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S Luvisetto

Publications and source records attributed to S Luvisetto.

At least 19 recordsLinked to original sources
PubMed1999

Functional consequences of mutations in the human alpha1A calcium channel subunit linked to familial hemiplegic migraine.

Mutations in alpha1A, the pore-forming subunit of P/Q-type calcium channels, are linked to several human diseases, including familial hemiplegic migraine (FHM). We introduced the four missense mutations linked to FHM into human alpha1A-2 subunits and investigated their functional consequences after expression in human embryonic kidney 293 cells. By combining single-channel and whole-cell patch-clamp recordings, we show that all four mutations affect both the biophysical properties and the density of functional channels. Mutation R192Q in the S4 segment of domain I increased the density of functional P/Q-type channels and their open probability. Mutation T666M in the pore loop of domain II decreased both the density of functional channels and their unitary conductance (from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of domains II and IV shifted the voltage range of activation toward more negative voltages, increased both the open probability and the rate of recovery from inactivation, and decreased the density of functional channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by mutations T666M and V714A was not observed in some patches or periods of activity, suggesting that the abnormal channel may switch on and off, perhaps depending on some unknown factor. Our data show that the FHM mutations can lead to both gain- and loss-of-function of human P/Q-type calcium channels.

Amino Acid Substitution↗
PubMed1999

Anomalous L-type calcium channels of rat spinal motoneurons.

Single channel patch-clamp recordings show that embryonic rat spinal motoneurons express anomalous L-type calcium channels, which reopen upon repolarization to resting potentials, displaying both short and long reopenings. The probability of reopening increases with increasing voltage of the preceding depolarization without any apparent correlation with inactivation during the depolarization. The probability of long with respect to short reopenings increases with increasing length of the depolarization, with little change in the total number of reopenings and in their delay. With less negative repolarization voltages, the delay increases, while the mean duration of both short and long reopenings decreases, remaining longer than that of the openings during the preceding depolarization. Open times decrease with increasing voltage in the range -60 to +40 mV. Closed times tend to increase at V > 20 mV. The open probability is low at all voltages and has an anomalous bell-shaped voltage dependence. We provide evidence that short and long reopenings of anomalous L-type channels correspond to two gating modes, whose relative probability depends on voltage. Positive voltages favor both the transition from a short-opening to a long-opening mode and the occupancy of a closed state outside the activation pathway within each mode from which the channel reopens upon repolarization. The voltage dependence of the probability of reopenings reflects the voltage dependence of the occupancy of these closed states, while the relative probability of long with respect to short reopenings reflects the voltage dependence of the equilibrium between modes. The anomalous gating persists after patch excision, and therefore our data rule out voltage-dependent block by diffusible ions as the basis for the anomalous gating and imply that a diffusible cytosolic factor is not necessary for voltage-dependent potentiation of anomalous L-type channels.

Animals↗
PubMed1997

Hyperthyroidism and mitochondrial uncoupling.

During the past years many efforts have been made to elucidate the origin of the uncoupling mechanisms induced by hyperthyroidism in mitochondria. Two main mechanisms have been proposed: a classical protonophoric uncoupler mechanism, considering the action of thyroid hormones at the level of the lipid membrane bilayer, and a slipping mechanism with more localized effects at the level of the redox proton pumps. This short review is devoted to comparing and discussing the evidence against and in favour of these two mechanisms.

Animals↗
PubMed1996

The nature of uncoupling by n-hexane, 1-hexanethiol and 1-hexanol in rat liver mitochondria.

We have analyzed the effects of n-hexane, 1-hexanethiol, and 1-hexanol on the coupled respiration of rat liver mitochondria. Incubation of mitochondria with n-hexane, 1-hexanethiol and 1-hexanol resulted in a stimulation, at low concentrations, and an inhibition, at high concentrations, of the state 4 mitochondrial respiration. Three criteria, all based on the comparison with the effect of DNP, have been used to establish whether the stimulation of respiration, at low concentrations of n-hexane, 1-hexanethiol, and 1-hexanol, depends on protonophoric mechanisms. First, the quantitative relationship between the extents of respiratory stimulation and membrane potential depression: a strong decrease of membrane potential was induced by increasing concentrations of DNP and a negligible depression by increasing concentrations of n-hexane or 1-hexanethiol. Only a slight decrease was induced by 1-hexanol. Second, the quantitative relationship between the extents of respiratory stimulation and of proton conductance increase: at equivalent rates of respiration, the enhancement of the proton conductance induced by DNP was very marked, by n-hexane and 1-hexanethiol practically negligible, and by 1-hexanol much smaller than that induced by DNP. Third, in titrations with redox inhibitors of the proton pumps, the pattern of the relationship between proton pump conductance and membrane potential was markedly different from protonophoric and non-protonophoric uncouplers: almost linear in the case of DNP, highly non-linear in the case of n-hexane, 1-hexanethiol and 1-hexanol. These three criteria support the view that n-hexane, 1-hexanethiol, and partially 1-hexanol, uncouple mitochondrial respiration by a non-protonophoric mechanism.

