV light flash produced a decrease of K_ATP channel current that was too slow ( 300 ms) to be explained by the expected timecourse of ATP release ( 3 ms) and the time-course of channel blockade by ATP ( 20 ms). In isolated membrane patches, caged-ATP itself caused partial blockade of K_ATP channels. Under these conditions, photorelease of ATP caused channel activity to decline further. The results suggest that caged-ATP can bind to the K_ATP channel but, on binding, decreases the open probability to a lesser extent than does ATP. Additionally, the observations indicate that for photolytically-generated ATP to bind to the channel, caged-ATP must first unbind (slowly) from the channel. We conclude that caged-ATP is not fully caged with respect to its allosteric action on the K_ATP channel.     

Comparison of membrane properties of the cell body and the initial part of the axon of phasic motoneurones in the spinal cord of the cat

Summary Membrane properties of the cell body and the initial part of the axon of cat phasic motoneurones were analyzed.The mean input resistance was 9.1 M in the axon and 2.0 M in the cell body. A great scattering of values, however, was observed in the axons.The passive membrane time constants were 0.35 msec ( m) in the axon and 3.2 msec ( m) and 0.63 msec ( ₁) in the soma. A close electrotonic coupling between the soma and the axon was shown by means of synaptic potentials.In the cell body the threshold for excitation was 1.46 times higher than in the axon (14.2 mV and 9.7 mV respectively).Stimulating with current pulses an S-shaped spike inactivation curve was found in the axon. At threshold current intensity, spike inactivation reached 37.2%. In the cell body either no or only slight inactivation (7%) occurred at rheobase.During suprathreshold current stimulation the axon discharged only a few spikes, whereas the soma fired steadily. The current-frequency curve of the soma consisted of two linear ranges and, in addition, a logarithmic range quite similar to the logarithmic frequency curve of the axon was observed.The differences of the discharge behaviour of the cell body and the axon were discussed.This research was supported by the Sonderforschungsbereich 38, Membranforschun.     

Chloramine-T effect on sodium conductance of neuroblastoma cells as studied by whole-cell clamp and single-channel analysis

Patch-clamp experiments were done on sodium channels of neuroblastoma cells (N1E-115) in the presence of tetraethylammonium ions to block potassium channels. In Ringer solution whole-cell records revealed a diphasic I _Na inactivation with the fast (₀) component being clearly larger than the slow (₁ 3 ₀) component. In single-channel studies on inside-out patches the mean open time, ¯t₀, turned out to be only a fraction of ₀ and almost independent of membrane potential. After external application of chloramine-T I _Na inactivation of whole cells was delayed with both ₀ and ₁ increased, and incomplete, i.e. a persistent current component emerged. The latter was maximal at a more positive membrane potential than the peak current. Also, after chloramine-T treatment the peak I _Na increased, particularly at weak depolarizations. In inside-out patches the equally effective internal application of chloramine-T led to bursting channel openings with mean burst times (¯t_b) 6 ms, and gap times (¯t_g) 20 ms, where gap is defined as a closure of 1.5 ms. Within the bursts (¯t_o) was approximately 2 ms, again clearly shorter than ₀; the mean close time, ¯t_c was approximately 0.5 ms. The single-channel conductance was approximately 13 pS and unaffected by chlopramine-T. Diphasic I _Na inactivation and the fact that ¯t_o < ₀ led to an extension of the model of Aldrich and Stevens [J Neurosci 7:418–431 (1987)], in which overall kinetics is determined by the openings rather than closures of the sodium channels. The extension comprises two inactivated states in series with the open state. Estimates are given of all rate constants of the transition between states that describe the single-channel results as well as whole-cell I _Na (t), both in the control and in chloramine-T.     

