Possible effect of structural phase transition in the reactive center of Rhodobacter sphaeroides on the rate of dark adaptation of photooxidized bacteriochlorophyll from primary quinone

The dependence of the rate of dark recombination between the photooxidized primary donor--dimer bacteriochlorophyll molecule (P) and reduced primary quinone acceptor (QA), P+QA(-)-->PQA was studied in photosynthetic reaction centers (RC) from Rhodobacter sphaeroides in the temperature range of 100-320 K. Control RC preparations, RC species with the removed H-subunit as well as RC samples with the hydrogen bonds network modified by isotopic D2O-H2O substitution were investigated. An anomalous temperature dependence of the recombination time (tau rec) of dark reaction P+QA(-)-->PQA was found for all RC samples. It was found that upon heating from 120 to 290 K tau rec increased 2.5 fold. However, upon further heating to 320 K, tau rec decreased again. The temperature dependences of the P+QA(-)-->PQA recombination time were compared with those of the thermodepolarization current of RC preparations in the same temperature range. The temperature curve of the thermodepolarization current was also nonmonotonous. The theoretical interpretation of the temperature dependence of tau rec as well as of the thermodepolarization current was made in the framework of the theory of structural phase transitions within the hydrogen bond network in the water-protein surrounding of the redox centers participating in the electron transfer reactions.     

A network of hydrogen bonds in the reaction centers of Rhodobacter sphaeroides serves as a regulatory factor of the temperature dependence of the recombination rate constant of photooxidized bacteriochlorophyll and primary quinone acceptors

The dark recombination rate constant for the photooxidized bacteriochlorophyll (P) and reduced primary quinone acceptor (QA) in the photosynthetic reaction centers (RC) from purple bacterium Rhodobacter sphaeroides depends nonmonotonically on temperature. The time of this reaction is approximately 100 ms at 270-300 K and decreases as the temperature both increases and decreases beyond this temperature range. It is known that the dome-shaped dependence of the thermodynamic stability on temperature is an intrinsic feature of many proteins in solution. The experimental results on the nonmonotonous temperature dependence of P+ and QA- recombination rate constant are discussed in terms of general thermodynamic approaches. The dynamic properties of the network of hydrogen bonds that are involved in the relaxation processes accompanying the electron transport are considered as a regulatory factor of the efficiency of electron transfer.     

Block by internal Mg2+ causes voltage-dependent inactivation of Kv1.5

Internal Mg2+ blocks many potassium channels including Kv1.5. Here, we show that internal Mg2+ block of Kv1.5 induces voltage-dependent current decay at strongly depolarised potentials that contains a component due to acceleration of C-type inactivation after pore block. The voltage-dependent current decay was fitted to a bi-exponential function (tau(fast) and tau(slow)). Without Mg2+, tau(fast) and tau(slow) were voltage-independent, but with 10 mM Mg2+, tau(fast) decreased from 156 ms at +40 mV to 5 ms at +140 mV and tau(slow) decreased from 2.3 s to 206 ms. With Mg2+, tail currents after short pulses that allowed only the fast phase of decay showed a rising phase that reflected voltage-dependent unbinding. This suggested that the fast phase of voltage-dependent current decay was due to Mg2+ pore block. In contrast, tail currents after longer pulses that allowed the slow phase of decay were reduced to almost zero suggesting that the slow phase was due to channel inactivation. Consistent with this, the mutation R487V (equivalent to T449V in Shaker) or increasing external K+, both of which reduce C-type inactivation, prevented the slow phase of decay. These results are consistent with voltage-dependent open-channel block of Kv1.5 by internal Mg2+ that subsequently induces C-type inactivation by restricting K+ filling of the selectivity filter from the internal solution.     

Dioxygen replacement reaction in myoglobin

The replacement reaction of myoglobin (Mb), MbCO + O2 leads to MbO2 + CO leads to MbCO + O2, has been studied with flash photolysis in the temperature range from 140 to 320 K and the time range from 2 mus to 200 s. In a fraction of the Mb, the photodissociated CO remains within the protein; rebinding is not affected by the presence of O2 and occurs with rates that are identical with the ones observed earlier in solvents containing only CO. In the remaining fraction CO migrates into the solvent and Mb combines preferentially with oxygen. The rate of the subsequent replacement of O2 by CO permits calculation of the oxygen dissociation rate ko2; ko2 has been determined from 260 to 320 K. The measurements support a multibarrier model.     

