Ion selectivity and competition in channelrhodopsins.

Channelrhodopsins are light-gated ion channels of green algae. They are widely used for the analysis of neuronal networks using light in the emerging field of optogenetics. Under steady-state light conditions, the two open states, O1 and O2, mediate the photocurrents with different ion conductance and selectivity. To understand the conducting process as well as its optogenetic applications, it is important to study ion binding and transport of this promiscuous cation channel. Here, we present an enzyme kinetic algorithm that allowed us to calculate the ion composition of the initial and steady-state photocurrents for multication media. The approach is based on current-voltage relations determined for the individual ions H(+), Na(+), Ca(2+), and Mg(2+). We identify and quantify the widely different competition of the ions in wild-type channelrhodopsin-2 and two high-performing channelrhodopsin variants CatCh+ and C1V1. Both variants show enhanced Ca(2+) conductance, but only CatCh+ displays high steady-state Ca(2+) currents at neutral pH due to reduced H+ competition and low inactivation. We demonstrate that for optogenetic applications, one should always take into account that the variable equilibria of the two open states depend on light intensity, voltage, and the ionic composition of the medium.

Rectification of the channelrhodopsin early conductance.

We analyzed the nonlinear current-voltage relationships of the early conducting state of channelrhodopsin-2 expressed in Xenopus oocytes and human embryonic kidney cells with respect to changes of the electrochemical gradients of H(+), Na(+)/K(+), and Ca(2+)/Mg(2+). Several models were tested for wild-type ChR2 and mutations at positions E90, E123, H134, and T159. Voltage-gating was excluded as cause for the nonlinearity. However, a general enzyme kinetic model with one predominant binding site yielded good fits throughout. The empty site with an apparent charge number of about -0.3 and strong external cation binding causes some inward rectification of the uniport function. Additional inward rectification is due to asymmetric competition from outside between the transported ion species. Significant improvement of the fits was achieved by introducing an elastic voltage-divider formed by the voltage-sensitive barriers.

Two open states with progressive proton selectivities in the branched channelrhodopsin-2 photocycle.

Channelrhodopsins are light-gated ion channels that mediate vision in phototactic green algae like Chlamydomonas. In neurosciences, channelrhodopsins are widely used to light-trigger action potentials in transfected cells. All known channelrhodopsins preferentially conduct H(+). Previous studies have indicated the existence of an early and a late conducting state within the channelrhodopsin photocycle. Here, we show that for channelrhodopsin-2 expressed in Xenopus oocytes and HEK cells, the two open states have different ion selectivities that cause changes in the channelrhodopsin-2 reversal voltage during a light pulse. An enzyme kinetic algorithm was applied to convert the reversal voltages in various ionic conditions to conductance ratios for H(+) and divalent cations (Ca(2+) and/or Mg(2+)), as compared to monovalent cations (Na(+) and/or K(+)). Compared to monovalent cation conductance, the H(+) conductance, alpha, is approximately 3 x 10(6) and the divalent cation conductance, beta, is approximately 0.01 in the early conducting state. In the stationary mixture of the early and late states, alpha is larger and beta smaller, both by a factor of approximately 2. The results suggest that the ionic basis of light perception in Chlamydomonas is relatively nonspecific in the beginning of a light pulse but becomes more selective for protons during longer light exposures.

Dynamics of voltage profile in enzymatic ion transporters, demonstrated in electrokinetics of proton pumping rhodopsin.

H(+)-pumping rhodopsins mediate a primordial conversion of light to metabolic energy. Bacteriorhodopsin from Halobacterium salinarium is the first identified and (biochemically) best-studied H(+)-pumping rhodopsin. The electrical properties of H(+)-pumping rhodopsins, however, are known in more detail for the homolog Acetabularia rhodopsin, isolated from the eukaryotic green alga Acetabularia acetabulum. Based on data from Acetabularia rhodopsin we present a general reaction kinetic model of H(+)-pumping rhodopsins with only seven independent parameters, which fits the kinetic properties of photocurrents as functions of light, transmembrane voltage, internal and external pH, and time. The model describes fast photoisomerization of retinal with simultaneous H(+) transfer to an H(+) acceptor, reprotonation of retinal from the intracellular face via an H(+) donor, and proton release to the extracellular space via an H(+) release complex. The voltage sensitivities of the individual reaction steps and their temporal changes are treated here by a novel approach, whereby--as in an Ohmic voltage divider--the effective portions of the total transmembrane voltage decrease with the relative velocities of the individual reaction steps. This analysis quantitatively infers dynamic changes of the voltage profile and of the pK values of the H(+)-binding sites involved.

Channelrhodopsin-1 initiates phototaxis and photophobic responses in chlamydomonas by immediate light-induced depolarization.

Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H+-channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H+, CHR1 also conducts Na+, K+, and Ca2+. The kinetic selectivity analysis demonstrates that H+ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1505 and CHR1463, that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions.

H+ -pumping rhodopsin from the marine alga Acetabularia.

