Effect of Temperature on the Potential and Current Thresholds of Axon Membrane

The effect of temperature on the potential and current thresholds of the squid giant axon membrane was measured with gross external electrodes. A central segment of the axon, 0.8 mm long and in sea water, was isolated by flowing low conductance, isoosmotic sucrose solution on each side; both ends were depolarized in isoosmotic KCl. Measured biphasic square wave currents at five cycles per second were applied between one end of the nerve and the membrane of the central segment. The membrane potential was recorded between the central sea water and the other depolarized end. The recorded potentials are developed only across the membrane impedance. Threshold current values ranged from 3.2 µa at 267deg;C to 1 µa at 7.5°C. Threshold potential values ranged from 50 mv at 26°C to 6 mv at 7.5°C. The mean Q₁₀ of threshold current was 2.3 (SD = 0.2), while the Q₁₀ for threshold potentials was 2.0 (SD = 0.1).     

Temperature Dependence of Accommodation and Excitation in Space-Clamped Axons

Accommodation and excitation in space-clamped squid axons were studied with the double sucrose gap technique, using linear current ramps, short (50 µsec) square wave pulses, and rheobasic square wave pulses as stimuli. The temperature was varied from 5° to 35°C. Experimental results showed a Q₁₀ for accommodation which was 44% higher than that for excitation. Yet calculations on the basis of the Hodgkin-Huxley equations predict equal Q₁₀'s for excitation and accommodation. Although the Hodgkin-Huxley equations are spectacularly successful for so many nerve phenomena, the differences between calculations of accommodation and these experiments, which were designed to test the equations, show that the equations need modification in this area.     

Temperature dependence of oscillation in squid axons: comparison of experiments with computations

The temperature dependence of the oscillatory behavior of experimental and computed axons was compared. In the experimental study of space-clamped giant axons of the squid, small oscillations after a single threshold spike were measured using the double glucose gap over the temperature range 10°-30°C during treatment with three concentrations of external CaCl₂ solutions. Calcium concentration had little effect on frequency, as was found also by Huxley in his computations at 18.5°C for a fiber at rest. The Q₁₀ both for the experimental and for the computed axon of FitzHugh was 2.25. The experimental measurements of the frequency of oscillations near threshold agree extremely well with the Hodgkin-Huxley calculations.     

Effect of Low Sodium, Tetrodotoxin, and Temperature Variation upon Excitation

The lowering of external sodium raised both the constant quantity threshold, Q_o, and the rheobase, I_o, in both real space-clamped squid axons and the theoretical axon as computed on the basis of the standard Hodgkin-Huxley equations. In both real and theoretical axons the minimum intensity for excitability for short pulses, which occurs at about 15°C, was still present when low sodium replaced seawater. Low sodium did not affect the temperature dependence of the strength-duration relationship in the range, 5° to 25°C. The excitability of tetrodotoxin-treated real axons was found to be more temperature-dependent than that of normal real axons. Also the data on dosage-response to TTX of real axons fit the dose-response relationship of a hypothetical system in which one TTX ion binds reversibly to its receptor to produce a fraction of the inhibitory effect, the curve being identical to a simple adsorption isotherm. The Hodgkin-Huxley equations describe the broad outline of events occurring during excitation quite well.     

Effect of Calcium, Temperature, and Polarizing Currents upon Alternating Current Excitation of Space-Clamped Squid Axons

Alternating current threshold excitation of space-clamped squid giant axons was measured as a function of frequency, external calcium concentration, temperature (from 10° to 35°C), and hyper- and depolarizing steps. In normal axons there is usually an optimum frequency at about 120 Hz, at which the threshold is a minimum. The threshold rises at both lower and higher frequencies to give a resonance curve. Low calcium causes an increase in optimum frequency, a decrease in current threshold, and an increase in sharpness of tuning in both real axons and axons computed according to the Hodgkin-Huxley formulation; high calcium causes opposite effects. An increase in temperature causes an increase of optimum frequency, an increase in sharpness of tuning, and an increase in threshold current in both real and computed axons. The Q₁₀ for the effect of temperature upon optimum frequency is 1.8 in real and computed axons at moderate temperatures. Hyperpolarization causes (a) a decrease in optimum frequency, (b) a decrease in sharpness of tuning, and (c) an increase in threshold. Depolarization causes opposite effects.     

