Roles of Ser130 and Thr126 in chloride binding and photocycle of pharaonis halorhodopsin

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump in Natronobacterium pharaonis. In order to clarify the roles of the Ser130(phR) and Thr126(phR) residues, which correspond to Ser115(shR) and Thr111(shR) of salinarum hR (shR), with regard to their Cl(-)binding affinity and the photocycle, the wild-type phR, and S130 and T126 mutants were expressed in Escherichia coli cells. The photocycles of the wild-type phR, and S130 and T126 mutants were investigated in the presence of 1 M NaCl. Based on results of kinetic analysis involving singular value decomposition and global fitting, typical photointermediates K, L and O were identified, and the kinetic constants of decay or formation varied depending on the mutant. The photocycle scheme was linear for the wild-type phR, and S130C, S130T and T126V mutants. On the other hand, the S130A mutant showed a branched pathway between the L-hR and L-O steps. The present study revealed the following two facts with respect to the Ser130(phR) residue: 1) The OH group of this residue is important for Cl(-) ion binding next to the Schiff base nitrogen, and 2) replacement of an Ala residue, which is unable to form a hydrogen bond, results in a branched photocycle. The implication of this branching was discussed.     

Chloride concentration dependency of the electrogenic activity of halorhodopsin

The capacitive photoelectric current responses of the halorhodopsins from Halobacterium salinarum (shR) and from Natronobacterium pharaonis (phR) were studied using membrane fragments adsorbed onto a thin polyester film. The electric current of shR was not much affected by ionic strength or cations present in the medium (Na+, K+, Li+, Mg2+, or Ca2+), but was greatly influenced by the Cl- concentration. It increased biphasically as the Cl- concentration increased from 0 to 5 M, then decreased and almost vanished at around 10 or 12 M. Apparent Kd's of about 0.1 and 6 M were deduced for the Kd of Cl- uptake sites. We had to assume a sigmoidal increase of Cl- binding with a Hill coefficient of about 8 at the cytoplasmic, Cl- release site(s). The half-maximum Cl- concentration for the sigmoidal binding was about 7.5 M. The electric current of phR had a maximum around 30 mM Cl- and biphasically decreased at higher Cl- concentrations. The apparent Kd for the Cl- uptake site was 5 mM. The biphasic decrease in the transport activity was explained by assuming a sum of simple hyperbolic type binding (Kd = 0.2 M) and sigmoidally increasing binding with a Hill coefficient of 10 on the cytoplasmic side. The half-maximum concentration of the latter cooperative binding was 5.6 M. This great difference between the apparent affinity of the release site of shR and that of phR can explain the previously reported difference between the Cl- dependency of their photocycles. These results also suggest that there may be multiple Cl- binding sites in the Cl- transport pathway. A simple sequence of Cl- transport steps based on a multiion channel model is proposed.     

Ser-130 of Natronobacterium pharaonis halorhodopsin is important for the chloride binding

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130(phR) residue which corresponds to Ser-115(shR) for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl beta-D-maltopyranoside The absorption maximum (lambda(max)) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift of lambda(max) upon Cl(-) addition, from which the dissociation constants of Cl(-) were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 degrees C. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorhodopsin is important for the chloride binding.     

Proton release and uptake of pharaonis phoborhodopsin (sensory rhodopsin II) reconstituted into phospholipids

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a photo-receptor for negative phototaxis in Natronobacterium pharaonis. During the photoreaction cycle (photocycle), ppR exhibits intraprotein proton movements, resulting in proton pumping from the cytoplasmic to the extracellular side, although it is weak. In this study, light-induced proton uptake and release of ppR reconstituted with phospholipid were analyzed using a SnO(2) electrode. The reconstituted ppR exhibited properties in proton uptake and release that are different from those of dodecyl maltoside solubilized samples. It showed fast proton release before the decay of ppR(M) (M-photointermediate) followed by proton uptake, which was similar to that of bacteriorhodopsin (BR), a light-driven proton pump. Mutant analysis assigned Asp193 to one (major) of the members of the proton-releasing group (PRG). Fast proton release was observed only when the pH was approximately 5-8 in the presence of Cl(-). When Cl(-) was replaced with SO(4)(2-), the reconstituted ppR did not exhibit fast proton release at any pH, suggesting Cl(-) binding around PRG. PRG in BR consists of Glu204 (Asp193 in ppR) and Glu194 (Pro183 in ppR). Replacement of Pro183 by Glu/Asp, a negatively charged residue, led to Cl(-)-independent fast proton release. The transducer binding affected the properties of PRG in ppR in the ground state and in the ppR(M) state, suggesting that interaction with the transducer extends to the extracellular surface of ppR. Differences and similarities in the molecular mechanism of the proton movement between ppR and BR are discussed.     