An opsin-encoding cDNA was cloned from the marine alga Acetabularia acetabulum. The cDNA was expressed in Xenopus oocytes into functional Acetabularia rhodopsin (AR) mediating H+ carried outward photocurrents of up to 1.2 microA with an action spectrum maximum at 518 nm (AR518). AR is the first ion-pumping rhodopsin found in a plant organism. Steady-state photocurrents of AR are always positive and rise sigmoidally from negative to positive transmembrane voltages. Numerous kinetic details (amplitudes and time constants), including voltage-dependent recovery of the dark state after light-off, are documented with respect to their sensitivities to light, internal and external pH, and the transmembrane voltage. The results are analyzed by enzyme kinetic formalisms using a simplified version of the known photocycle of bacteriorhodopsin (BR). Blue-light causes a shunt of the photocycle under H+ reuptake from the extracellular side. Similarities and differences of AR with BR are pointed out. This detailed electrophysiological characterization highlights voltage dependencies in catalytic membrane processes of this eukaryotic, H+ -pumping rhodopsin and of microbial-type rhodopsins in general.

Oscillations in plant membrane transport: model predictions, experimental validation, and physiological implications.

Although oscillations in membrane-transport activity are ubiquitous in plants, the ionic mechanisms of ultradian oscillations in plant cells remain largely unknown, despite much phenomenological data. The physiological role of such oscillations is also the subject of much speculation. Over the last decade, much experimental evidence showing oscillations in net ion fluxes across the plasma membrane of plant cells has been accumulated using the non-invasive MIFE technique. In this study, a recently proposed feedback-controlled oscillatory model was used. The model adequately describes the observed ion flux oscillations within the minute range of periods and predicts: (i) strong dependence of the period of oscillations on the rate constants for the H+ pump; (ii) a substantial phase shift between oscillations in net H+ and K+ fluxes; (iii) cessation of oscillations when H+ pump activity is suppressed; (iv) the existence of some 'window' of external temperatures and ionic concentrations, where non-damped oscillations are observed: outside this range, even small changes in external parameters lead to progressive damping and aperiodic behaviour; (v) frequency encoding of environmental information by oscillatory patterns; and (vi) strong dependence of oscillatory characteristics on cell size. All these predictions were successfully confirmed by direct experimental observations, when net ion fluxes were measured from root and leaf tissues of various plant species, or from single cells. Because oscillatory behaviour is inherent in feedback control systems having phase shifts, it is argued from this model that suitable conditions will allow oscillations in any cell or tissue. The possible physiological role of such oscillations is discussed in the context of plant adaptive responses to salinity, temperature, osmotic, hypoxia, and pH stresses.

Electrokinetics of miniature K+ channel: open-state V sensitivity and inhibition by K+ driving force.

Kcv, isolated from a Chlorella virus, is the smallest known K+ channel. When Kcv is expressed in Xenopus oocytes and exposed to 50 mM: [K+](o), its open-state current-voltage relationship (I-V) has the shape of a "tilted S" between -200 and +120 mV. Details of this shape depend on the conditioning voltage (V (c)) immediately before an I-V recording. Unexpectedly, the I-V relationships, recorded in different [K+](o), do intersect. These characteristics are numerically described here by fits of a kinetic model to the experimental data. In this model, the V (c) sensitivity of I-V is mainly assigned to an affinity increase of external K+ association at more positive voltages. The general, tilted-S shape as well as the unexpected intersections of the I-V relationships are kinetically described by a decrease of the cord conductance by the electrochemical driving force for K+ in either direction, like in fast V-dependent blocking by competing ions.

Multiple photocycles of channelrhodopsin.

Two rhodopsins with intrinsic ion conductance have been identified recently in Chlamydomonas reinhardtii. They were named "channelrhodopsins" ChR1 and ChR2. Both were expressed in Xenopus laevis oocytes, and their properties were studied qualitatively by two electrode voltage clamp techniques. ChR1 is specific for H+, whereas ChR2 conducts Na+, K+, Ca2+, and guanidinium. ChR2 responds to the onset of light with a peak conductance, followed by a smaller steady-state conductance. Upon a second stimulation, the peak is smaller and recovers to full size faster at high external pH. ChR1 was reported to respond with a steady-state conductance only but is demonstrated here to have a peak conductance at high light intensities too. We analyzed quantitatively the light-induced conductance of ChR1 and developed a reaction scheme that describes the photocurrent kinetics at various light conditions. ChR1 exists in two dark states, D1 and D2, that photoisomerize to the conducting states M1 and M2, respectively. Dark-adapted ChR1 is completely arrested in D1. M1 converts into D1 within milliseconds but, in addition, equilibrates with the second conducting state M2 that decays to the second dark state D2. Thus, light-adapted ChR1 represents a mixture of D1 and D2. D2 thermally reconverts to D1 in minutes, i.e., much slower than any reaction of the two photocycles.

Fast, triangular voltage clamp for recording and kinetic analysis of an ion transporter expressed in Xenopus oocytes.