Oscillation and Repetitive Firing in Squid Axons : Comparison of experiments with computations

Space-clamped squid axons treated with low calcium and computed Hodgkin-Huxley (HH) axons were stimulated by steps of superthreshold current from 101 to 400% of the rheobasic value over a temperature range of 5–27°C. The natural frequency of sustained repetitive firing of real and computed axons depended weakly upon stimulus intensity and strongly upon temperature, with a Q₁₀ of 2.7 (experimental) and 2.6 (computed). For real axons, but not the computed axon, the intervals between the first two spikes were shorter than between subsequent spikes. Constant spike frequencies from 75 Hz at low intensities and temperatures to 330 Hz at high intensities and temperatures were soon achieved. Subthreshold and superthreshold responses were sometimes intermixed in a train of responses from a real axon responding to a constant step of current, but not predicted by HH. The time interval following a spike was always longer than that following a subthreshold oscillation in slightly decalcified real axons, as Huxley and FitzHugh also found for computed axons. There was a bias toward spikes at the beginning of the train and toward subthreshold responses later on. Some repeated patterns were found, every second, third, or fourth response being a spike. Neither the HH equations nor the computed or experimental threshold behaviors show a critical temperature to support a membrane phase transition.     

Effect of temperature on electrolyte metabolism of isolated frog skin

A study is presented on the effect of temperature on unidirectional active ion transport, resting electrolyte equilibrium (electrolyte composition), and oxygen consumption in isolated frog skin. The aims were twofold: first, to find out whether the rate of active transport can be changed without affecting the Na⁺ and K⁺ balance of skin itself; second, to arrive at minimal ΔNa/ΔO₂ values by correlating quantitatively inhibition of active ion transport with inhibition of O₂ consumption. NaCl transport was maximal at 20°C. At 28° and at temperatures below 20°, rate of NaCl transport was diminished. In many instances NaCl transport was diminished in skins which maintained their normal Na⁺ and K⁺ content. In several cases, however, neither rate of transport nor resting electrolyte equilibrium was affected; in other cases, both were. O₂ consumption decreased when lowering the temperature over the range from 28 to 10°C. From a plot of log Q_OO2 against 1/T an activation energy of µ 13,700 cal. was calculated, valid for the range from 10 to 20°C. It appeared that µ was smaller for temperatures above 20°C. Working between 10 and 20°, it was found that, on the average, 4 to 5 equivalents of Na⁺ were transported for one mole of O₂ consumed in skins with undisturbed resting electrolyte equilibrium.     

Temperature Compensation of Circadian Period Length in Clock Mutants of Neurospora crassa

Temperature compensation of circadian period length in 12 clock mutants of Neurospora crassa has been examined at temperatures between 16 and 34°C. In the wild-type strain, below 30°C (the “breakpoint” temperature), the clock is well-compensated (Q₁₀ = 1), while above 30°C, the clock is less well-compensated (Q₁₀ = 1.3). For mutants at the frq locus, mutations that shorten the circadian period length (frq-1, frq-2, frq-4, and frq-6) do not alter this temperature compensation response. In long period frq mutants (frq-3, frq-7, frq-8), however, the breakpoint temperature is lowered, and the longer the period length of the mutants the lower the breakpoint temperature. Long period mutants at other loci exhibit other types of alterations in temperature compensation—e.g. chr is well-compensated even above 30°C, while prd-3 has a Q₁₀ significantly less than 1 below 30°C. Prd-4, a short period mutant, has several breakpoint temperatures. Among four double mutants examined, the only unusual interaction between the individual mutations occurred with chr prd, which had an unusually low Q₁₀ value of 0.86 below 27°C. There was no correlation between circadian period length and growth rate. These strains should be useful tools to test models for the temperature compensation mechanism.     