Anion uptake in halorhodopsin from Natromonas pharaonis studied by FTIR spectroscopy: consequences for the anion transport mechanism

The uptake of chloride, bromide, iodide, nitrate, and azide by anion-depleted blue halorhodopsin from Natronobacterium pharaonis has been followed by FTIR difference spectroscopy using an ATR sampling device. The spectra are compared with the spectrum of the O intermediate obtained by time-resolved FTIR studies of the photocycle. It is demonstrated that anion-free blue halorhodopsin can be identified with the O intermediate and, thus, that the decay of O is due to the passive uptake of the anion. The great similarity of the anion-binding spectra and their identity in the case of the monoatomic anions indicate a rather unspecific binding site for the different anions dominated by electrostatic interactions. Comparing spectra obtained with 15N nitrate and unlabeled nitrate, the NO-stretching bands could be identified. The small splitting and the small IR intensity of those bands indicate a rather nonpolar binding site with a rather isotropic influence on the nitrate, in contrast to aqueous nitrate. In further experiments on the photocycle of blue halorhodopsin, the all-trans --> 13-cis isomerization can be clearly identified. Up to 100 micros, the isomerization-induced structural changes deduced from amide I changes are similar to those occurring during the anion-transporting photocycle. Compared to these, the molecular changes involved in the release and their reversion during the uptake of anions are considerably larger. They can be reached via two pathways: (1) by reducing the anion concentration and (2) transiently during the anion-transporting photocycle with the formation of the precursor of O with O conformation. Consequences of the anion transport mechanism are discussed.     

Cl(-)-HCO3- exchange in rat renal basolateral membrane vesicles

Pathways for HCO3- transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl(-)-HCO3- exchange was assessed directly by 36Cl- tracer flux measurements and indirectly by determinations of acridine orange absorbance changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake compared to Cl- uptake under 100% N2 in the presence of a pH gradient alone. Mediated exchange of Cl- for HCO3- was suggested by the HCO3- gradient-induced concentrative accumulation of intravesicular Cl-. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO3- gradient-driven Cl- uptake further suggesting chemical as opposed to electrical Cl(-)-HCO3- exchange coupling. Although basolateral membrane vesicle Cl- uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl- conductive pathway served to distinguish this mode of Cl- translocation from HCO3- gradient-driven Cl- uptake. No evidence for K+/Cl- cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pHo 7.5/pHi 6), a HCO3- dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl- concentration gradient. The basolateral membrane vesicle origin of the observed Cl(-)-HCO3- exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl- on HCO3- gradient-driven Na+ uptake suggesting a basolateral membrane Na+-HCO3- for Cl- exchange mechanism, no effect of Na+ on Cl-HCO3- exchange was observed in the present study.     

Properties and photochemistry of a halorhodopsin from the haloalkalophile, Natronobacterium pharaonis

Pharaonis halorhodopsin is a light-driven transport system for chloride, similarly to the previously described halorhodopsin, but we find that it transports nitrate as effectively as chloride. We studied the photoreactions of the purified, detergent-solubilized pharaonis pigment with a gated multichannel analyzer. At a physiological salt concentration (4 M NaCl), the absorption spectra and rate constants of rise and decay for intermediates of the photocycle were similar to those for halorhodopsin. In buffer containing nitrate, halorhodopsin exhibits a second, truncated photocycle; this difference in the photoreaction of the pigment occurs when an anion is bound in such a way as to preclude transport. As expected from the lack of anion specificity in the transport, the photocycle of pharaonis halorhodopsin was nearly unaffected by replacement of chloride with nitrate. All presumed buried positively charged residues, which might play a role in anion binding, are conserved in the two pigments. At the extracellular end of the presumed helix C, however, an arginine residue is found in halorhodopsin, but not in pharaonis halorhodopsin, and an arginine-rich segment between the presumed helices A and B in halorhodopsin is replaced by a less positively charged sequence in pharaonis halorhodopsin (Lanyi, J. K., Duschl, A., Hatfield, G. W., May, K., and Oesterhelt, D. (1990) J. Biol. Chem. 265, 1253-1260). One or both of these alterations may explain the difference in the anion selectivity of the two proteins.     

