Mechanism of interaction of the cyanine dye DiS−C3-(5) with renal brush-border vesicles

Summary The equilibrium binding mechanism and kinetics of binding of diS–C₃-(5) (3,3-dipropylthiodicarbocyanine iodide) to rabbit renal brush-border membrane vesicles (BBMV) were examined using steady-state and time-resolved fluorescence, and fluorescence stopped-flow methods. In aqueous solution, diS–C₃-(5) exists as a monomer at concentrations <5 m with fluorescence emission peak at 670 nm (excitation 622 nm), anisotropyr=0.102, and lifetime =1.2 nsec (23°C). Upon addition of increasing BBMV (voltage clamped to 0 mV using K⁺/valinomycin), the 670 nm emission peak decreases, corresponding to formation of a nonfluorescent membrane dimer, and subsequently a new emission peak at 695 nm increases, corresponding to membrane monomer. Dynamic depolarization studies show that aqueous diS–C₃-(5) rotation is unhindered with a rotational rateR=0.57 nsec^–1 while membrane monomer is hindered with steady-state anisotropyr=0.190, lifetime =2.1 nsec,R=0.58 nsec^–1 and limiting anisotropyr =0.11. Based on equilibrium fluorescence titrations, the membrane monomer-dimer (M-D) dissociation constant,K _d=[M]²/[D][BBMV], is 0.0013, where BBMV is expressed as membrane phospholipid concentration. Three distinct kinetic processes are identified by stopped-flow experiments in which BBMV are mixed with diS–C₃-(5). There is rapid binding of diS–C₃-(5) to the membrane to form bound monomer with a 6-msec exponential time constant. The membrane monomer at the membrane outer surface then aggregates to form bound dimer at the outer surface with a concentration independent time constant of 30 msec. The overall dimerization reaction probably consists of a rate-limiting reorientation process (30 msec) followed by a rapid dimerization which occurs on a nanosecond time scale. Finally, there is a 0.8 to 1 sec translocation of membrane dimer between symmetric sites at the inner and outer membrane surfaces. The translocation reaction is the step which is probably sensitive to changes in transmembrane electrical potential.     

Calcium-dependent anion channel in the water mold,Blastocladiella emersonii

Summary Injection of depolarizing current into vegetative cells of the water moldBlastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about –40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near –100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr²⁺ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr²⁺. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor.     

Tissue water relations of four co-occurring chaparral shrubs

Summary Chaparral shrubs of California have a suite of morphological and physiological adaptations to withstand the prolonged summer droughts of a mediterranean climate. Not all species of chaparral have the same rooting depth and there is some evidence that those with shallow roots have tissue that is most tolerant to water stress. We tested this notion by comparing the tissue water relations of four co-occurring chaparral shrubs: Quercus durata, Heteromeles arbutifolia, Adenostoma fasciculatum, and Rhamnus californica. We used a pressure-volume technique and a dew-point hygrometer to metsure seasonal changes in osmotic potential when plant tissue was fully hydrated and osmotic potential at predawn, midday, and the turgor loss point. We also calculated seasonal changes in the minimum daily turgor potential, saturated weight/dry weight ratio of leaf tissue, and the bulk modulus of elasticity. We had information on the seasonal water use patterns and apparent rooting depths of these same four shrubs from a previous study (Davis and Mooney 1986). All evidence indicated that Rhamnus had shallow roots and Quercus deep roots. Our results indicated that the tissue water relations of our four co-occurring chaparral shrubs were not alike. Even though Rhamnus had shallow roots, it had the least xerophytic tissue. Seasonal osmotic potential and saturated weight/dry weight ratios were relatively high and bulk modulus of elasticity and minimum daily turgor potentials were low. Furthermore, even though Quercus had deep roots and experienced no seasonal water stress at our study site, its tissue water relations indicated relatively high tolerance to water stress. We conclude that seasonal drought tolerance of stem and leaf tissue of co-occurring chaparral shrubs does not necessarily correspond to rooting depth, to soil moisture resources available to the shrub, or to the degree of seasonal water stress experienced by the shrub.     

Effects of the calcium channel blocker diltiazem on the excitability of mouse oocytes

Effects of the organic Ca antagonist diltiazem on Ca channels were studied in ovulated and unfertilized oocytes of the mouse by using intracellular recording techniques. The resting potential was not affected by diltiazem. The threshold level of the Ca action potential shifted slightly toward positive voltages with diltiazem concentration, but the shift was not statistically significant. The overshoot and maximum rate of rise of the Ca action potential were inhibited by the drug in a dose-dependent manner, but higher amounts of diltiazem were necessary to cause similar blocking effects on Ca channels in mouse oocytes than in other differentiated cells. Increases of external concentration of Ca²⁺ antagonized the degree of diltiazem inhibition. However, the sequence of block of Ca²⁺, Sr²⁺, and Ba²⁺ currents was different for diltiazem vs Cd²⁺. It is suggested that diltiazem inhibition can not be explained by simple competitive scheme, ie, antagonism between diltiazem and permeant cations does not occur at the same binding site associated with the Ca channel in mouse oocytes.     

