Torque Generation of Enterococcus hirae V-ATPase

V-ATPase (V_oV₁) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V_oV₁ for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V_oV₁ (EhV_oV₁) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV_oV₁ exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV_oV₁ only showed the “clear” state without apparent backward steps, whereas EhV₁ showed two states, “clear” and “unclear.” Furthermore, EhV_oV₁ showed slower rotation than EhV₁ without the three distinct pauses separated by 120° that were observed in EhV₁. When using a large probe, EhV_oV₁ showed faster rotation than EhV₁, and the torque of EhV_oV₁ estimated from the continuous rotation was nearly double that of EhV₁. On the other hand, stepping torque of EhV₁ in the clear state was comparable with that of EhV_oV₁. These results indicate that rotor-stator interactions of the V_o moiety and/or sodium ion transport limit the rotation driven by the V₁ moiety, and the rotor-stator interactions in EhV_oV₁ are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV₁. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV_oV₁.     

A study of lipid bilayer membrane stability using precise measurements of specific capacitance

A method is described for measuring the specific capacitance (C_m) of lipid bilayer membranes with an estimated experimental error of only 1%. The gross capacitance was measured with an AC Wheatstone bridge and a photographic technique was used to determine the area of thin membrane. The results of measurements on oxidized cholesterol-decane membranes formed in 1 × 10^-2 M KCl show that C_m depends upon temperature, voltage, time, and the age of the bulk membrane solutions. For a freshly thinned membrane (from 5 week old solution), C_m increases exponentially from an initial value of 0.432 ±0.021 (SD) μF/cm² with a time constant of ~15 min. A 100 mv potential applied across the membrane for 10-20 min prior to making measurements eliminated this time dependence and produced final-state membranes. C_m of final-state membranes depends upon applied voltage (V_a) and obeys the equation C_m = C₀ + βV_a² where V_a V_DC + V^rms_AC. C₀ and β depend upon temperature; C₀ decreases linearly with temperature while β increases linearly. At 20°C, C₀ = 0.559 ±0.01 (SD) μF/cm² and β = 0.0123 ±0.0036 (SD) (μF/cm²)/(mv²) and at 34°C, C₀ = 0.472 ±0.01 and β = 0.0382 ±0.0039. These variations in C_m are interpreted as resulting from thickness changes. The possibility that they result from diffuse layer and/or membrane dielectric phenomena is discussed and found to be unlikely. The results are discussed in terms of membrane stability by constructing hypothetical potential energy vs. thickness curves.     

Membrane voltage as a dynamic platform for spatiotemporal signaling,physiological, and developmental regulation

Membrane voltage arises from the transport of ions through ion-translocating ATPases, ion-coupled transport of solutes, and ion channels, and is an integral part of the bioenergetic “currency” of the membrane. The dynamics of membrane voltage—so-called action, systemic, and variation potentials—have also led to a recognition of their contributions to signal transduction, both within cells and across tissues. Here, we review the origins of our understanding of membrane voltage and its place as a central element in regulating transport and signal transmission. We stress the importance of understanding voltage as a common intermediate that acts both as a driving force for transport—an electrical “substrate”—and as a product of charge flux across the membrane, thereby interconnecting all charge-carrying transport across the membrane. The voltage interconnection is vital to signaling via second messengers that rely on ion flux, including cytosolic free Ca²⁺, H⁺, and the synthesis of reactive oxygen species generated by integral membrane, respiratory burst oxidases. These characteristics inform on the ways in which long-distance voltage signals and voltage oscillations give rise to unique gene expression patterns and influence physiological, developmental, and adaptive responses such as systemic acquired resistance to pathogens and to insect herbivory.

Membrane voltage serves as a platform coordinating ion flux to transmit and transduce biological signals.

Advances

• The biophysics of transport that determine membrane voltage are well-described with quantitative flux equations.
• In the models of the guard cell and the giant algae Chara and Nitella these charge-transporting processes accurately describe and predict physiological behavior, including the coupling of membrane voltage oscillations with ion flux, [Ca²⁺]_i, pH, their consequences for cellular osmotic adjustments, and their spatial propagation.
• Unlike neuronal and other animal tissues, action potentials in plants are mediated by a temporal sequence of ion flux through Ca²⁺ and Cl^- channels with voltage recovery driven by ion flux through K⁺ channels. The interplay of channel-mediated ion flux and changes in H⁺-ATPase activity are likely responsible for the slower propagation of variation and systemic potentials.
• In terrestrial plants, membrane voltage transients may propagate along vascular traces, both through the parenchymal cells lining the xylem and through the phloem. Propagation of such voltage transients is associated with glutamate receptor-like channels that may contribute to plasma membrane Ca²⁺ flux and [Ca²⁺]_i elevations.
• Changes in [Ca²⁺]_i, pH, and reactive oxygen species are key mediators that translate voltage signals into physiological, developmental, and adaptive responses in plant tissues.