An Investigation of Electrical Conductivity of Steroids

The electrical conductivity of sixteen steroids in the solid state was measured as a function of temperature. While too small to measure at room temperature, the exponential increase in conductivity with temperature can be determined at elevated temperatures (70° - 130°C). Most steroids tested had a conductivity of 10^-13 to 10^-15 ohm^-1 cm^-1 at 100°C and an activation energy of 2 to 4 ev. It it concluded that the observed conductivity is due to impurities. Exposure of keto steroids to iodine vapor results in unstable colored compounds, probably charge transfer complexes, which in some cases have a conductivity as high as 10^-4 ohm^-1 cm^-1.     

Properties of sodium and potassium channels of the squid giant axon far below 0°C

Summary Squid giant axon could be excited in concentrated glycerol solutions containing normal concentrations of electrolytes, when osmolalities of solutions inside and outside the axon were matched. These glycerol solutions did not freeze at the temperature as low as –19°C. The nerve excitation in these solutions were observed at this low temperature. The excitation process at this low temperature was slowed down and time constants of the excitation kinetics were several hundredfold larger than those in normal seawater at 10°C, under which temperature the squid habituated. The temperature coefficients for the electrophysiological membrane parameters under this condition were larger than those in normal seawater above 0°C. The Q₁₀ value for the conduction velocity was 2.0 and that of the duration of the action potential was around 8.5. The time course of the membrane currents was also slowed with the Q₁₀ value of around 5 and the magnitude decreased with the Q₁₀ value of around 2 as the temperature was lowered. The Q₁₀ values for the kinetics of the on process of the Na-channel were around 4.5 and were almost the same as those of the off process of the Na-channel in the wide range of the temperature below 0°C. The Q₁₀ value of the on process of K-channel was around 6.5 and was larger than those for Na-channel. The Q₁₀ values increased gradually as the temperature was lowered.     

Kinetics of Sulfur Oxidation at Suboptimal Temperatures

Chemolithoautotrophic bacteria were enriched from mine water at incubation temperatures ranging from 4 to 46°C, using elemental sulfur as a substrate in acid mineral salts media. Thiobacillus-type bacteria were successfully enriched for at all test temperatures except 46°C. Changes in pH (−dpH/dt) were used to estimate the rate constants for the enrichment cultures. The rate constants yielded a linear Arrhenius plot, an activation energy of 65 kJ/mol, and a temperature coefficient (Q₁₀) of 2.1 for the 4 to 37°C temperature interval.     

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46°54′ S, 37°45′ E) increased from −5°C to a maximum at 25 to 27°C. Q₁₀ values between 0 and 25°C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35°C than at 0°C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 μmol m⁻² s⁻¹, saturation occurring at ca. 1,000 μmol m⁻² s⁻¹. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.     

Temperature dependence of proton permeation through a voltage-gated proton channel

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (I_ratio) were determined. The use of I_ratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q₁₀s for the proton currents were evaluated from the I_ratios. Q₁₀ exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q₁₀. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q₁₀ was decomposed into Q₁₀ of the channel and of the access resistances. Finally, the Q₁₀ for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.     

Acclimation to low temperature by microsomal membranes from tomato cell cultures

Sealed vesicles were prepared from microsomal membranes from cell suspension cultures of tomato (Lycopersicon esculentum Mill cv VF36). ATP-dependent proton transport activity by the vesicles was measured as quenching of fluorescence of acridine orange. Measurements of proton transport were correlated with the activity of a nitrate-inhibitable ATPase. The initial rate of proton influx into the vesicles was strongly temperature dependent with a Q₁₀ of 2 and a maximum rate near 35°C. The data suggest that passive permeability did not increase at chilling temperatures but did increase rapidly with temperatures above 30°C. A comparison was made between membranes from cell cultures grown at 28°C and 9°C. The temperature optimum for proton transport broadened and shifted to a lower temperature range in membranes from cells maintained at 9°C.     