Catalysis by heteropentalenes of electron transfer to oxygen from ferredoxin-NADP oxidoreductase reduced by NADPH

A range of heteropentalene and bipyridinium compounds have been tested as catalysts of electron transfer to oxygen from spinach ferredoxin-NADP⁺ oxidoreductase reduced by NADPH. For a particular class of compound, the rate of oxygen reduction increased with increasing midpoint potential of the compound under conditions in which reduction of the compound was rate-limiting. Compounds with similar midpoint potentials from different structural classes showed marked differences in rate, attributed to specificity in the interaction with ferredoxin-NADP⁺ oxidoreductase.     

Determination of membrane potential with lipophilic cations: correction of probe binding

The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined in the absence and presence of membrane potential. The lipophilic ions used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0–4) and tetraphenylphosphonium (TPP⁺). In the absence of membrane potential, the amounts of binding were proportional to the probe concentration in the medium when the concentration is dilute. Upon illumination, interior negative membrane potential is generated which induces the uptake of phosphonium cation probe. 2 μM were employed as the initial probe concentration. The real membrane potential was evaluated by means of extrapolation to the state of no binding: The values of for various probes are plotted against the binding coefficient. Here, C_i^app is the apparent intra-vesicular concentration of the probes which is calculated without consideration of bound probes. The ordinate intercept of the plot gives the true concentration ratio, and from this the membrane potential is evaluated. The membrane potential-dependent binding was analysed with a model: the membrane is split into two halves, outer and inner half, and the amounts of bound probes in each region are governed by the concentration in the contiguous solution. We obtained a formula which describes amounts of binding as a function of the membrane potential.     

Effects of the anion transport inhibitor,SITS, on the proximal straight tubule of the rabbit perfusedin vitro

Summary Conventional microelectrodes were used to study the effects of SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonate) on the basolateral membrane potentialVbl of the superficial proximal straight tubule (PST) of the rabbit kidney perfusedin vitro. Addition of 0.1mm SITS to the bathing solution resulted in a slow and irreversible hyperpolarization ofVbl from –42.5±1.17 (37) mV to –77.3±0.83 (52) mV. The new steady-state potential was reached in 10 to 15 min and was accompanied by visible cell swelling. Associated with thisVbl hyperpolarization was: 1) an increased steady-state depolarization (from 6.2±0.77 (17) mV to 25.7±0.83 (29) mV) in response to increasing bath potassium concentration from 5 to 16.7mm (HK); 2) a decreased transient depolarization (from 19.8±1.88 (8) mV to 0.43±0.37 (8) mV) in response to decreasing bath bicarbonate concentration from 22 to 6.6mm at constant bath pH (L-HCO₃); and 3) inhibition of a depolarizing overshoot and a decreased steady-state depolarization (from 35.9±1.84 (12) mV to 4.7±1.37 (13) mV) in response to reducing bath sodium concentration from 144 to zero (0-Na). Sodium, chloride and NMDG (N-methyl-d-glucamine) were used as the substituting ions, respectively. These results are consistent with the presence of a coupled sodium-bicarbonate carrier in the basolateral membrane which is electrogenic and SITS inhibitable. Comparison of the time course of SITS effects on these ion-substitution responses suggests that the inhibition of the bicarbonate exit pathway(s) is the primary event and that the changes inVbl and in the steady-stateVbl responses to HK and 0-Na are secondary events which may be related to changes in intracellular composition and/or basolateral membrane properties.     

Molecular motion underlying activation and inactivation of sodium channels in squid giant axons

Summary Measurements of the changes in birefringence associated with changes in membrane potential were made with internally perfused squid giant axons in low sodium solutions at 0–8°C. The time course of the birefringence changes share many properties of the gating (polarization) currents previously studied in this nerve. Both can be demonstrated as an asymmetry in the response to voltage pulses symmetrical about the resting potential which is not present about a hyperpolarized holding potential. Both have a rapid relaxation, which precedes the sodium permeability change. Both exhibit an initial delay or rising phase. Both are reversibly blocked by perfusion with 30mm colchicine; neither are altered by changes on sodium concentrations or 300nm tetrodotoxin. The birefringence response has a decrease in the amplitude of the rapid relaxation associated with the appearance of a slow relaxation. This is similar to the immobilization of fast gating charges which parallels sodium current inactivation.The amplitude of the birefringence and the gating current responses is consistent with a change in the alignment of several hundred peptide bonds per sodium channel.