2,4-Dinitrophenol↗
PubMed1995

Nature of respiratory stimulation in hyperthyroidism: the redox behaviour of cytochrome c.

Hyperthyroid mitochondria show an increased Km and Vmax in the high affinity phase of cytochrome oxidase kinetics. During inhibitor titrations, cytochrome c shows a different redox behaviour in hyperthyroid with respect to protonophore-treated euthyroid mitochondria. The observations are discussed in terms of a different regulation of electron input and output into the respiratory chain during slip and leak types of uncoupling. In hyperthyroid mitochondria during inhibitor titrations, the pattern of the relationship between uncoupler-induced extra-respiration and membrane potential is highly non-linear. The complex nature of the respiratory stimulation in hyperthyroid mitochondria is discussed.

Animals↗
PubMed1995

The nature of mitochondrial respiration and discrimination between membrane and pump properties.

A new criterion is utilized for the interpretation of flow-force relationships in rat liver mitochondria. The criterion is based on the view that the nature of the relationship between the H+/O ratio and the membrane potential can be inferred from the relationship between ohmic-uncoupler-induced extra respiration and the membrane potential. Thus a linear relationship between extra respiration and membrane potential indicates unequivocally the independence of the H+/O ratio from the membrane potential and the leak nature of the resting respiration [Brand, Chien, and Diolez (1994) Biochem. J. 297, 27-29]. On the other hand, a non-linear relationship indicates that the H+/O ratio is dependent on the membrane potential. The experimental assessment of this relationship in the presence of an additional ohmic leak, however, is rendered difficult by both the uncoupler-induced depression of membrane potential and the limited range of dependence of the H+/O ratio on the membrane potential. We have selected conditions, i.e. incubation of mitochondria at low temperatures, where the extent of non-linearity is markedly increased. It appears that the nature of the resting respiration of mitochondria in vitro is markedly dependent on the temperature: at low temperatures the percentage of resting respiration due to membrane leak decreases and that due to intrinsic uncoupling of the proton pumps increases.

Animals↗
PubMed1994

The effect of the protonmotive force on the redox state of mitochondrial cytochromes.

In the absence of kinetic limitations, as determined either by high substrate concentrations or by absence of respiratory chain inhibitors, we have observed that: (a) the relationship between the percentage reduction of the cytochromes and the protonmotive force is linear in the case of cytochrome c and biphasic in the case of cytochrome b, (b) the redox state of cytochrome c depends only on the membrane potential and not on the total proton motive force and (c) the alkalinization of the matrix enhances the extent of cytochrome c reduction because of the marked inhibitory effect on the cytochrome oxidase activity. Thus, although the redox states of the b, c and aa3 mitochondrial cytochromes depend on the protonmotive force, the quantitative correlation between the two parameters and the relative effects of the electrical and chemical components of the force differ among the various cytochromes.

Animals↗
PubMed1994

The effect of respiration on the permeability of the mitochondrial membrane to ions.

1. The rates of cation uptake, for either organic cations such as tetrapropylammonium, TPA+, at variable tetraphenylboron concentrations, TPB-, or inorganic cations such as Mn2+, or K+ plus valinomycin, have been measured in mitochondria either respiring, under uncoupler titrations, or non-respiring, under variable K+ diffusion potentials. 2. The flow-force relationship for the respiration-coupled ion fluxes during titrations with uncouplers is almost identical to that obtained for the K(+)-diffusion driven fluxes. Similar results are obtained when TPA+ is replaced with inorganic cations, either monovalent such as K+ (+valinomycin), or divalent such as Mn2+. 3. By applying the Eyring analysis, as developed by Garlid et al. (Garlid, K.D., Beavis, A.D. and Ratkje, S.K. (1989) Biochim. Biophys. Acta 976, 109-121), from the flux-voltage relationships the values for the permeability coefficients and for the energy barriers have been obtained for the transport of the ion pair TPA(+)-TPB-, of Mn2+ and of K+ plus valinomycin, in non-respiring and in respiring, coupled and uncoupled, mitochondria. 4. The findings that the rates of respiration-coupled ion fluxes, at all values of membrane potential, are similar to the rates of the K+ diffusion potential-coupled ion fluxes and the similar pattern of the flux-voltage relationships during the titrations with uncouplers and artificial gradients indicate that the membrane permeability for ions is not modified by respiration.