     

Activation of Na+-permeant Cation Channel by Stretch and Cyclic AMP-dependent Phosphorylation in Renal Epithelial A6 Cells

It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na⁺ absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na⁺, K⁺, Li⁺, and Cs⁺, but little selectivity for Ca²⁺ (P_Ca/P_Na < 0.005) or Cl⁻ (P_Cl/P_Na < 0.01), identifying it as an NSC channel. Stretch had no additional effects on the open probability (P _o) of the IBMX-activated channel. This channel had one open (“O”) and two closed (short “C _S” and long “C _L”) states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the P _o of the channel without any detectable changes in the mean open or closed times. These observations led us to the conclusion that a kinetic model “C _L ↔ C _S ↔ O” was the most suitable among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of the channel for C _L from C _S, resulting in an increase in P _o. Cytochalasin D pretreatment abolished the response to stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.     

The voltage-dependent step of the chloride transporter of Valonia utricularis encounters a Nernst-Planck and not an Eyring type of potential energy barrier

Voltage-clamp experiments were performed on cells of the giant marine alga Valonia utricularis to study the voltage dependence of the previously postulated chloride transporter (Wang, J., G. Wehner, R. Benz, and U. Zimmermann. 1991. Biophys. J. 59:235-248). Only one exponential current relaxation (apart from the capacitive spike) could be resolved up to a clamp voltage of ~120 mV within the time resolution of our experimental instrumentation (100 μs). This means that the rate constants of the heterogeneous complexation, k_R (association) and k_D (dissociation), were too fast to be resolved. Therefore, the “Läuger” model for carrier-mediated ion transport with equilibrium heterogeneous surface reaction was used to fit the experimental results. The voltage dependence of the initial membrane conductance was used for the evaluation of the voltage dependence of the translocation rate constant of the complexed carriers, k_AS. The initial conductance was found to be independent on the clamp voltage, which means that the translocation rate constant k_AS is a linear function of the applied voltage and that the voltage dependence of the translocation of charged carriers through the plasmalemma could be explained by a square-type Nernst-Planck barrier. The movement of the complexed form of the carrier through the membrane may be explained by a diffusion process rather than by simple first-order kinetic jump across an Eyring-type potential well. The current relaxation after a voltage clamp was studied as a function of the external chloride concentration. The results allowed an estimation of the stability constant, K, of the heterogeneous complexation reaction and a calculation of the translocation rate constants of the free and the complexed carriers, k_s and k_AS, respectively.     

Nature of the Inorganic Carbon Species Actively Taken Up by the Cyanobacterium Anabaena variabilis

The nature of the inorganic carbon (C_i) species actively taken up by cyanobacteria CO₂ or HCO₃⁻ has been investigated. The kinetics of CO₂ uptake, as well as that of HCO₃⁻ uptake, indicated the involvement of a saturable process. The apparent affinity of the uptake mechanism for CO₂ was higher than that for HCO₃⁻. Though the calculated V_max was the same in both cases, the maximum rate of uptake actually observed was higher when HCO₃⁻ was supplied. C_i uptake was far more sensitive to the carbonic anhydrase inhibitor ethoxyzolamide when CO₂ was the species supplied. Observations of photosynthetic rate as a function of intracellular C_i level (following supply of CO₂ or HCO₃⁻ for 5 seconds) led to the inference that HCO₃⁻ is the species which arrives at the inner membrane surface, regardless of the species supplied. When the two species were supplied simultaneously, mutual inhibition of uptake was observed.

On the basis of these and other results, a model is proposed postulating that a carboic anhydrase-like subunit of the C_i transport apparatus binds CO₂ and releases HCO₃⁻ at or near a membrane porter. The latter transports HCO₃⁻ ions to the cell interior.