A halorhodopsin-overproducing mutant isolated from an extremely haloalkaliphilic archaeon Natronomonas pharaonis

The halorhodopsin (hR)-overproducing mutant strain KM-1 was isolated from the extremely haloalkaliphilic archaeon Natronomonas pharaonis type strain DSM2160(T). hR-enriched membranes were easily obtained by washing the cells with distilled water. The membranes were claret colored owing to two pigments: hR and bacterioruberin. The hR component in the absorption spectra changed from blue to purple upon the addition of Cl(-) and had a K(m) value of 1.7mM. Overexpression of hR in strain KM-1 might be caused by the point mutation Asp324-->Asn in the bacteriorhodopsin activator homologues of N. pharaonis. The mutation changed the hR-expression pattern from inducible to constitutive in the late exponential phase.     

Characterization of the azide-dependent bacteriorhodopsin-like photocycle of salinarum halorhodopsin

The photocycle of salinarum halorhodopsin was investigated in the presence of azide. The azide binds to the halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum halorhodopsin and pharaonis halorhodopsin are discussed.     

C1/HCO3 exchange in human placental brush border membrane vesicles

Membrane transport pathways for transplacental transfer of CO2/HCO3 were investigated by assessing the possible presence of a Cl/HCO3 exchange mechanism in the maternal-facing membrane of human placental epithelial cells. Cl/HCO3 exchange was tested for in preparations of purified brush border membrane vesicles by 36Cl tracer flux measurements and determinations of acridine orange fluorescence changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake to levels approximately 2-fold greater than observed at equilibrium. Maneuvers designed to offset the development of ion gradient-induced diffusion potentials (valinomycin, Ko = Ki) significantly reduced HCO3- gradient-driven Cl- uptake but concentrative accumulation of Cl- persisted. Early time point determinations performed in the presumed absence of membrane potential suggests the reduced level of HCO3- gradient-driven Cl- uptake resulted from a more rapid dissipation of the HCO3- concentration gradient. Concentrative accumulation of Cl- was not observed in the presence of a pH gradient alone under 100% N2, suggesting a preference of HCO3- over OH- as a substrate for transport. As monitored by acridine orange fluorescence the Cl- gradient-dependent collapse of an imposed pH gradient (pHo 8.5/pHi 6) was accelerated in the presence of CO2/HCO3 when compared with its absence, indicating coupling of HCO3- influx to Cl- efflux. Increasing concentrations of the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid were observed to cause a stepwise reduction in HCO3- gradient-driven Cl- uptake (I50 approximately 25 microM) further suggesting the presence of a Cl/HCO3 exchange mechanism. The results of this study provide evidence for a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl/HCO3 exchange mechanism in the maternal-facing membrane of human placental epithelial cells. The identification of an ion-coupled HCO3- transport pathway in placental epithelia may suggest functional roles in mediating transplacental transfer of CO2 as well as maintenance of fetal acid/base balance.     

Characterization of the proton-transporting photocycle of pharaonis halorhodopsin

The photocycle of pharaonis halorhodopsin was investigated in the presence of 100 mM NaN(3) and 1 M Na(2)SO(4). Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR' state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide.     

ATP-driven,Na(+)-independent inward Cl- pumping in neuroblastoma cells

In immature neurones, the steady-state intracellular Cl- concentration [Cl-](i) is generally higher than expected for passive distribution, and this is believed to be due to Na(+)-K(+)-2Cl(-) co-transport. Here, we show that N2a neuroblastoma cells, incubated in HEPES-buffered NaCl medium maintain a [Cl-](i) around 60 mm, two- to threefold higher than expected for passive distribution at a membrane potential of - 49 mV. When the cells were transferred to a Cl(-) -free medium, [Cl-](i) decreased quickly (t(1/2) < 5 min), suggesting a high Cl- permeability. When the intracellular ATP concentration was reduced to less than 1 mm by metabolic inhibitors, the initial rate of (36) Cl- uptake was strongly inhibited (60-65%) while steady-state [Cl-](i) decreased to 24 mm, close to the value predicted from the Nernst equilibrium. Moreover, after reduction of [ATP](i) and [Cl-](i) by rotenone, the subsequent addition of glucose led to a reaccumulation of Cl-, in parallel with ATP recovery. Internal bicarbonate did not affect Cl- pumping, suggesting that Cl-/HCO(3)(-) exchange does not significantly contribute to active transport. Likewise, Na(+) -K(+) -2Cl(-) co-transport also appeared to play a minor role: although mRNA for the NKCC1 form of the co-transporter was detected in N2a cells, neither the initial rate of (36)Cl- uptake nor steady-state [Cl-](i) were appreciably decreased by 10 microm bumetanide or replacement of external Na(+) by choline. These results suggest that a highly active ATP-dependent mechanism, distinct from Na(+) -K(+) -2Cl(-) co-transport, is responsible for most of the inward Cl- pumping in N2a cells.     