Animals↗
PubMed1992

Mechanism of loss of thermodynamic control in mitochondria due to hyperthyroidism and temperature.

Incubation of normal mitochondria at 45 degrees C results in increases of respiration and of total apparent proton conductance (TAPC, respiration/proton motive force) and in an upward shift of the flow-force relationships. Similar effects are observed during operation of the redox proton pumps at different sites of the respiratory chain. These effects are accompanied by an almost equivalent increase of the passive proton conductance (PPC, proton leakage/proton motive force). In mitochondria from 3,3,5-triiodo-L-thyronine (T3)-treated rats there are also increases of respiration and of TAPC and an upward shift of flow-force relationships, more pronounced at the level of the cytochrome oxidase proton pump. However, at variance from the incubation at 45 degrees C, in mitochondria from T3-treated rats there is only a slight increase of PPC. Addition of bovine serum albumin to normal mitochondria incubated at 45 degrees C results in a marked depression of TAPC in the nonlinear range of the flow-force relationships. An equivalent effect is not observed in mitochondria from T3-treated rats. The experimental results have been compared with computer simulations obtained on the basis of a chemiosmotic model of energy transduction. The increase of TAPC following incubation at high temperature is apparently due to changes of the proton conductance mainly at the level of PPC, while the increase of TAPC following T3 administration is rather due to changes presumably at the level of the redox or ATPase proton pumps.

Adenosine Triphosphate↗
PubMed1991

Tracking of proton flow during transition from anaerobiosis to steady state. 1. Response of matrix pH indicators.

1. The kinetics of acidification and realkalinization of the matrix after addition of nigericin to respiring and non-respiring mitochondria, recorded by intramitochondrial pH indicators such as neutral red and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), is complementary to that recorded by extramitochondrial pH indicators. The extent of acidification decreases with the logarithm of the KCl concentration and is inhibited by Pi and ammonium ions. 2. Proton translocation during respiration has been compared with proton extraction from matrix bulk water. During oxygen pulses to EGTA-untreated mitochondria, BCECF records an extraction of protons from matrix bulk water of about 2-3 nmol H+/mg, reduced to 1-2 nmol H+/mg in EGTA-treated mitochondria. Since the amount of proton translocation required to achieve steady state is of the order of 6-7 nmol H+/mg, it appears that 75-90% of the protons are not extracted from matrix bulk water. Only a slight response is recorded by neutral red. 3. The effect of permeant cations and of uncouplers on the distribution of proton extraction between membrane and matrix bulk water has been studied in presteady state. During Sr2+ uptake, proton extrusion into cytosolic bulk water, as well as proton extraction from matrix bulk water, corresponds almost to 100% of the protons translocated by the redox proton pumps. In the absence of Sr2+, parallel to the disappearance of the proton extrusion in cytosolic bulk water, the proton extraction from matrix bulk water diminishes to about 20% of the proton translocation. 4. The mechanism by which divalent cation uptake and protonophoric uncouplers affect the distribution of proton extraction between matrix bulk water and membrane domains and the nature of the membrane domains are discussed.

2,4-Dinitrophenol↗
PubMed1991

Tracking of proton flow during transition from anaerobiosis to steady state. 2. Effect of cation uptake on the response of a hydrophobic membrane bound pH indicator.

1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification). 2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization. 3. The comparison of the bromothymol blue response with the arsenazo III, 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b) phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation efflux and proton reuptake. All the above processes reach completion during phase 4. 4. Although bromothymol blue always behaves as a membrane-bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of the membrane-potential-determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer. 5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported.

Anaerobiosis↗
PubMed1991

Activation of respiration and loss of thermodynamic control in hyperthyroidism. Is it due to increased slipping in mitochondrial proton pumps?