     

Effects of Epinephrine on the Pacemaker Potassium Current of Cardiac Purkinje Fibers

Epinephrine promotes spontaneous activity in cardiac Purkinje fibers through its action on the pacemaker potassium current (i_KK2). The mechanism of the acceleratory effect was studied by means of a voltage clamp technique. The results showed that the hormone speeds the deactivation of i_KK2 during pacemaker activity by displacing the kinetic parameters of i_KK2 toward less negative potentials. This depolarizing voltage shift is the sole explanation of the acceleratory effect since epinephrine did not alter the rectifier properties of i_KK2, or the underlying inward leakage current, or the threshold for i_NNa. The dose dependence of the voltage shift in the i_KK2 activation curve was similar in 1.8 and 5.4 mM [Ca]_o. The maximal voltage shift (usually ~20 mV) was produced by epinephrine concentrations of > 10^-6 M. The half-maximal effect was evoked by 60 nM epinephrine, nearly an order of magnitude lower than required for half-maximal effect on the secondary inward current (Carmeliet and Vereecke, 1969). The β-blocker propranolol (10^-6 M) prevented the effect of epinephrine (10^-7M) but by itself gave no voltage shift. Epinephrine shifted the activation rate coefficient α₈ to a greater extent than the deactivation rate coefficient β₈, and often steepened the voltage dependence of the steady-state activation curve. These deviations from simple voltage shift behavior were discussed in terms of possible mechanisms of epinephrine's action on the i_KK2 channel.     

Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore

Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be “transplanted” to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl⁻ conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby “rescuing” the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl⁻ conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO₂)₄²⁻ in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl⁻ channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl⁻ transport through a combination of failure to increase Cl⁻ conductance and increased susceptibility to channel block by cytosolic substances.     

Resolving the Negative Potential Side (n-side) Water-accessible Proton Pathway of F-type ATP Synthase by Molecular Dynamics Simulations

The rotation of F₁F_o-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane F_o domain, which is coupled to the ATP-producing F₁ domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the “locked” and “open” conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F₁F_o-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode.     

Amplification of small signals by voltage-gated sodium channels in drone photoreceptors

Summary Photoreceptor cells of the drone,Apismellifera , have a voltage-gated Na⁺ membrane conductance that can be blocked by tetrodotoxin (TTX) and generates an action potential on abrupt depolarization: an action potential is triggered by the rising phase of a receptor potential evoked by an intense light flash (Autrum and von Zwehl 1964; Baumann 1968). We measured the intracellular voltage response to a small (9%), brief (30 ms) decrease in light intensity from a background, and found that its amplitude was decreased by 1M TTX. The response amplitude was maximal when the background intensity depolarized the cell to –38 mV. With intensities depolarizing the cell membrane to –45 to –33 mV the average response amplitude was decreased by TTX from 1.2mV to 0.5mV. TTX is also known to decrease the voltage noise during steady illumination (Ferraro et al. 1983) but, despite this, the ratio of peak-to-peak signal to noise was, on average, decreased by TTX. The results suggest that drone photoreceptors use voltage-gated Na⁺ channels for graded amplification of responses to small, rapid changes in light intensity.Abbreviations TTX tetrodotoxin - V _i intracellular potential with respect to the bath - V _o extracellular potential - V _m,V _i-V _o approximate transmembrane potential - S amplitude of the voltage response to an 8.9% decrease in light intensity - N voltage noise, usually measured as root mean square voltage deviation as described in Methods     

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

Summary The uptake and binding of the lipophilic cations ethidium⁺, tetraphenylphosphonium⁺ (TPP⁺), triphenylmethylphosphonium⁺ (TPMP⁺), and tetraphenylarsonium⁺ (TPA⁺) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K' _o) and the internal matrix volume and matrix face of the inner membrane (K' _i) were determined and were utilized to estimate the membrane potential from the cation accumulation factorR _c according to the relation =RT/ZF ln [(R _cV_o–K'_o)/(V_i+K'_i)] whereV _o andV _i are the volume of the external medium and the mitochondrial matrix, respectively, andR _c is the ratio of the cation content of the mitochondria and the medium. The values of estimated from this equation are in remarkably good agreement with those estimated from the distribution of⁸⁶Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations.     