Effects of aerobic endurance training status and specificity on oxygen uptake kinetics during maximal exercise

The main purpose of this study was to analyze the effects of exercise mode, training status and specificity on the oxygen uptake (O₂) kinetics during maximal exercise performed in treadmill running and cycle ergometry. Seven runners (R), nine cyclists (C), nine triathletes (T) and eleven untrained subjects (U), performed the following tests on different days on a motorized treadmill and on a cycle ergometer: (1) incremental tests in order to determine the maximal oxygen uptake (O_2max) and the intensity associated with the achievement of O_2max (IO_2max); and (2) constant work-rate running and cycling exercises to exhaustion at IO_2max to determine the effective time constant of the O₂ response (O₂). Values for O_2max obtained on the treadmill and cycle ergometer [R=68.8 (6.3) and 62.0 (5.0); C=60.5 (8.0) and 67.6 (7.6); T=64.5 (4.8) and 61.0 (4.1); U=43.5 (7.0) and 36.7 (5.6); respectively] were higher for the group with specific training in the modality. The U group showed the lowest values for O_2max, regardless of exercise mode. Differences in O₂ (seconds) were found only for the U group in relation to the trained groups [R=31.6 (10.5) and 40.9 (13.6); C=28.5 (5.8) and 32.7 (5.7); T=32.5 (5.6) and 40.7 (7.5); U=52.7 (8.5) and 62.2 (15.3); for the treadmill and cycle ergometer, respectively]; no effects of exercise mode were found in any of the groups. It is concluded that O₂ during the exercise performed at IO_2max is dependent on the training status, but not dependent on the exercise mode and specificity of training. Moreover, the transfer of the training effects on O₂ between both exercise modes may be higher compared with O_2max.     

Analysing ion channels with hidden Markov models

Ion channel current amplitudes () and open probabilities (P _o) have been analysed so far by defining a 50% threshold to distinguish between open and closed states of the channels. With this standard method (SM) it is very difficult or even impossible to analyse channels of different size in one membrane patch correctly. A stochastical model, named the hidden Markov model (HMM), separates between observation noise and the stochastic process of opening and closing of ion channels. The HMM allows the independent analysis of , P _o, and mean dwell times () of different channels in one membrane patch, without defining threshold levels. Using this method errors in the analysis are not summarized like in the SM because all different analysing procedures (e. g. filtering, setting of threshold, fitting processes) are done in one step. Two different K⁺ channels in excised basolateral membranes of the cortical collecting duct of rat (CCD) were analysed by the SM and the HMM. The value of the intermediate-conductance K⁺ channel (i-K⁺) was 3.9±0.1 pA (SM) and 3.8±0.2 pA (HMM) for 11 observations. The P _o value of this channel was 10.2±4.2% (SM) and 10.1±4.0% (HMM). The mean values were 5.4±0.6 ms for the open state and 9.6±2.2 ms and 145±21 ms for the closed states (SM) and 7.8±1.1 ms, 7.7±0.9 ms and 148±24 ms (HMM), respectively. For seven small-conductance K⁺ (s-K⁺) channels, which were found in the same membrane patches as the i-K⁺, an accurate analysis of P _o and was not possible with the SM. The value was 1.0±0.1 (SM), 0.9±0.1 (HMM) pA. P _o was 16.6±4.6%, the open value was 11.1±2.8 ms, and the closed value was 34.9±8.5 ms. The HMM allows the analysis of single-channel currents, P _o, and mean values when different or more than one ion channel(s) are colocalized in one membrane patch. Where analysis with the SM was possible results did not significantly differ from those obtained with the HMM. Thus for this kind of analysis the method of setting a 50% threshold appears justified.     

Temperature experiments on nerve and muscle membranes of frogs

The influence of temperature changes in the range of 25°C to –6°C on the time constants of Na activation (m) and inactivation (h) was studied in twitch muscle fibers and the node of Ranvier under voltage-clamp conditions. Arrhenius plots of m and h exhibit a change in activation enthalpy at temperatures below 10°C. Cooling and subsequent heating induce a hystersis in the temperature dependence of m and h Ni²⁺ and UO ₂ ²⁺ increase the hysteresis width. With fast temperature changes the gating kinetics relax to their new values more slowly than the temperature change. Hence, temperature must be changed more slowly than 5°C/min if an additional apparent hysteresis due simply to this relaxation is to be avoided. The data are explained by the hypothesis of a phase transition in the membrane lipids. This conception is favoured over a temperature-induced change in protein conformation, since the neutral local anaesthetic benzocaine shows use-dependent block as if low temperature restricted the access of the drug through the lipid phase to its receptor.Supported by grant NS 08174 from the U.S. Public Health Service and SFB 38 from Deutsche Forschungsgemeinschaft     