Temperature dependence of kinetic parameters of single potential dependent K+ channels in mollusc neurons. Similarity in the action of potential and temperature]

Using the patch-voltage-clamp method on excised membrane fragments from molluscan neurones temperature dependences of kinetic parameters of the fast and slow K(+)-channels were investigated in the temperature range 1 to 40 degrees C. Temperature dependences of probability of the channel open state (P0) for the slow and fast K(+)-channels are, generally, opposite, that is P0 increases for the slow channel and decreases for the fast channel with temperature. Similar dependences characterize durations of single channel open intervals (tau 0) and burst durations (t(p)). Durations of interburst and interpulse intervals (respectively, t(i) and tau) decrease for the slow channel and increase, in contrast, for the fast channel with temperature. For the channels of both types temperature dependences of P0 (as for other parameters) are essentially nonmonotonous. There are two local extrema, at least: for the slow K(+)-channel-maximum at 15 degrees C (minimum for the fast channel) and minimum at 20-25 degrees C (maximum for the fast channel). In some cases the number of local extrema may be greater than two. Some similarity in the action of temperature and membrane potential on the kinetic parameters was observed. For the slow K(+)-channel P0, tau 0 and t p increase with temperature and membrane potential. For the fast channel these parameters decrease at the same conditions. Moreover, for the channels of both types temperature dependences of the kinetic parameters are slightly pronounced at the potentials where potential dependences of the parameters are least. As a whole, temperature measurements showed that there are, possibly, several points of structural transitions (similar to phase transitions) in the temperature range 0 to 40 degrees C. Primarily, the kinetic parameters are determined by these transitions.     

Molecular basis for the different activation kinetics of the pacemaker channels HCN2 and HCN4

The pacemaker channels HCN2 and HCN4 have been identified in cardiac sino-atrial node cells. These channels differ considerably in several kinetic properties including the activation time constant (tau act), which is fast for HCN2 (144 ms at -140 mV) and slow for HCN4 (461 ms at -140 mV). Here, by analyzing HCN2/4 chimeras and mutants we identified single amino acid residues in transmembrane segments 1 and 2 and the connecting loop between S1 and S2 that are major determinants of this difference. Replacement of leucine 272 in S1 of HCN4 by the corresponding phenylalanine present in HCN2 decreased tau act of HCN4 to 149 ms. Conversely, activation of the fast channel HCN2 was decreased 3-fold upon the corresponding mutation of F221L in the S1 segment. Mutation of N291T and T293A in the linker between S1 and S2 of HCN4 shifted tau act to 275 ms. While residues 272, 291, and 293 of HCN4 affected the activation speed at basal conditions they had no obvious influence on the cAMP-dependent acceleration of activation kinetics. In contrast, mutation of I308M in S2 of HCN4 abolished the cAMP-dependent decrease in tau act. Surprisingly, this mutation also prevented the acceleration of channel activation observed after deletion of the C-terminal cAMP binding site. Taken together our results indicate that the speed of activation of the HCN4 channel is determined by structural elements present in the S1, S1-S2 linker, and the S2 segment.     

Orientational ordering and dynamics of the hydrate and exchangeable hydrogen atoms in crystalline crambin