We present a procedure for determination of 11 system parameters of an ion transporter expressed in Xenopus oocytes. The experiments consist of fast triangular voltage-clamp experiments in the presence and absence of external substrate. A four-state enzymatic cycle operating between an external and an internal section of electrodiffusion is used for analysis. The explicit example treats experiments with the fungal 2H+-NO3- symporter EnNRT, a member of the major superfamily transporters. The results comprise a density of approximately 150 fmol functional transporter molecules per oocyte, a gross charge number z(E) approximately -0.3 of the empty binding site of the enzyme, individual rate constants for reorientation of the empty and occupied binding site in the range of 5-500 s(-1), electrical access sections between bulk solutions and reaction cycle of approximately 3% inside and 15% outside, an increase of internal NO3- at the plasma membrane from approximately 0.5 to approximately 2 mM during exposure to external NO3-, and K(D) approximately 0.3 microM3 inside and K(D) approximately 3 microM3 outside in binding the triplicate substrate (2H+ +NO3-). The results compare well with the known structure of the lactose permease, another major superfamily transporter.

Apparent charge of binding site in ion-translocating enzymes: kinetic impact.

Recently, we presented a general scope for the nonlinear electrical properties of enzymes E which catalyze translocation of a substrate S with charge number z(S) through lipid membranes (Boyd et al. J. Membr. Biol. 195:1-12, 2003). In this study, the voltage sensitivity of the enzymatic reaction cycle has been assigned to one predominant reversible reaction step, i.e. the reorientation of either E or ES in the electric field, leaving the reorientation of the alternate state (ES or E) electroneutral, respectively. With this simplification, the steady-state current-voltage relationships (IV) assumed saturation kinetics like in Michaelis-Menten systems. Here, we introduce an apparent charge number z(E) of the unoccupied binding site of the enzyme, which accounts for the impact of all charged residues in the vicinity of the physical binding site. With this more realistic concept, the occupied binding site assumes an apparent charge of z(ES) = z(E) + z(S), and IV does not saturate any more in general, but exponentially approaches infinite or zero current for large voltage displacements from equilibrium. These nonlinear characteristics are presented here explicitly. They are qualitatively explained in a mechanistic way, and are illustrated by simple examples. We also demonstrate that the correct determination of the model parameters from experimental data is still possible after incorporating z(E) and its corollaries into the previous model of enzyme-mediated ion translocation.

Current-voltage-time records of ion translocating enzymes.

Membrane currents, as non-linear functions of membrane voltage, V, and time, t, can be recorded quickly by triangular V protocols. From the differences, d I(V, t), of these relationships upon addition of a putative substrate of a charge-translocating membrane protein, the I(V, t) relationships of the transporter itself can be determined. These relationships likely comprise a steady-state component, I(a)(V), of the active transporter, and a dynamic component, p(a)(V, t), of its V- and time-dependent activity, p(a). Here, the steady-state component is modeled by a central reaction cycle, which senses a fraction delta(tr) of the total V, whereas 1-delta(tr) can be assigned to an inner and outer pore section with delta(i) and delta(o), respectively (delta(i)+delta(tr)+delta(o) = 1). For the enzymatic cycle, fast binding/debinding is assumed, plus V-sensitive and -insensitive reaction steps which may become rate limiting for charge translocation. At given substrate concentrations, I(a)(V) is defined by eight independent system parameters, including a coefficient for the barrier shape of charge translocation. In ordinary cases, the behavior of p(a)(V, t) can be described by two rate constants (for activation and inactivation) and their respective V-sensitivity coefficients. Here, the effects of the individual system parameters on I(V, t) from triangular V-clamp experiments are investigated systematically. The results are illustrated by panels of typical curve shapes for non-gated and gated transporters to enable a first classification of mechanisms. We demonstrate that all system parameters can be determined fairly well by fitting the model to "experimental" data of known origin. Applicability of the model to channels, pumps and cotransporters is discussed.

Evidence for a light-induced H(+) conductance in the eye of the green alga Chlamydomonas reinhardtii.

Rhodopsin-mediated photoreceptor currents, I(P), of the unicellular alga Chlamydomonas reinhardtii were studied under neutral and acidic conditions. We characterized the kinetically overlapping components of the first, flash-induced inward current recorded from the eye, I(P1), as a low- and high-intensity component, I(P1a) and I(P1b), respectively. They peak between 1 and 10 ms after the light-flash and are both likely to be carried by Ca(2+). I(P1a) and I(P1b) exhibit half-maximal photon flux densities, Q(1/2), of approximately 0.14 and 58 microE m(-2), and maximal amplitudes of approximately 4.9 and 38 pA, respectively. At acidic extracellular pH values (pH 3-5), both I(P1) currents are followed by distinct H(+) currents, I(P2a) and I(P2b), with maxima after approximately 5 and 100 ms, respectively. Because the Q(1/2) values of I(P1b) and I(P2b) virtually coincide with Q(1/2) of rhodopsin bleaching, we suggest that the respective conductances G(1b) and G(2b) are closely coupled to the rhodopsin, whereas the low light-saturating conductances G(1a) and G(2a) reflect transducer-activated states of a second rhodopsin photoreceptor system.