End-plate currents evoked by paired stimuli in frog muscle fibres

Using voltage-clamp techniques spontaneously occuring miniature end-plate currents (mepc) and nerve-evoked end-plate currents (epc) were recorded in frog glycerol-treated or cut muscle preparations. Epcs were induced by pairs of stimuli (the delay of the 2nd stimulus, t being 6 ms–30 s; one pair was delivered every 60–90 s). The decay time constant of the epc (_epc) was longer, the larger its quantal content despite the presence of active acetylcholinesterase (AChE). After treatment with anticholinesterases (prostigmine or armin, an irreversible inhibitor) this increase in _epc became more pronounced. When AChE was fully active the decay of the 1st epc ₁ was slightly faster than the decay of the 2nd epc ₂ only when the interstimulus interval was rather short (t<20 ms). Following treatment with anticholinesterases this difference between ₂ and ₁ could be determined even when t was as long as 30 s. In anticholinesterase-treated preparations was found to be inversely proportional to log t: a 50% increase in the decay time-constant of the 2nd epc occurred with t=120 ms. During continuous stimulation (10 impulses/s) _epc increased from the 1st to the 5–6th responses, but then decreased in parallel with the fall in the epc amplidude. The phenomenon of postsynaptic potentiation we observed could be readily abolished when quantal content was decreased by the presence of magnesium ions, but it was relatively unaffected when the receptor density was decreased by -bungarotoxin (-BuTX).The possible existence is discussed of two kinds of repetitive binding of ACh molecules, first, to free cholinoreceptors (a process which could be inhibited by -BuTX) and, second, to a complex of the cholinoreceptor plus one molecule of ACh (a process which is less sensitive to -BuTX blocking action).     

Arginine-vasopressin induces mode-2 gating in L-type Ca2+ channels (smooth muscle cells of the urinary bladder of the guinea-pig)

The effect of arginine-vasopressin (AVP, 0.1 M) on elementary Ca²⁺ channel currents (L-type) was studied in cell-attached patches with 10 mM BaCl₂ as the charge carrier. At a constant potential of –30 mV, bath applied AVP increased the channel openness (NP _o) by a factor of 4.7±3.0 (mean±SD, n=9), the effect resulted from an increase in the frequency of opening (factor 2.5±0.8) and from a longer mean open time. Under control, openings longer than 5 ms contributed only 4% of the total, however, with the application of AVP this contribution increased to 29%. Under control, the open times were distributed along a single exponential (_o1=0.8±0.4 ms), a double exponential distribution was obtained during AVP (_o1=0.8±0.5 ms, _o2=7.5±0.7 ms). The Ca²⁺ agonist BAYk8644 (1 M) changed the open time distribution similarly to AVP (_o1=1.0±0.5 ms, _o2=9±2.8 ms). With 1 M BAYk8644 in the bath, AVP did not significantly increase the relative contribution of long openings, however, AVP increased the frequency of openings by a factor of 2.0±1 (n=6). The results are compatible with the idea that AVP can change the gating of L-type Ca²⁺ channels from mode 1 to mode 2.     

Effects of hypoxia on passive electrical properties of canine ventricular muscle

The effect of hypoxia, either in the presence or in the absence of glucose, on the passive electrical properties of canine ventricular muscle fibers was examined, employing a single sucrose gap method. The significant changes after 30 min of hypoxia (PO ₂=35–45mm Hg) were an increase in the specific internal longitudinal resistance (R _i) and a decrease in the space constant (). The values during the control (PO ₂>450mm Hg) were 198±77 cm forR _i and 0.81±0.15 mm for , and they changed to 245±90 cm and 0.70±0.10mm, respectively, after 30min of hypoxia. Hypoxia decreased the specific membrane resistance (R _m), but the changes were not statistically significant. The membrane time constant ( _m ) and capacity (C{imm}) were not affected significantly. The absence of glucose during hypoxia was found to cause more profound changes than hypoxia alone in the passive electrical properties, especiallyR _i and , suggesting that glucose might counteract the effects of hypoxia on these parameters of ventricular muscles.     