Deuterium nuclear magnetic resonance studies of crambin crystals grown from deuterated solvent (2H2O/CH3CH2O2H or H2O/C2H3CH2OH) are reported. The extent to which the hydrate and exchangeable hydrogen atoms are dynamically disordered are then determined from the size of the residual deuterium quadrupole couplings, qcc. Rapid molecular reorientation (tau c-1 greater than 10(5) s-1) reduces the magnitude of the quadrupole coupling from its static value (216 kHz for solid water). We find that the room temperature spectrum of crambin is dominated by two features: a sharp line with very small residual quadrupolar coupling less than 3 kHz, and a broad pattern with a quadrupolar coupling in the range 185 to 195 kHz. The former is indicative of very nearly isotropically reorienting deuterons, whereas the latter is somewhat narrower than that observed for the amide deuterons of poly-gamma-benzyl-L-glutamate and thus indicative of deuterons that are almost but not completely stationary. By considering the nuclear magnetic resonance spectrum intensities along with the amino acid sequence, X-ray structure and the manner in which quadrupole couplings are reduced by dynamics, we conclude that the nuclear magnetic resonance signal from most of the water molecules of hydration are contained in the sharp line, i.e. reorient nearly isotropically in the crystalline protein. Unlike bulk water, which freezes abruptly in the manner of a phase transition, the water of hydration in crambin has a broad freezing range from 180 to 250K, as evidenced by the decreasing intensity of the sharp line that disappears at 180K. At temperatures between 150 and 200K, a typical hydrate molecule reorients at a rate comparable to the quadrupole coupling, 10(4) s-1 to 10(5) s-1, a process that occurs in hexagonal ice in the range of 240 to 270K. At 140K, the hydrate is stationary, tau c-1 less than 10(3) s-1. Studies of the protein crystallized from solvent deuterated only at the non-exchangeable methyl group of ethanol confirm that ethanol is in the lattice and show that this solvate behaves in much the same way as the hydrate. The refined X-ray structure has identified four ethanol solvate molecules. The deuterium spectrum at room temperature has a well-defined residual pattern with qcc = 2.2 kHz, i.e. a small-order parameter consistent with nearly isotropically reorienting molecules. The spectrum width broadens substantially only at temperatures below 200K and achieves the characteristic spectrum of a rotating methyl group with stationary C-C axis at 140K.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intramolecular dynamics and electron transfer in photosynthetic reaction centers. A study using luminescence

The temperature dependences of fluorescence and phosphorescence spectra maxima of chromophor labels--endogenic (tryptophan) and exogenic (eosinisothiocyanate)--were measured for the preparations of photosynthetic membranes and reaction centers from Rhodospirillum rubrum. It was found that the dipole mobility of protein-lipid matrix in the vicinity of the chromophores intensified markedly with a temperature rise from 150 to 300K resulting in the corresponding relaxation time tau r decrease from 10(0) to 10(-8) s. The efficiency of direct transfer of the photomobilized electron in the system of quinone acceptors (A1- leads to A2) of reaction centers (characteristic half-times of the process being 10(-3) divided by 10(-4) s) was shown also to increase sharply at temperatures higher than 200K parallel to the enhancement of molecular motions with tau r approximately 10(-8) s. Meanwhile, changes observed in the rate of recombination of primary photoproducts, i.e. an oxidized bacteriochlorophyll dimer, P+ and a reduced acceptor, A1- (characteristic half-time of 10(-1) divided by 10(-2) s) and the activization of low-frequency motions with tau r approximately 10(-3) s in the external layers and tau r less than 1 s in the internal parts of the reaction centers protein develop over the same range of low temperatures (150-220 K). The nature of interactions which determine the dependence of the photosynthetic electron transport on the molecular mobility of the membrane proteins is discussed.     

Effect of energy status of hydrogen bond protons on the rate of electron transfer in photosynthetic reaction centers

We present here a theoretical interpretation of the temperature dependence of the rate of dark recombination which takes place in Rhodobacter sphaeroides reaction centers between a primary quinone (Q(A)) and a bacteriochlorophyll dimer. Taking the energy of interaction between hydrogen bond protons and an excessive electron into account, we described qualitative by this nonmonotonous dependence. We considered a molecular model of the primary quinone from Rb. sphaeroides reaction centers. In addition to the primary quinone, the model includes two reaction center fragments that form hydrogen bonds with Q(A). One of these fragments is His(M219), and the other is the peptide [Asn(M259) - Ala(M260)]. We used the two-center approach with regard for electron-phonon interaction in order to calculate the characteristic time of electron tunneling during the recombination reaction. The energy of the phonon emitted/absorbed during the electron tunneling is determined by a relative shift of the donor and the acceptor energy levels, the detuning of levels. The value of level detuning was shown to be temperature dependent in a nonmonotonous manner in the case of hydrogen bonds with double-well potential energy surface. The characteristic time (or the reaction rate) depends on temperature parametrically. The dependence is nonmonotonous and is in qualitative agreement with the experimental one.     

