Dietrich Gradmann

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.

Electrocoupling of ion transporters in plants.

In the plasmalemma of plants, the major ion transporters are voltage gated. Hence, they are intrinsically coupled via the membrane voltage. Theoretical predictions and electrophysiological recordings on guard cells demonstrate nonlinear oscillations of a dynamic system which provides longterm osmotic adjustment by switching between periods of net uptake and net release of salt, rather than by a steady-state.

Membrane-potential responses following gravistimulation in roots of Lepidium sativum L.

Membrane potentials were measured in lateral statocytes of vertically and nonvertically growing roots of Lepidium sativum L. using conventional glass-microelectrode techniques. Statocytes in vertically growing roots showed a stable resting potential of-118±5.9 mV without spontaneous fluctuations. Upon tilting the root 45° from the vertical, an electrical asymmetry was observed. Statocytes on the physically lower side of the root depolarized by approx. 25 mV. This depolarization occurred following a latent period of 8 s reaching a minimum (approx.-93 mV) after 170 s. This depolarization is the earliest event in graviperception ever recorded. After this depolarization, the cell repolarized within 60 s to a potential approx. 10 mV more positive than the original resting potential. Statocytes on the upper flank showed a slow hyperpolarization (t1/2h=half time for hyperpolarization=168 s) reaching a final, stable potential at a level 10 mV more negative. These effects of gravistimulation were statenchyma-specific, since cells in the cortex and rhizodermis showed no similar effects. The gravi-electrical responses were observed in 25% of all roots tested. Roots which showed no gravi-electrical response had a reduced elongation growth, lacked gravity-induced bending and lacked the typical structural polarity in punctured statocytes. This observed transition from a symmetrical pattern of resting potential in the statenchyma to an asymmetrical pattern following gravistimulation supports the results observed with external current measurements (Behrens et al., Plant Physiol. 70, 1079–1083, 1982) and extends these results to the cellular level and to considerably improved temporal resolution. The asymmetry in the gravi-electrical response extends the graviperception model of Sievers and Volkmann (Planta 102, 160–172, 1972) which comprises an asymmetrical sedimentation of the amyloplasts on the distal endoplasmic reticulum of statocytes. This generates an intraorgan signal which then must be transmitted to the growth zone.

Gating and conductance in an outward-rectifying K+ channel from the plasma membrane of Saccharomyces cerevisiae.

SummaryThe plasma membrane of the yeast Saccharomyces cerevisiae has been investigated by patch-clamp techniques, focusing upon the most conspicuous ion channel in that membrane, a K+-selective channel. In simple observations on inside-out patches, the channel is predominantly closed at negative membrane voltages, but opens upon polarization towards positive voltages, typically displaying long flickery openings of several hundred milliseconds, separated by long gaps (G). Elevating cytoplasmic calcium shortens the gaps but also introduces brief blocks (B, closures of 2–3 msec duration). On the assumption that the flickery open intervals constitute bursts of very brief openings and closings, below the time resolution of the recording system, analysis via the beta distribution revealed typical closed durations (interrupts, I) near 0.3 msec, and similar open durations. Overall behavior of the channel is most simply described by a kinetic model with a single open state (O), and three parallel closed states with significantly different lifetimes: long (G), short (B) and very short (I). Detailed kinetic analysis of the three open/closed transitions, particularly with varied membrane voltage and cytoplasmic calcium concentration, yielded the following stability constants for channel closure: KI=3.3 · e−zu in which u=eVm/kT is the reduced membrane voltage, and z is the charge number; KG = 1.9 · 10−4([Ca2+] · ezu )−1; and KB =2.7 · 103([Ca2+] · ezu )2. Because of the antagonistic effects of both membrane voltage (Vm ) and cytoplasmic calcium concentration ([Ca2+]cyt) on channel opening from the B state, compared with openings from the G state, plots of net open probability (P0) vs. either Vm or [Ca2+] are bell-shaped, approaching unity at low calcium (μm) and high voltage (+150 mV), and approaching 0.25 at high calcium (10 mm) and zero voltage. Current-voltage curves of the open channel are sigmoid vs. membrane voltage, saturating at large positive or large negative voltages; but time-averaged currents, along the rising limb of P0 (in the range 0 to +150 mV, for 10 μm [Ca2+]) make this channel a strong outward rectifier. The overall properties of the channel suggest that it functions in balancing charge movements during secondary active transport in Saccharomyces.