Inhibitory synaptic channels activated by γ-aminobutyric acid (GABA) in crayfish muscle

Small crayfish muscle fibres were voltage clamped and synaptic current elicited by superfused GABA solutions was measured. Analysis of the fluctuations of synaptic current and of relaxations of the current after voltage steps yielded analogous results. The current has two components. The first component is characterized by the opening of synaptic channels with a single channel conductance =9 pS and an average open time =5 ms, measured at 23°C and-100 mV. depends on the membrane potential, _E = ₀ · e ^E/, and was about +100 mV in the average. The channel open time agrees with the time constant of decay of the inhibitory postsynaptic current (IPSC) elicited by a nerve stimulus. The current is carried by chloride ions. The second current component is much slower, the average channel open time was _s = 33 ms at 23°C and-60 mV. The open time _s of the slow component also was shortened on hyperpolarization. The reversal potential for the current component was more positive than-50 mV. This slow component also seems to be a synaptic one.This investigation was supported by a grant of the Deutsche Forschungsgemeinschaft     

Gating current experiments on frog nodes of Ranvier treated withCentruroides sculpturatus toxins or aconitine

(1) Gating currents were recorded from frog nodes of Ranvier treated either with toxins III or IV from the venom of the scorpionCentruroides sculpturatus or with the alkaloid toxin aconitine. (2) Toxins III or IV fromCentruroides sculpturatus (which drastically reduce the sodium permeabilityP _Na and slightly shift its voltage dependence in the depolarizing direction) caused a small depolarizing shift of the relation between charge (Q _on) and membrane potential (E) without affecting the maximum chargeQ _{on max}. (3) On nodes treated with toxins III or IV fromCentruroides sculpturatus, a depolarizing conditioning pulse (which transiently shifts the descending branch of theI _Na(E) curve by up to 60 mV in the hyperpolarizing direction) shifted the midpoint potential (E_mid) of theQ _on(E) curve by –17 mV and slightly increased the slope of the curve; it also decreasedQ _{on max} markedly but had little effect onQ _on measured with small depolarizing pulses. By contrast, massive treatment with aconitine (which irreversibly shifts sodium activation in the hyperpolarizing direction) irreversibly shifted the midpoint potential of theQ _on(E) curve from –28.5 to –69 mV and significantly increasedQ _on andQ _off measured with small depolarizing pulses; concomitantly, the voltage dependence of the on time constant of the charge movement [_on(E)] was shifted by –44 mV. (4) The sodium currentI _Na was exponential both in nodes treated with toxins III or IV ofCentruroides sculpturatus and subjected to a depolarizing conditioning pulse and in aconitine-treated nodes; in the latter,I _Na started after a delay of 30–40 s. The time constant of the sodium current, _{on Na}, was larger than the time constant of the charge movement, _{on Q}; the ratio _{on Q}/_{on Na} was 0.61 and 0.73 in the experiments withCentruroides sculpturatus toxins and aconitine, respectively. (5) The off time constant of the sodium current (_{off Na}) was slightly increased in nodes treated withCentruroides sculpturatus toxins and subjected to a depolarizing conditioning pulse, whereas it was markedly increased in aconitine-treated nodes. With the former treatment, the off time constant of the charge movement (_{off Q}) was unaffected but with aconitine treatment it was considerably increased although it remained smaller than _{off Na}. Consequently, the ratio _{off Q}/_{off Na} (which is 1 in untreated nodes) became smaller than one, reaching values as low as 0.58 and 0.44 in the experiments withCentruroides sculpturatus toxins and aconitine, respectively. The small _{off Q}/_{off Na} ratio suggests that the channels remain open for an appreciable time after most of the gating charges have returned to their resting position. (6) The results obtained with aconitine resemble the findings on batrachotoxin-treated nodes (Dubois and Schneider 1985), except that in the latter the time constants _{on Na} and _{off Na} of the sodium current are smaller than the corresponding time constants _{on Q} and _{off Q} of the charge movement.     

Plasma catecholamines and heart rate at the beginning of muscular exercise in man

Summary The relationship between the time course of heart rate and venous blood norepinephrine (NE) and epinephrine (E) concentrations was studied in 7 sedentary young men before and during 3 bicycle exercises of 5 min each (respectively 23±2.8%, 45±2.6% and 65±2.4% , mean ±SE). During the low level exercise the change in heart rate is monoexponential ( =5.7±1.2s) and no increment above the resting level of NE (NE) or of E (E) occurs. At the medium and highest intensity of exercise: a) the change in heart rate is biexponential, for the fast and the slow component averaging about 3 and 80 s respectively; b) NE (but not E) increases continuously with time of exercise; c) at the 5th min of exercise heart rate increments are related to NE; d) between 20s and 5 min, at corresponding sampling times, the heart rate of the slow component is linearly related to NE. At exercise levels higher than 33% the increase in heart rate described by the slow component of the biexponential kinetic could be due to an augmented sympathetic activity revealed by increased NE blood levels.     