Low molecular weight poly(A)+ mRNA species encode factors that modulate gating of a non-Shaker A-type K+ channel

Voltage-dependent K+ channels control repolarization of action potentials and help establish firing patterns in nerve cells. To determine the nature and role of molecular components that modulate K+ channel function in vivo, we coinjected Xenopus oocytes with cRNA encoding a cloned subthreshold A-type K+ channel (mShal1, also referred to as mKv4.1) and a low molecular weight (LMW) fraction (2-4 kb) of poly(A)+ mRNA (both from rodent brain). Coinjected oocytes exhibited a significant (fourfold) increase in the surface expression of mShal1 K+ channels with no change in the open-channel conductance. Coexpression also modified the gating kinetics of mShal1 current in several respects. Macroscopic inactivation of whole oocyte currents was fitted with the sum of two exponential components. Both fast and slow time constants of inactivation were accelerated at all membrane potentials in coinjected oocytes (tau f = 47.2 ms vs 56.5 ms at 0 mV and tau s = 157 ms vs 225 ms at 0 mV), and the corresponding ratios of amplitude terms were shifted toward domination by the fast component (Af/As = 2.71 vs 1.17 at 0 mV). Macroscopic activation was characterized in terms of the time-to-peak current, and it was found to be more rapid at all membrane potentials in coinjected oocytes (9.9 ms vs 13.5 ms at 0 mV). Coexpression also leads to more rapid recovery from inactivation (approximately 2.4-fold faster at -100 mV). The coexpressed K+ currents in oocytes resemble currents expressed in mouse fibroblasts (NIH3T3) transfected only with mShal1 cDNA. These results indicate that mammalian regulatory subunits or enzymes encoded by LMW mRNA species, which are apparently missing or expressed at low levels in Xenopus oocytes, may modulate gating in some native subthreshold A-type K+ channels.     

Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila.

The photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm. Both time-gated spectra and single wavelength traces were measured. Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T.E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423; Meyer, T. E., G. Tollin, T. P. Causgrove, P. Cheng, and R. E. Blankenship. 1991. Biophys. J. 59:988-991). The first, red-shifted intermediate decays biexponentially (60% with tau = 0.25 ms and 40% with tau = 1.2 ms) to a blue-shifted intermediate. The last step of the photocycle is the biexponential (93% with tau = 0.15 s and 7% with tau = 2.0 s) recovery to the ground state of the protein. Reconstruction of the absolute spectra of these photointermediates yielded absorbance maxima of about 465 and 355 nm for the red- and blue-shifted intermediate with an epsilon max at about 50% and 40% relative to the epsilon max of the ground state. The quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.     

Double-quantum-filtered 23Na NMR study of intracellular sodium in the perfused liver.

We acquired double-quantum-filtered 23Na NMR spectra from perfused liver, using a range of tau values from 0.2 to 24 ms, where tau is the separation between the first and second pi/2 pulses in the radio-frequency pulse sequence. For each tau value we compared the amplitude of the double-quantum-filtered 23Na NMR signal acquired from intracellular sodium ions when the liver was perfused with buffer containing the "shift reagent" Dy(PPP)2 to the amplitude of the total double-quantum-filtered 23Na NMR signal acquired when the liver was perfused with buffer containing no Dy(PPP)2. For tau < or = 4 ms, the average ratio of the two amplitudes was 0.98 +/- 0.03 (mean +/- SEM). For tau > or = 8 ms, the average ratio was significantly less than 1. These results demonstrate that double-quantum-filtered 23Na NMR signals acquired from perfused liver using short tau values arise almost exclusively from intracellular sodium ions, but double-quantum-filtered 23Na NMR signals acquired from perfused liver using long tau values contain contributions from both intracellular and extracellular sodium ions. This conclusion suggests that multiple-quantum-filtered 23Na NMR spectroscopy will be useful in studying intracellular sodium levels in the perfused liver, and possibly in the intact liver in vivo.     