Dynamic Light-Scattering Evidence for the Flexibility of Native Muscle Thin Filaments

We have obtained clear evidence for the flexibility of native scallop adductor thin filaments by studying the temperature and ionic strength dependence of the average decay constants obtained from intensity fluctuation spectroscopic (IFS) measurements. The low-angle (10-25°), average decay constants obtained from time autocorrelation functions of scattered light were independent of concentration (0.08-1.3 mg/ml), scaled with the ratio of temperature to solvent viscosity, T/η, over a range of 4-45°C, and yielded a value for the translational diffusion coefficient of D_T^5°C = (1.24 ± 0.06) × 10^-8 cm²/s. From this value and the Broersma relation for rigid rods, we find an average filament length of 1.06 ± 0.06 μm. Quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that at high temperatures (> 35°C) or in 0.6 M NaCl, tropomyosin completely dissociates from native thin filaments. Decay constants from high-angle (60-150°C) IFS temperature dependence measurements do not scale with T/η and hence do not show the temperature dependence expected for rigid rods. The differences are not due to any change in length distribution of filaments with temperature or to the free tropomyosin in solution, but are attributed to nonrigid motions of the filaments. Similar experiments on samples in high- and low-salt solvents gave results consistent with this interpretation.     

Structure of the Red Fluorescence Band in Chloroplasts

Using Weber's method of "matrix analysis" for the estimation of the number of fluorescent species contributing to the emission of a sample, it is shown that the fluorescence¹ band in spinach chloroplast fragments at room temperature originates in two species of chlorophyll a. Emission spectra obtained upon excitation with different wavelengths of light (preferentially absorbed in chlorophyll a or b) are presented. Upon cooling to - 196°C, the fluorescence efficiency increases about twentyfold. Two additional bands, that now appear at 696 and 735 mµ, suggest the participation of four molecular species. Emission spectra observed at different concentrations of chloroplast fragments with excitation in chlorophyll a and b and excitation spectra for different concentrations of chloroplast fragments and measurements at 685 and 760 mµ are presented. Two of the four emission bands may belong to pigment system I and two to system II. The 685, 696, and 738 mµ bands respond differently to temperature changes. In the -196°C to -150°C range, the intensity of the 685 mµ band remains constant, and that of the 696 mµ band decreases twice as fast as that of the 738 mµ band.     

Thermal Acclimation of Photosynthetic Electron Transport Activity by Thylakoids of Saxifraga cernua

Thermal acclimation by Saxifraga cernua to low temperatures results in a change in the optimum temperature for gross photosynthetic activity and may directly involve the photosynthetic apparatus. In order to test this hypothesis photosynthetic electron transport activity of S. cernua thylakoids acclimated to growth temperatures of 20°C and 10°C was measured in vitro. Both populations exhibited optimum temperatures for whole chain and PSII electron transport activity at temperatures close to those at which the plants were grown. Chlorophyll a fluorescence transients from 10°C-acclimated leaves showed higher rates in the rise and subsequent quenching of variable fluorescence at low measuring temperatures; 20°C-acclimated leaves showed higher rates of fluorescence rise at higher measuring temperatures. At these higher temperatures, fluorescence quenching rates were similar in both populations. The kinetics of State 1-State 2 transitions in 20°C- and 10°C-acclimated leaf discs were measured as changes in the magnitude of the fluorescence emission maxima measured at 77K. Leaves acclimated at 10°C showed a larger F730/F695 ratio at low temperatures, while at higher temperatures, 20°C-acclimated leaves showed a higher F730/F695 ratio after the establishment of State 2. High incubation temperatures also resulted in a decrease in the F695/F685 ratio for 10°C-acclimated leaves, suggesting a reduction in the excitation transfer from the light-harvesting complex of photosystem II to photosystem II reaction centers. The relative amounts of chlorophyll-protein complexes and thylakoid polypeptides separated electro-phoretically were similar for both 20°C- and 10°C-acclimated leaves. Thus, photosynthetic acclimation to low temperatures by S. cernua is correlated with an increase in photosynthetic electron transport activity but does not appear to be accompanied by major structural changes or different relative amounts in thylakoid protein composition.     