T3 administration increases the extent of non-linearity in the flow-force relationship between pump proton conductance and protonmotive force. The effect is present also at the ATPase proton pump. These effects are not accompanied by changes in passive proton conductance. Incubation of mitochondria at 45 degrees C also causes an increased non-linearity, accompanied by a partial increase of proton conductance. It appears that the increase of respiratory activity following T3 administration is due to loss of thermodynamic control within or at the proton pumps, an effect which might be attributed to increased slipping.

Adenosine Triphosphatases↗
PubMed1991

Flux ratios and pump stoichiometries at sites II and III in liver mitochondria. Effect of slips and leaks.

Addition of bovine serum albumin to state 4 mitochondria results in a depression of the proton leak and of the resting respiration of 70 and 25%, respectively. The conductance membrane potential diagram, both in the ohmic and in the non-ohmic region, shows that in the presence of bovine serum albumin the level of ohmic conductance is lowered while that of non-ohmic conductance is increased toward higher delta psi values. The same effect is observed during operation of the different proton pumps. Addition of chloroform affects the conductance membrane potential diagram in the following manner: there is no effect in the ohmic region with all pumps, while there is an effect in the non-ohmic region either at site III or at sites II plus III but not at site II. This suggests a possible effect of chloroform at the level of the cytochrome oxidase proton pump. During titration with oligomycin of the ATPase proton pump the conductance potential diagram shows a region of non-ohmicity only in the presence but not in the absence of an ATP-regenerating system. Protonophoric uncouplers such as carbonyl cyanide p(trifluoromethoxy)phenylhydrazone and intrinsic uncouplers such as chloroform have different effects on the relationship between rates of charge translocation and of oxygen consumption, and thus on the pump stoichiometries, in that the slope of the diagram is modified by the latter but not by the former. The differential effects of protonophores and of intrinsic uncouplers on the stoichiometries have been analyzed by computer simulations and represent an additional criterion to distinguish between extrinsic and intrinsic mechanisms of uncoupling.

Adenosine Triphosphatases↗
PubMed1990

Tracking of proton flow during transition from anaerobiosis to steady state in rat liver mitochondria.

(1) The hydrophobic pH indicator Bromthymol blue and the hydrophilic pH indicator Phenol red have been used to follow the redox-pump-linked proton flows during transition from anaerobiosis to static head. The domains monitored by the pH indicators, whether external or internal, and the localization of the dye, whether free or membrane bound, have been identified by recording the absorbance changes following addition of nigericin or valinomycin to anaerobic or aerobic mitochondria and the effects of permeant and impermeant buffers. (2) After addition of the H+/K+ exchanger, nigericin, to anaerobic mitochondria. Phenol red and Bromthymol blue record an alkalinization and an acidification, respectively, indicating that while the hydrophilic pH indicator faces an external domain, the hydrophobic pH indicator faces, at least partly, an internal domain. The latter effect is sensitive to phosphate and to phosphate carrier inhibitors. On the other hand, addition of nigericin to aerobic mitochondria leads to an increased Bromthymol blue absorbance, which reflects an alkalinization, indicating that the pH indicator faces an external domain. The reorientation of the dye from the internal to the external domain is a function of the uncoupler concentration and thus of the membrane potential (cf. Mitchell et al. (1968) Eur. J. Biochem. 4, 9-19). (3) The amount of oxygen required for the transition from anaerobiosis to static head has been determined by following in parallel the extent of oxidation of cytochrome aa3 and the rise of delta mu H+. With succinate as substrate, 50% levels of cytochrome oxidation are obtained at 0.125 ngatom oxygen/mg and 50% of Safranine response at about 0.2 ngatom oxygen/mg. These amounts of oxygen correspond to an H+ displacement of about 0.8-1.2 ngatom/mg on the basis of the H+/O stoichiometry. It is concluded that mitochondria are in presteady state below, and in static head above, displacement of 2-3 ngatom H+/mg. This figure is very close to the original calculation of Mitchell (Mitchell, P. (1966) Biol. Rev. 41, 445-502). (4) Transition, by oxygen pulses, of EGTA-supplemented mitochondria from anaerobiosis to either presteady state or static head state results in a response of the hydrophilic pH indicator, Phenol red, which is negligible in amount and/or kinetically unrelated to the delta mu H+ rise. The fact that H+ extrusion in the bulk aqueous phase is negligible also in presteady state excludes proton cycling as an explanation. Addition of oxygen pulses to Sr2(+)-supplemented anaerobic mitochondria results in an H+ extrusion whose amount and rate is proportional to the Sr2+ concentration.(ABSTRACT TRUNCATED AT 400 WORDS)

Anaerobiosis↗
PubMed1990

On the nature of the uncoupling effect of fatty acids.