Mechanism of Photosynthetic Carbon Dioxide Uptake by the Red Macroalga, Chondrus crispus

The aim of this study was to determine how Chondrus crispus, a marine red macroalga, acquires the inorganic carbon (C_i) it utilizes for photosynthetic carbon fixation. Analyses of C_i uptake were done using silicone oil centrifugation (using multicellular fragments of thallus), infrared gas analysis, and gas chromatography. Inhibitors of carbonic anhydrase (CA), the band 3 anion exchange protein and Na⁺/K⁺ exchange were used in the study. It was found that: (a) C. crispus does not accumulate C_i internally above the concentration attainable by diffusion; (b) the initial C_i fixtion rate of C. crispus fragments saturates at approximately 3 to 4 millimolar C_i; (c) CA is involved in carbon uptake; its involvement is greatest at high HCO₃⁻ and low CO₂ concentration, suggesting its participation in the dehydration of HCO₃⁻ to CO₂; (d) C. crispus has an intermediate C_i compensation point; and (e) no evidence of any active or facilitated mechanism for the transport of HCO₃⁻ was detected. These data support the view that photosynthetic C_i uptake does not involve active transport. Rather, CO₂, derived from HCO₃⁻ catalyzed by external CA, passively diffuses across the plasma membrane of C. crispus. Intracellular CA also enhances the fixation of carbon in C. crispus.     

Recording of single K+ channels in the membrane of cytoplasmic drop ofChara australis

Summary The cytoplasmic drop formed of effused cytoplasm fromChara internodes is enclosed by a membrane. Patch clamp experiments have been carried out on this membrane, revealing a K⁺ channel as the most frequently detected ion translocator. The K⁺ channel is saturated at a level of about 20 pA inward and 10 pA outward current. The channel conductance is dependent on the accessability of K⁺ ions, its maximum value amounts to about 165 pS. The discrimination of Na⁺ and Cl^– is significant, permeability ratios P_Na/P_K and P_Cl/P_K were estimated to be 0.01 either. Binding experiments with the fluorescent probe concanavalin A/FITC suggest that the membrane is derived from the tonoplast.Abbreviations E_K K⁺ equilibrium potential - FITC fluorescein isothiocyanat - V_m membrane voltage - V_pip pipette clamp voltage - V_r reversal voltage     

Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector

The membrane sector (V_o) of the proton pumping vacuolar ATPase (V-ATPase, V₁V_o-ATPase) from Saccharomyces cerevisiae was purified to homogeneity, and its structure was characterized by EM of single molecules and two-dimensional crystals. Projection images of negatively stained V_o two-dimensional crystals showed a ring-like structure with a large asymmetric mass at the periphery of the ring. A cryo-EM reconstruction of V_o from single-particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of three-dimensional reconstructions of free V_o and V_o as part of holo V₁V_o revealed that the cytoplasmic N-terminal domain of subunit a (a_NT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from V_o, V₁, and subunit C. Isothermal titration calorimetry using recombinant subunit d and a_NT revealed that the two proteins bind each other with a K_d of ∼5 μm. Treatment of the purified V_o sector with 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] resulted in selective release of subunit d, allowing purification of a V_oΔd complex. Passive proton translocation assays revealed that both V_o and V_oΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V₁ from V_o during reversible enzyme dissociation plays a role in blocking passive proton translocation across free V_o and that the interaction between a_NT and d seen in free V_o functions to stabilize the V_o sector for efficient reassembly of V₁V_o.     

Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel,hHV1

Part of the “signature sequence” that defines the voltage-gated proton channel (H_V1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H_V1 genes. Replacing Trp²⁰⁷ in human H_V1 (hH_V1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, E_a, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp²⁰⁷ establishes the major energy barrier between closed and open hH_V1. Cation–π interaction between Trp²⁰⁷ and Arg²¹¹ evidently latches the channel closed. Trp²⁰⁷ mutants lost proton selectivity at pH_o >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H_V1 that is essential to its biological functions, was compromised. In the WT hH_V1, ΔpH-dependent gating is shown to saturate above pH_i or pH_o 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp²⁰⁷ mutants, ΔpH-dependent gating saturated at lower pH_o but not at lower pH_i. That Trp²⁰⁷ mutation selectively alters pH_o sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H_V1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pH_o and pH_i in H_V1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.     