Voltage-gated transient currents in bovine adrenal fasciculata cells. I. T-type Ca2+ current

The whole cell version of the patch clamp technique was used to identify and characterize voltage-gated Ca2+ channels in enzymatically dissociated bovine adrenal zona fasciculata (AZF) cells. The great majority of cells (84 of 86) expressed only low voltage-activated, rapidly inactivating Ca2+ current with properties of T-type Ca2+ current described in other cells. Voltage-dependent activation of this current was fit by a Boltzmann function raised to an integer power of 4 with a midpoint at -17 mV. Independent estimates of the single channel gating charge obtained from the activation curve and using the "limiting logarithmic potential sensitivity" were 8.1 and 6.8 elementary charges, respectively. Inactivation was a steep function of voltage with a v1/2 of -49.9 mV and a slope factor K of 3.73 mV. The expression of a single Ca2+ channel subtype by AZF cells allowed the voltage-dependent gating and kinetic properties of T current to be studied over a wide range of potentials. Analysis of the gating kinetics of this Ca2+ current indicate that T channel activation, inactivation, deactivation (closing), and reactivation (recovery from inactivation) each include voltage-independent transitions that become rate limiting at extreme voltages. Ca2+ current activated with voltage- dependent sigmoidal kinetics that were described by an m4 model. The activation time constant varied exponentially at test potentials between -30 and +10 mV, approaching a voltage-independent minimum of 1.6 ms. The inactivation time constant (tau i) also decreased exponentially to a minimum of 18.3 ms at potentials positive to 0 mV. T channel closing (deactivation) was faster at more negative voltages; the deactivation time constant (tau d) decreased from 8.14 +/- 0.7 to 0.48 +/- 0.1 ms at potentials between -40 and -150 mV. T channels inactivated by depolarization returned to the closed state along pathways that included two voltage-dependent time constants. tau rec-s ranged from 8.11 to 4.80 s when the recovery potential was varied from - 50 to -90 mV, while tau rec-f decreased from 1.01 to 0.372 s. At potentials negative to -70 mV, both time constants approached minimum values. The low voltage-activated Ca2+ current in AZF cells was blocked by the T channel selective antagonist Ni2+ with an IC50 of 20 microM. At similar concentrations, Ni2+ also blocked cortisol secretion stimulated by adrenocorticotropic hormone. Our results indicate that bovine AZF cells are distinctive among secretory cells in expressing primarily or exclusively T-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

A tyrosine substitution in the cavity wall of a k channel induces an inverted inactivation

Ion permeation and gating kinetics of voltage-gated K channels critically depend on the amino-acid composition of the cavity wall. Residue 470 in the Shaker K channel is an isoleucine, making the cavity volume in a closed channel insufficiently large for a hydrated K(+) ion. In the cardiac human ether-a-go-go-related gene channel, which exhibits slow activation and fast inactivation, the corresponding residue is tyrosine. To explore the role of a tyrosine at this position in the Shaker channel, we studied I470Y. The activation became slower, and the inactivation faster and more complex. At +60 mV the channel inactivated with two distinct rates (tau(1) = 20 ms, tau(2) = 400 ms). Experiments with tetraethylammonium and high K(+) concentrations suggest that the slower component was of the P/C-type. In addition, an inactivation component with inverted voltage dependence was introduced. A step to -40 mV inactivates the channel with a time constant of 500 ms. Negative voltage steps do not cause the channel to recover from this inactivated state (tau > 10 min), whereas positive voltage steps quickly do (tau = 2 ms at +60 mV). The experimental findings can be explained by a simple branched kinetic model with two inactivation pathways from the open state.     

Ligand binding and the catalytic reaction of cytochrome caa(3) from the thermophilic bacterium Rhodothermus marinus