Charge motions during the photocycle of pharaonis halorhodopsin

Oriented gel samples were prepared from halorhodopsin-containing membranes from Natronobacterium pharaonis, and their photoelectric responses to laser flash excitation were measured at different chloride concentrations. The fast component of the current signal displayed a characteristic dependency on chloride concentration, and could be interpreted as a sum of two signals that correspond to the responses at high-chloride and no-chloride, but high-sulfate, concentration. The chloride concentration-dependent transition between the two signals followed the titration curve determined earlier from spectroscopic titration. The voltage signal was very similar to that reported by another group (Kalaidzidis, I. V., Y. L. Kalaidzidis, and A. D. Kaulen. 1998. FEBS Lett. 427:59-63). The absorption kinetics, measured at four wavelengths, fit the kinetic model we had proposed earlier. The calculated time-dependent concentrations of the intermediates were used to fit the voltage signal. Although no negative electric signal was observed at high chloride concentration, the calculated electrogenicity of the K intermediate was negative, and very similar to that of bacteriorhodopsin. The late photocycle intermediates (O, HR', and HR) had almost equal electrogenicities, explaining why no chloride-dependent time constant was identified earlier by Kalaidzidis et al. The calculated electrogenicities, and the spectroscopic information for the chloride release and uptake steps of the photocycle, suggest a mechanism for the chloride-translocation process in this pump.     

Role of putative anion-binding sites in cytoplasmic and extracellular channels of Natronomonas pharaonis halorhodopsin

Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl(-) ion pump. For efficient Cl(-) transport, the existence of Cl(-)-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl(-) to anion-free NpHR. The binding affinity was affected in mutants in which certain EC residues had been replaced; this finding revealed the importance of Arg123. On the other hand, mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants, L2-photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl(-) to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.     

Effect of anions on the photocycle of halorhodopsin. Substitution of chloride with formate anion

Halorhodopsin from Natronomonas pharaonis is a light-driven chloride pump which transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. It was shown that the chloride anion bound in the vicinity of retinal PSB can be replaced by several inorganic anions, including azide which converts the chloride pump into a proton pump and induces formation of an M-like intermediate detected in the bR photocycle but not in native halorhodopsin. Here we have studied the possibility of replacing the chloride anion with organic anions and have followed the photocycle under several conditions. It is revealed that the chloride can be replaced with a formate anion but not with larger organic anions such as acetate. Flash photolysis experiments detected in the formate pigment an M-like intermediate characterized by a lifetime much longer than that of the O intermediate. The lifetime of the M-like intermediate depends on the pH, and its decay is significantly accelerated at low pH. The decay rate exhibited a titration-like curve, suggesting that the protonation of a protein residue controls the rate of M decay. Similar behavior was detected in N. pharaonis pigments in which the chloride anion was replaced with NO(2)(-) and OCN(-) anions. It is suggested that the formation of the M-like intermediate indicates branching pathways from the L intermediate or basic heterogeneity in the original pigment.     

Temperature and halide dependence of the photocycle of halorhodopsin from Natronobacterium pharaonis

The photocycle kinetics of halorhodopsin from Natronobacterium pharaonis (pHR(575)) was analyzed at different temperatures and chloride concentrations as well as various halides. Over the whole range of modified parameters the kinetics can be adequately modeled with six apparent rate constants. Assuming a model in which the observed rates are assigned to irreversible transitions of a single relaxation chain, six kinetically distinguishable states (P(1-6)) are discernible that are formed from four chromophore states (spectral archetypes S(j): K(570), L(N)(520), O(600), pHR'(575)). Whereas P(1) coincides with K(570) (S(1)), both P(2) and P(3) have identical spectra resembling L(520) (S(2)), thus representing a true spectral silent transition between them. P(4) constitutes a fast temperature-dependent equilibrium between the chromophore states S(2) and S(3) (L(520) and O(600), respectively). The subsequent equilibrium (P(5)) of the same spectral archetypes is only moderately temperature dependent but shows sensitivity toward the type of anion and the chloride concentration. Therefore, S(2) and S(3) occurring in P(4) as well as in P(5) have to be distinguished and are assigned to L(520)<--> O(1)(600) and O(2)(600)<--> N(520) equilibrium, respectively. It is proposed that P(4) and P(5) represent the anion release and uptake steps. Based on the experimental data affinities of the halide binding sites are estimated.     