K+-Sensitive Gating of the K+ Outward Rectifier in Vicia Guard Cells

Abstract. The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K+ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K+ current was monitored under voltage clamp in 0.1–30 mm K+ and in equivalent concentrations of Rb+, Cs+ and Na+. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K+ activated current through outward-rectifying K+ channels (IK,out) at the plasma membrane in a voltage-dependent fashion. Increasing [K+]o shifted the voltage-sensitivity of IK,out in parallel with the equilibrium potential for K+ across the membrane. A similar effect of [K+]o was evident in the kinetics of IK,out activation and deactivation, as well as the steady-state conductance- (gK−) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K+, −80 mV in 1.0 mm K+, and −20 mV in 10 mm K+. Similar data were obtained with Rb+ and Cs+, but not with Na+, consistent with the relative efficacy of cation binding under equilibrium conditions (K+≥ Rb+ > Cs+ > > Na+). Changing Ca2+ or Mg2+ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of gK or on IK,out activation kinetics, although 10 mm [Ca2+]o accelerated current deactivation at voltages negative of −75 mV. At any one voltage, increasing [K+]o suppressed gK completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K+ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively. These, and additional data indicate that extracellular K+ acts as a ligand and alters the voltage-dependence of IK,out gating; the results implicate K+-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K+ ions outside affects the distribution between closed states of the channel.

Reaction kinetic parameters for ion transport from steady-state current-voltage curves

This study demonstrates possible ways to estimate the rate constants of reaction kinetic models for ion transport from steady-state current-voltage data as measured at various substrate concentrations. This issue is treated theoretically by algebraic reduction and extension of a reaction kinetic four-state model for uniport. Furthermore, an example for application is given; current-voltage data from an open K+ selective channel (Schroeder, J.I., R. Hedrich, and J.M. Fernandez, 1984, Nature (Lond.), 312:361-362) supplemented by some new data have been evaluated. The analysis yields absolute numerical estimates of the 14 rate constants of a six-state model, which is discussed in a wider context.

Enzyme kinetics of the prime K+ channel in the tonoplast of Chara: selectivity and inhibition.