The ligand-binding dynamics and the reaction with O(2) of the fully (five-electron) reduced cytochrome caa(3) from the thermohalophilic bacterium Rhodothermus (R.) marinus were investigated. The enzyme is a proton pump which has all the residues of the proton-transfer pathways found in the mitochondrial-like enzymes conserved, except for one of the key elements of the D-pathway, the helix-VI glutamate [Glu(I-286), R. sphaeroides numbering]. In contrast to what has been suggested previously as general characteristics of thermophilic enzymes, during formation of the R. marinus caa(3)-CO complex, CO binds weakly to Cu(B), and is rapidly (k(Ba) = 450 s(-1)) trapped by irreversible (K(Ba) = 4.5 x 10(3)) binding to heme a(3). Upon reaction of the fully reduced enzyme with O(2), four kinetic phases were resolved during the first 10 ms after initiation of the reaction. On the basis of a comparison to reactions observed with the bovine enzyme, these phases were attributed to the following transitions between intermediates (pH 7.8, 1 mM O(2)): R --> A (tau congruent with 8 micros), A --> P(r) (tau congruent with 35 micros), P(r) --> F (tau congruent with 240 micros), F --> O (tau congruent with 2.5 ms), where the last two phases were associated with proton uptake from the bulk solution. Oxidation of heme c was observed only during the last two reaction steps. The slower transition times as compared to those observed with the bovine enzyme most likely reflect the replacement of Glu(I-286) of the helix-VI motif -XGHPEV- by a tyrosine in the R. marinus enzyme in the motif -YSHPXV-. The presence of an additional, fifth electron in the enzyme was reflected by two additional kinetic phases with time constants of approximately 20 and approximately 720 ms during which the fifth electron reequilibrated within the enzyme.     

On the structure and conformational dynamics of yeast phenylalanine-accepting transfer ribonucleic acid in solution.

The solution structure of yeast tRNAPhe was investigated by using ethidium as a fluorescent probe in the D loop and the anticodon loop. For this purpose the dihydrouracils in position 16/17 and wybutine in position 37 were substituted by ethidium. The lifetimes and the time-dependent anisotropy of ethidium fluorescence were measured by pulsed nanosecond fluorometry. The kinetics of the transitions between different states of the tRNAPheEtd derivatives were determined by chemical relaxation measurements. It was found that the ethidium label irrespective of its position exhibits three different states called T1, T2 and T3 characterized by lifetimes tau 1 = 30 ns, tau 2 = 12 ns, and tau 3 = 3 ns. The lifetime differences are due to different accessibilities of ethidium for solvent quenching in the three states. Thus, there are three different defined structural environments of the ethidium in both the anticodon and the D loop. The distribution of the three states was measured as a function of Mg2+ concentration and temperature; it was found that state T3 is favored over states T2 and T1 by both increasing Mg2+ concentration and increasing temperature. The chemical relaxation kinetics exhibit a fast transition between T1 and T2 (10--100 ms) and a slow transition between T2 and T3 (100--1000 ms). The rates of both transitions depend likewise on Mg2+ concentration and temperature. The equilibrium and kinetic data clearly show the presence of strong and weak interactions between Mg2+ and tRNA. A cooperative model accounting for this behavior is developed. The ethidium probe behaves identically when located in different regions of the tRNA regarding both its distribution of states and its transition kinetics. This suggests that the different spectroscopic states report different conformations of the tRNA structure. The dependence of the three states on Mg2+ and spermine indicates that conformation T3 is closely related to or identical with the crystal structure. The rotational diffusion constants indicate that of all three states T3 is most extended while T2 is most compact. The thermodynamic analysis reveals that the strongly bound Mg2+ ions reduce both the activation entropy and enthalpy of all transitions. The weakly bound Mg2+ ions increase both the activation enthalpy and entropy of the slow transition between T2 and T3. It is suggested that the breaking of several intramolecular bonds, e.g., hydrogen bonds, is involved in this transition.     

Role of impaired myocardial relaxation in the production of elevated left ventricular filling pressure

Although present in many patients with heart failure and a normal ejection fraction, the role of isolated impairments in active myocardial relaxation in the genesis of elevated filling pressures is not well characterized. Because of difficulties in determining the effect of prolonged myocardial relaxation in vivo, we used a cardiovascular simulated computer model. The effect of myocardial relaxation, as assessed by tau (exponential time constant of relaxation), on pulmonary vein pressure (PVP) and left ventricular end-diastolic pressure (LVEDP) was investigated over a wide range of tau values (20-100 ms) and heart rate (60-140 beats/min) while keeping end-diastolic volume constant. Cardiac output was recorded over a wide range of tau and heart rate while keeping PVP constant. The effect of systolic intervals was investigated by changing time to end systole at the same heart rate. At a heart rate of 60 beats/min, increases in tau from a baseline to extreme value of 100 ms cause only a minor increase in PVP of 3 mmHg. In contrast, at 120 beats/min, the same increase in tau increases PVP by 23 mmHg. An increase in filling pressures at high heart rates was attributable to incomplete relaxation. The PVP-LVEDP gradient was not constant and increased with increasing tau and heart rate. Prolonged systolic intervals augmented the effects of tau on PVP. Impaired myocardial relaxation is an important determinant of PVP and cardiac output only during rapid heart rate and especially when combined with prolonged systolic intervals. These findings clarify the role of myocardial relaxation in the pathogenesis of elevated filling pressures characteristic of heart failure.     