The role of chloride in taurine transport across the human placental brush-border membrane.

Taurine, a sulfated beta-amino acid, is conditionally essential during development. A maternal supply of taurine is necessary for normal fetal growth and neurologic development, suggesting the importance of efficient placental transfer. Uptake by the brush-border membrane (BBM) in several other tissues has been shown to be via a selective Na(+)-dependent carrier mechanism which also has a specific anion requirement. Using BBM vesicles purified from the human placenta, we have confirmed the presence of Na(+)-dependent, carrier-mediated taurine transport with an apparent Km of 4.00 +/- 0.22 microM and a Vmax of 11.72-0.36 pmol mg-1 protein 20 s-1. Anion dependence was examined under voltage-clamped conditions, in order to minimize the contribution of membrane potential to transport. Uptake was significantly reduced when anions such as thiocyanate, gluconate, or nitrate were substituted for Cl-. In addition, a Cl(-)-gradient alone (under Na(+)-equilibrated conditions) could energize uphill transport as evidenced by accelerated uptake (3.13 +/- 0.8 pmol mg-1 protein 20 s-1) and an overshoot compared to Na+, Cl- equilibrated conditions (0.60 +/- 0.06 pmol mg-1 protein 20 s-1). A Cl(-)-gradient (Na(+)-equilibrated) also stimulated uptake of [3H]taurine against its concentration gradient. Analysis of uptake in the presence of varying concentrations of external Cl- suggested that 1 Cl- ion is involved in Na+/taurine cotransport. We conclude that Na(+)-dependent taurine uptake in the placental BBM has a selective anion requirement for optimum transport. This process is electrogenic and involves a stoichiometry of 2:1:1 for Na+/Cl-/taurine symport.     

Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel

The gating of ClC-0, the voltage-dependent Cl- channel from Torpedo electric organ, is strongly influenced by Cl- ions in the external solution. Raising external Cl- over the range 1-600 mM favors the fast- gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl- than to external Cl-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl- or not, in a weakly voltage-dependent fashion, (c) that the Cl(-)-liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl(-)- liganded closed state opens with a very high rate. According to this picture, Cl- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC-0. Moreover, since the Cl- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessarily coupled to ion conduction, a nonequilibrium process.     

The characteristics of Ca -activated Cl- channels of the salt-tolerant charophyte Lamprothamnium

The dependence of the Ca++-activated Cl- channels on potential difference (PD) was extracted from current-voltage (I/V) profiles recorded at the time of hypotonic regulation while the large conductance (G) K+ channels were blocked by tetraethylammonium (TEA). The total clamp current (I) was dominated by the Cl- I, i(Cl), with small contribution from the background I (i(background)). The i(Cl) was fitted by the Goldman-Hodgkin-Katz (GHK) model with enhanced PD dependence simulated by Boltzmann probability distributions. The i(background) was modelled by an empirical equation. The i(Cl) responded to PD changes within tens of milliseconds. The G maxima were located between -20 and -150 mV. The Cl- channel number and channel permeability parameter, N(Cl)P(Cl), decreased as a function of time in a hypotonic medium (from 0.45 x 10(-7) to 0.17 x 10(-7) ms(-1) in 19 min), with the positive half activation PD, V50+, shifting from +35 to -65 mV, and the negative half activation PD, V50-, shifting from -134 to -310 mV. The fitted Cl- concentration [Cl-]cyt at the time of hypotonic regulation indicated rapid equalization of vacuolar and cytoplasmic concentrations. Excellent data obtained under similar experimental conditions in a previous study enabled us to infer [Ca++]cyt influences on the Cl- channel characteristics. Thick sulphated polysaccharide mucilage, found on Lamprothamnium cells acclimated to more saline media, eliminated the activation of the i(Cl) at the time of the hypotonic regulation. This effect was reversed by the application of the enzyme heparinase. The characteristics of the i(Cl) were found to be consistent with a component of the excitation Is at the time of the action potential (AP). The short duration of the excitation transients was contrasted with that of the hypotonic regulation. The mechanisms for Cl- channel activation (and hence the Ca++ channel activation) were considered.     

FTIR spectroscopy of the M photointermediate in pharaonis rhoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate (ppR(M)). The present FTIR spectroscopy of ppR(M) revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR). In addition, the C==O stretching vibrations of Asn-105 were assigned for ppR and ppR(M). The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate). These results implicate similar protein structural changes between ppR and BR. However, BR(M) decays to BR(N) accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure. In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M(N)) at alkaline pH. In ppR, such an N-like (M(N)-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.