The effect of palmitic acid on the electrical potential differences delta psi across the inner mitochondrial membrane appears to depend on the medium in which mitochondria are incubated. In medium A (cf. Luvisetto et al. (1987), Biochemistry, 26, 7332-7338) delta psi decreases much more than in medium B (cf. Rottenberg and Hashimoto (1986), Biochemistry, 25, 1747-1755) at concentrations of fatty acid which equally stimulate the rate of respiration in state 4. Valinomycin and NaCl were both present in medium B and absent in medium A. However, in both media the pattern of the P/O ratio as a function of antimycin in the presence of a constant amount of palmitic acid or of FCCP shows similar behaviour. We conclude that in both media palmitic acid increases the membrane conductance to protons, but for unclear reasons the delta psi assay fails to measure the decline of delta psi in medium B. However, the increase in membrane conductance induced by palmitic acid does not quantitatively account for the stimulation of the rate of respiration.

Adenosine Triphosphate↗
PubMed1989

Nature of proton cycling during gramicidin uncoupling of oxidative phosphorylation.

Addition of gramicidin D to liver mitochondria, incubated in low- or high-salt media, results in stimulation of respiration in the absence or presence of depression of delta muH, respectively. Gramicidin D concentrations 2 orders of magnitude higher are required in the low-salt media with full uncoupling at 1 nmol of gramicidin.mg-1. The stimulation of respiration is not accompanied by increased passive proton influx in low-salt media. In high-salt media, the extent of respiratory stimulation and the extent of delta muH depression differ according to the nature and concentration of cation. The flow-force relationship is very steep when gramicidin D induced uncoupling occurs in low-salt media and much less steep in high-salt media. A multiplicity of flow-force relationship, respiratory rate vs delta muH, is obtained, the slope of which depends on the nature and concentration of cation, and which can be reproduced by computer simulation by introducing a variable extent of proton cycling either in the membrane or in the pump. The apparent proton conductance, as analyzed in the relationship of Je/delta muH vs delta muH, increases in the so-called ohmic and nonohmic regions according to whether gramicidin D is added in high-salt or low-salt media, respectively. Titration with antimycin of the respiratory control ratio (RCR) in gramicidin D treated mitochondria leads to a depression of the RCR in high-salt but not in low-salt media. The view is discussed that in low-salt media the gramicidin D induced uncoupling is due to a cycling of protons within a proton domain operationally located at or near the proton pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Local protons and uncoupling of aerobic and artificial delta muH-driven ATP synthesis.

Gramicidin D causes inhibition of ATP synthesis either in the absence or in the presence of depression of delta muH, in low-salt and in high-salt media, respectively, at concentrations 2 orders of magnitude higher in the former with respect to the latter case. When the number of active redox pumps is reduced by increasing the antimycin concentration, the P/O ratio of respiring, gramicidin-treated mitochondria either is slightly increased in low-salt media or is first decreased and then constant in high-salt media. Addition of gramicidin D in low-salt media to mitochondria synthesizing ATP by means of artificially imposed delta muH gradients results in (a) no effect on the K+ efflux ratio +/- ADP (equivalent to the aerobic respiratory control ratio) and (b) no effect on the ATP/K+ ratio (equivalent to the P/O ratio) except at the low gramicidin D concentrations where there is also a slight enhancement of the rate of ATP hydrolysis. During respiration-driven ATP synthesis, addition of valinomycin plus K+ causes depression of delta muH with little inhibition of ATP synthesis while addition of gramicidin D causes inhibition of ATP synthesis with little depression of delta muH. The view is discussed that the gramicidin-accessible protons which uncouple aerobic ATP synthesis in a delta muH-independent manner are of a different class from the gramicidin-inaccessible protons which uncouple diffusion potential driven ATP synthesis in a delta muH-dependent manner. The gramicidin-accessible protons are suggested to be pump associated and to reflect primary events in energy transduction.

Adenosine Triphosphate↗