A linkage analysis toolkit for studying allosteric networks in ion channels

A thermodynamic approach to studying allosterically regulated ion channels such as the large-conductance voltage- and Ca²⁺-dependent (BK) channel is presented, drawing from principles originally introduced to describe linkage phenomena in hemoglobin. In this paper, linkage between a principal channel component and secondary elements is derived from a four-state thermodynamic cycle. One set of parallel legs in the cycle describes the “work function,” or the free energy required to activate the principal component. The second are “lever operations” activating linked elements. The experimental embodiment of this linkage cycle is a plot of work function versus secondary force, whose asymptotes are a function of the parameters (displacements and interaction energies) of an allosteric network. Two essential work functions play a role in evaluating data from voltage-clamp experiments. The first is the conductance Hill energy W_H[g], which is a “local” work function for pore activation, and is defined as kT times the Hill transform of the conductance (G-V) curve. The second is the electrical capacitance energy W_C[q], representing “global” gating charge displacement, and is equal to the product of total gating charge per channel times the first moment (V_M) of normalized capacitance (slope of Q-V curve). Plots of W_H[g] and W_C[q] versus voltage and Ca²⁺ potential can be used to measure thermodynamic parameters in a model-independent fashion of the core gating constituents (pore, voltage-sensor, and Ca²⁺-binding domain) of BK channel. The method is easily generalized for use in studying other allosterically regulated ion channels. The feasibility of performing linkage analysis from patch-clamp data were explored by simulating gating and ionic currents of a 17-particle model BK channel in response to a slow voltage ramp, which yielded interaction energies deviating from their given values in the range of 1.3 to 7.2%.     

A kinetic analysis of the electrogenic pump ofChara corallina: I. Inhibition of the pump by DCCD

Summary The current-voltage curve of theChara membrane was obtained by applying a slow ramp depo- and hyperpolarization by use of voltage clamp. With the progress of poisoning by DCCD (dicyclohexylcarbodiimide) theI–V curve moved by about 50 mV (depolarization) along the voltage axis, reducing its slope, and finally converged to thei _d -V curve of the passive diffusion channel. Changes ofi _p -V curve of the electrogenic pump channel could be obtained by subtracting the latter from the former.The sigmoidali _p -V curve could be simulated satisfactorily by adopting a simple reaction kinetic model. Kinetic parameters of the successive changes of state of the H⁺ ATPase could be evaluated. Changes of these kinetic parameters during inhibition gave useful information about the molecular mechanism of the electrogenic pump.Depolarization of the membrane potential, decrease of membrane conductance, and decrease of pump current during inhibition of the pump with DCCD are caused mainly by the decrease of conductance of the pump channel. The decrease of this pump conductance is caused principally by a marked decrease of the rate constant for releasing H⁺ to the outside.     

Distinct roles for CaV1.1’s voltage-sensing domains

Study reveals how a slowly activating calcium channel is able to control rapid excitation–contraction coupling in skeletal muscle.