Ethylene Removal at Low Temperatures under Biofilter and Batch Conditions

Removal of the plant hormone ethylene (C₂H₄) is often required by horticultural storage facilities, which are operated at temperatures below 10°C. The aim of this study was to demonstrate an efficient, biological C₂H₄ removal under such low-temperature conditions. Peat-soil, acclimated to degradation of C₂H₄, was packed in a biofilter (687 cm³) and subjected to an airflow (~73 ml min⁻¹) with 2 ppm (μl liter⁻¹) C₂H₄. The C₂H₄ removal efficiencies achieved at 20, 10, and 5°C, respectively, were 99.0, 98.8, and 98.4%. This corresponded to C₂H₄ levels of 0.022 to 0.032 ppm in the biofilter outlet air. At 2°C, the average C₂H₄ removal efficiency dropped to 83%. The detailed temperature response of C₂H₄ removal was tested under batch conditions by incubation of 1-g soil samples in a temperature gradient ranging from 0 to 29°C with increments of 1°C. The C₂H₄ removal rate was highest at 26°C (0.85 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹), but remained at levels of 0.14 to 0.28 μg of C₂H₄ g (dry weight)⁻¹ h⁻¹ at 0 to 10°C. At 35 to 40°C, the C₂H₄ removal rate was negligible (0.02 to 0.06 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹). The Q₁₀ (i.e., the ratio of rates 10°C apart) for C₂H₄ removal was 1.9 for the interval 0 to 10°C. In conclusion, the present results demonstrated microbial C₂H₄ removal, which proceeded at 0 to 2°C and produced a moderately psychrophilic temperature response.     

Out of the frying pan into the air—emersion behaviour and evaporative heat loss in an amphibious mangrove fish (Kryptolebias marmoratus)

Amphibious fishes often emerse (leave water) when faced with unfavourable water conditions. How amphibious fishes cope with the risks of rising water temperatures may depend, in part, on the plasticity of behavioural mechanisms such as emersion thresholds. We hypothesized that the emersion threshold is reversibly plastic and thus dependent on recent acclimation history rather than on conditions during early development. Kryptolebias marmoratus were reared for 1 year at 25 or 30°C and acclimated as adults (one week) to either 25 or 30°C before exposure to an acute increase in water temperature. The emersion threshold temperature and acute thermal tolerance were significantly increased in adult fish acclimated to 30°C, but rearing temperature had no significant effect. Using a thermal imaging camera, we also showed that emersed fish in a low humidity aerial environment (30°C) lost significantly more heat (3.3°C min⁻¹) than those in a high humidity environment (1.6°C min⁻¹). In the field, mean relative humidity was 84%. These results provide evidence of behavioural avoidance of high temperatures and the first quantification of evaporative cooling in an amphibious fish. Furthermore, the avoidance response was reversibly plastic, flexibility that may be important for tropical amphibious fishes under increasing pressures from climatic change.     

Temperature Dependence of Voltage-gated H+ Currents in Human Neutrophils,Rat Alveolar Epithelial Cells,and Mammalian Phagocytes

H⁺ currents in human neutrophils, rat alveolar epithelial cells, and several mammalian phagocyte cell lines were studied using whole-cell and excised-patch tight-seal voltage clamp techniques at temperatures between 6 and 42°C. Effects of temperature on gating kinetics were distinguished from effects on the H⁺ current amplitude. The activation and deactivation of H⁺ currents were both highly temperature sensitive, with a Q ₁₀ of 6–9 (activation energy, E _a, ≈ 30–38 kcal/mol), greater than for most other ion channels. The similarity of E _a for channel opening and closing suggests that the same step may be rate determining. In addition, when the turn-on of H⁺ currents with depolarization was fitted by a delay and single exponential, both the delay and the time constant (τ_act) had similarly high Q ₁₀. These results could be explained if H⁺ channels were composed of several subunits, each of which undergoes a single rate-determining gating transition. H⁺ current gating in all mammalian cells studied had similarly strong temperature dependences. The H⁺ conductance increased markedly with temperature, with Q ₁₀ ≥ 2 in whole-cell experiments. In excised patches where depletion would affect the measurement less, the Q ₁₀ was 2.8 at >20°C and 5.3 at <20°C. This temperature sensitivity is much greater than for most other ion channels and for H⁺ conduction in aqueous solution, but is in the range reported for H⁺ transport mechanisms other than channels; e.g., carriers and pumps. Evidently, under the conditions employed, the rate-determining step in H⁺ permeation occurs not in the diffusional approach but during permeation through the channel itself. The large E _a of permeation intrinsically limits the conductance of this channel, and appears inconsistent with the channel being a water-filled pore. At physiological temperature, H⁺ channels provide mammalian cells with an enormous capacity for proton extrusion.