Characterization of slowly inactivating KV{alpha} current in rabbit pulmonary neuroepithelial bodies: effects of hypoxia and nicotine

Pulmonary neuroepithelial bodies (NEB) form innervated cell clusters that express voltage-activated currents and function as airway O(2) sensors. We investigated A-type K(+) currents in NEB cells using neonatal rabbit lung slice preparation. The whole cell K(+) current was slowly inactivating with activation threshold of approximately -30 mV. This current was blocked approximately 27% by blood-depressing substance I (BDS-I; 3 microM), a selective blocker of Kv3.4 subunit, and reduced approximately 20% by tetraethylammonium (TEA; 100 microM). The BDS-I-sensitive component had an average peak value of 189 +/- 14 pA and showed fast inactivation kinetics that could be fitted by one-component exponential function with a time constant of (tau1) 77 +/- 10 ms. This Kv slowly inactivating current was also blocked by heteropodatoxin-2 (HpTx-2; 0.2 microM), a blocker of Kv4 subunit. The HpTx-2-sensitive current had an average peak value of 234 +/- 23 pA with a time constant (tau) 82 +/- 11 ms. Hypoxia (Po(2) = 15-20 mmHg) inhibited the slowly inactivating K(+) current by approximately 47%, during voltage steps from -30 to +30 mV, and no further inhibition occurred when TEA was combined with hypoxia. Nicotine at concentrations of 50 and 100 microM suppressed the slowly inactivating K(+) current by approximately 24 and approximately 40%, respectively. This suppression was not reversed by mecamylamine suggesting a direct effect of nicotine on these K(+) channels. In situ hybridization experiments detected expression of mRNAs for Kv3.4 and Kv4.3 subunits, while double-label immunofluorescence confirmed membrane localization of respective channel proteins in NEB cells. These studies suggest that the hypoxia-sensitive current in NEB cells is carried by slowly inactivating A-type K(+) channels, which underlie their oxygen-sensitive potassium currents, and that exposure to nicotine may directly affect their function, contributing to smoking-related lung disease.     

Voltage dependence of proton pumping by bacteriorhodopsin is regulated by the voltage-sensitive ratio of M1 to M2.

The voltage dependence of light-induced proton pumping was studied with bacteriorhodopsin (bR) from Halobacterium salinarum, expressed in the plasma membrane of oocytes from Xenopus laevis in the range -160 mV to +60 mV at different light intensities. Depending on the applied field, the quenching effect by blue light, which bypasses the normal photo and transport cycle, is drastically increased at inhibiting (negative) potentials, and is diminished at pump current increasing (positive) potentials. At any potential, two processes with different time constants for the M --> bR decay of approximately 5 ms (tau1) and approximately 20 ms (tau2) are obtained. At pump-inhibiting potentials, a third, long-lasting process with tau3 approximately 300 ms at neutral pH is observed. The fast processes (tau1, tau2) can be assigned to the decay of M2 in the normal pump cycle, i.e., to the reprotonation of the Schiff base via the cytoplasmic side, whereas tau3 is due to the decay of M1 without net pumping, i.e., the reprotonation of the Schiff base via the extracellular side. The results are supported by determination of photocurrents induced by bR on planar lipid films. The pH dependence of the slow decay of M1 is fully in agreement with the interpretation that the reprotonation of the Schiff base occurs from the extracellular side. The results give strong evidence that an externally applied electrical field changes the ratio of the M1 and the M2 intermediate. As a consequence, the transport cycle branches into a nontransporting cycle at negative potentials. This interpretation explains the current-voltage behavior of bR on a new basis, but agrees with the isomerisation, switch, transfer model for vectorial transport.