Skeletal muscle contraction is initiated by action potentials that depolarize the muscle fiber and trigger the rapid release of Ca²⁺ from the SR via RYR1 channels. This process of excitation–contraction coupling depends on voltage-gated Ca_V1.1 channels in the plasma membrane, or sarcolemma, of muscle fibers. But Ca_V1.1 channels are only slowly activated by changes in the sarcolemma membrane potential, and it is therefore unclear how they are able to trigger the much faster activation of RYR1 channels. In this issue of JGP, Savalli et al. reveal that this paradox can be explained by the fact that each of Ca_V1.1’s four voltage-sensing domains (VSDs) have distinct biophysical properties (1).Nicoletta Savalli (left), Riccardo Olcese (center), and colleagues reveal the distinct physical properties of the Ca_V1.1 channel’s four voltage-sensing domains (VSD I–IV, right). VSD-I shows slow activation kinetics and is the main contributor to the opening of Ca_V1.1. The other VSDs activate much faster and may therefore be coupled to RYR1 to mediate the rapid release of Ca²⁺ from the SR during skeletal muscle contraction.RYR1 channels have no voltage-sensing machinery of their own and therefore rely on a physical connection to Ca_V1.1 channels to release Ca²⁺ and initiate muscle contraction in response to muscle fiber depolarization. But RYR1 channels open ∼25 times faster than Ca_V1.1 channels. “So, how can these slowly activating Ca_V1.1 channels trigger the rapid release of Ca²⁺ from the SR?” asks Riccardo Olcese, a professor at the David Geffen School of Medicine, UCLA.Olcese and colleagues, including Assistant Project Scientist Nicoletta Savalli, suspected that the answer might lie in the fact that, like many other voltage-gated ion channels, Ca_V1.1 has four VSDs that alter their conformation in response to voltage changes. These domains are similar, but not identical, to each other, potentially enabling them to have distinct biophysical properties and perform distinct functions. Indeed, Olcese and colleagues previously demonstrated that, in the closely related channel Ca_V1.2, only VSDs II and III are involved in pore opening (2, 3).Savalli et al. used voltage-clamp fluorometry to compare the properties of Ca_V1.1’s VSDs, expressing the channel in Xenopus oocytes and labeling each of its VSDs in turn with an environmentally sensitive fluorophore to report voltage-dependent changes in their conformation (1). “We found that the four VSDs were very heterogenous in both their kinetics and voltage dependencies,” says Olcese. “VSD-I had very slow kinetics, compatible with the slow activation of the Ca_V1.1 pore. The other three VSDs had much faster kinetics and could, therefore, be good candidates to be the voltage sensors for RYR1 activation.”Olcese and colleagues confirmed the importance of VSD-I for Ca_V1.1 activation by analyzing a naturally occurring, charge-neutralizing mutation in this domain, R174W, that is linked to malignant hyperthermia (4). The team found that this mutation reduced the voltage-sensitivity of VSD-I and abolished the ability of Ca_V1.1 to conduct Ca²⁺ at physiological membrane potentials, but had no effect on the behavior of the other three VSDs.Finally, Savalli et al. applied their data on both the wild-type and mutant VSDs to an allosteric model of Ca_V activation (2, 3), which predicted that VSD-I contributes most of the energy required to stabilize the open state of Ca_V1.1, while the other VSDs contribute little to nothing.Thus, Ca_V1.1 activation is mainly driven by a single VSD—a mechanism that hasn’t been seen in any other voltage-gated ion channel—leaving the other VSDs free to perform other functions, such as the rapid activation of RYR1. Olcese and colleagues now want to pinpoint exactly which VSD(s) are coupled to RYR1 and determine how they trigger rapid Ca²⁺ release from the SR.     

Cytoplasmic calcium stimulates exocytosis in a plant secretory cell

Although exocytosis is likely to occur in plant cells, the control of this process is the subject of speculation, as no direct measurements of vesicle fusion to the plasma membrane have been made. We used the patch clamp technique to monitor the secretory activity of single aleurone protoplasts by measuring membrane capacitance (C_m), while dialyzing the cytosol with different Ca²⁺ containing solutions. Secretory activity increased with [Ca²⁺]_i ~ 1 μM. This demonstrates directly the existence of exocytosis in plant cells, and suggests that both plant and animal cells share common mechanisms (cytosolic Ca²⁺) for the control of exocytotic secretion.     

Editorial

The voltage dependence of the rat renal type II Na⁺/P_i cotransporter (NaP_i-2) was investigated by expressing NaP_i-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (P_i) induced inward currents (I_p) in the presence of 96 mM Na⁺ over the potential range −140 ≤ V ≤ +40 mV. With P_i as the variable substrate, the apparent affinity constant (K _m ^Pi) was strongly dependent on Na⁺, increasing sixfold for a twofold reduction in external Na⁺. K _m ^Pi increased with depolarizing voltage and was more sensitive to voltage at reduced Na⁺. The Hill coefficient was close to unity and the predicted maximum I_p (I_pmax) was 40% smaller at 50 mM Na⁺. With Na⁺ as the variable substrate, K _m ^Na was weakly dependent on both P_i and voltage, the Hill coefficient was close to 3 and I_pmax was independent of P_i at −50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na⁺-dependent manner, indicating the existence of uncoupled Na⁺ slippage. Voltage steps induced pre–steady state relaxations typical for Na⁺-coupled cotransporters. NaP_i-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na⁺, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V_0.5) in the range −20 to −50 mV and an apparent valency of ∼0.5 e⁻. V_0.5 became more negative as Na⁺ was reduced. P_i suppressed relaxations in a dose-dependent manner, but had little effect on their voltage dependence. Reducing external pH shifted V_0.5 to depolarizing potentials and suppressed relaxations in the absence of Na⁺, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na⁺ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na⁺ binding step.