Characterisation, asymmetry and reproducibility of on- and off-transient pulmonary oxygen uptake kinetics in endurance-trained runners

The purpose of this study was three-fold: (1) to characterise both the on- and off-transient oxygen uptake (O₂) kinetics in endurance runners during moderate-intensity treadmill running; (2) to determine the degree of symmetry between on- and off-transients; and (3) to determine the reproducibility of O₂ kinetic parameters in endurance runners. Twelve endurance-trained runners [mean (SD) age 25.2 (4.7) years, body mass 70.1 (9.7) kg, height 179.5 (7.5) cm, ventilatory threshold (V_T), 3,429 (389) ml.min^–1, maximal O₂ (O_2max) 4,138 (625) ml.min^–1] performed two multiple square-wave transition protocols on separate days. The protocol consisted of six (three transitions, 15 min rest, three transitions) square-wave transitions from walking at 4 km.h^–1 to running at a speed equivalent to 80% of the O₂ at the V_T (80%V_T). To determine the reproducibility, the protocol was repeated on a separate day (i.e. a test-retest design). Pulmonary gas-exchange was measured breath-by-breath. The O₂ data were modelled [from 20 s post-onset (or offset) of exercise] using non-linear least squares regression by a mono-exponential model, incorporating a time delay. The on- and off-transient time constants (_on and _off), mean response times (MRT_on and MRT_off) and amplitudes (A_on and A_off) were obtained from the model fit. On- and off transient kinetics were compared using paired t-tests. The reproducibility of each kinetic parameter was explored using statistical (paired t-tests) and non-statistical techniques [95% limits of agreement (LOA, including measurement error and systematic bias) and coefficient of variation (CV)]. It was found that the _on [12.4 (1.9)] was significantly (P<0.001) shorter than _off [24.5 (2.3) s]. Similarly, MRT_on [27.1 (1.9) s] was shorter than MRT_off [33.4 (2.2) s]. With respect to the reproducibility of the parameters, paired t-tests did not reveal significant differences between test 1 and test 2 for any on- or off-transient O₂ kinetic parameter (P>0.05). The LOA for _on (1.9 s), _off (2.3 s), MRT_on (1.2 s), MRT_off (3.2 s), A_on (204 ml.min^–1) and A_off (198 ml.min^–1) were narrow and acceptable. Furthermore, the measurement error (range, 4.3 to 15.1%) and CV (1.3 to 4.8%) all indicated good reproducibility. There was a tendency for _off to be more reproducible than _on. However, MRT_on was the most reproducible kinetic parameter. Overall, the results suggest that: (1) a multiple square-wave transition protocol can be used to characterise, reproducibly, both on- and off-transient O₂ kinetic parameters during treadmill running in runners; (2) the phase II time constant is independent of O_{2 max}, and (3) asymmetry exists between on- and off transient O₂ kinetic parameters.     

Differential effects of halothane on adult and juvenile sodium channels in human muscle

Myoballs were cultured from biopsies of adult human skeletal muscle. Transient currents through the sodium channels were elicited by depolarizing a myoball membrane with the whole-cell patch-clamp technique. The properties of the sodium channels were determined from the Hodgkin-Huxley parameters (I _{Na max}, _m, _h,h -curve) derived from these transients. Halothane, when applied at 3.4 mmol/l (15 kPa), blocked about 50% of the current through the adult, TTX-sensitive sodium channels but had little effect on the current through the juvenile, TTX-insensitive sodium channels. At >12 mmol/l, halothane blocked both channel types completely. The time constants of activation and inactivation were decreased in the presence of 3.4 mmol/l halothane but not enough to account for the decrease of the current amplitude. Halothane shifted the h-curves of both channel types towards more negative potentials by an amount that was roughly proportional to its concentration. Myoballs from a man susceptible to malignant hyperthermia (MH) gave the same results as the controls indicating that the halothane effects on the action potential of MH-susceptible muscle are not mediated by a specific effect on the sodium channels.