SummaryThe prime potassium channel from the tonoplast of Chara corallina has been analyzed in terms of an enzyme kinetic model (Gradmann, Klieber & Hansen 1987, Biophys. J.53:287) with respect to its selectivity for K+ over Rb+ and to its blockage by Cs+ and by Ca2+. The channel was investigated by patchclamp techniques over a range of membrane voltages (Vm, referred to an extracytoplasmic electrical potential of zero) from −200 mV to + 200 mV under various ionic conditions (0 to 300 mM K+, Rb+, Cs+, Ca2+, and Cl−) on the two sides of isolated patches. The experimental data are apparent steady-state currentvoltage relationships under all experimental conditions used and amplitude histograms of the seemingly noisy open-channel currents in the presence of Cs+. The used model for K+ uniport comprises a reaction cycle of one binding site through four states, i.e., (1) K+-loaded and charged, facing the cytoplasm, (2) K+-loaded and charged facing the vacuole, (3) empty, facing the vacuole, and (4) empty, facing the cytoplasm. Vm enters the system in the form of a symmetric Eyring barrier between state 1 and 2. The numerical results for the individual rate constants are (in 106s−1 for zero voltage and 1 m substrate concentration): k12: 1,410, k21: 3,370, k23: 105,000, k32: 10,600, k34: 194, k43: 270, k41: 5,290, k14: 15,800. For the additional presence of an alternate transportee (here Rb+), the model can be extended in an analog way by another two states ((5) Rb+-loaded and charged, facing cytoplasm, and (6) Rb+-loaded and charged, facing vacuole) and six more rate constants (k45: 300, k54: 240, k56: 498, k65: 4,510, k63: 4,070, k36: 403). This six-state model with its unique set of fourteen parameters satisfies the complete set of experimental data. If the competing substrate can be bound but not translocated (here Cs+ and Ca2+), k56 and k65 of the model are zero, and the stability constants Kcyt (= k36/k63) and Kvac (= k45/k54) turn out to be Kcyt(Ca2+): 250 m−1 · exp(Vm/(64 mV)), kvac(Ca2+): 10 m−1 · exp(−Vm/(66 mV)), Kcyt(Cs+): 0, and Kvac(Cs+): 46 m−2 · exp(−Vm/(12.25 mV)). With the assumption that the current fluctuations in the presence of Cs+ consist of incompletely resolved, short periods of complete openings and complete closures, the amplitude histograms of the noisy open channel currents can be described by a beta distribution, yielding the rate constants for binding (92 · 106 sec−1 · m−2 · exp(−Vm/(22.5 mV)) and debinding (2, 106 sec−1 · m−2 · exp(Vm/(22.5 mV)) of Cs+ to the vacuolar side of the channel as functions of the [Cs+] and of Vm. Considering these data and those from the literature, an asymmetry of the channel can be assessed, with a high charge density at the cytoplasmic side (Eisenman-series Nr. XI) and a low charge density at the vacuolar side (Eisenman-series Nr. I). Furthermore, the results provide an example for intimate linkage between conduction and switching of a channel.

Microscopic elements of electrical excitation in Chara: Transient activity of Cl− channels in the plasma membrane

The plasma membrane of Chara corallina was made accessible for patch pipettes by cutting a small window through the cell wall of plasmolyzed internodal cells. With pipettes containing Cl− as Ca2+ or Ba2+ (50 or 100 mm), but not as Mg2+ or K+ salt, it was possible to record in the cell-attached mode for long periods with little channel activity, randomly interspersed with intervals of transient activation of two Cl− channel types (cord conductance at +50 mV: 52 and 16 pS, respectively). During these periods of transient channel activity, variable numbers (up to some 10) of the two Cl− channel types activated and again inactivated over several 100 msec in a coordinated fashion. Transient Cl channel activity was favored by voltages positive of the free running membrane voltage (> −45 mV); but positive voltage alone was neither a sufficient nor a necessary condition for activtion of these channels. Neither type of Cl− channel was markedly voltage dependent. A third, nonselective 4 pS channel is a candidate for Ca2+ translocation. The activity of this channel does not correlate in time with the transient activity of the Cl− channels. The entire set of results is consistent with the following microscopic mechanism of action potentials in Chara, concerning the role of Ca2+ and Cl− for triggering and time course: Ca2+ uptake does not activate Cl− channels directly but first supplies a membrane-associated population of Ca2+ storage sites. Depolarization enhances discharge of Ca2+ from these elements (none or few under the patch pipette) resulting in a local and transient increase of free Ca2+ concentration ([Ca2+]cyt) at the inner side of the membrane before being scavenged by the cytoplasmic Ca2+ buffer system. In turn, the transient rise in [Ca2+]cyt causes the transient activity of those Cl− channels, which are more likely to open at an elevated Ca2+ concentration.

Calcium release from InsP3‐sensitive internal stores initiates action potential in Chara

Neomycin and U73122 are known to suppress inositol 1,4,5‐trisphosphate (InsP3) production by inhibition of phospholipase C. We studied the effects of these inhibitors on the excitatory currents, I ex, in Chara corallina under voltage‐clamp conditions. Computer simulations of the experimental effects by a minimum model for the excitatory reaction pathway allow the assignment of the inhibitory effects to one specific reaction step, i.e. the release of Ca2+ from InsP3‐sensitive internal stores. In contrast, the inhibitory effect of La3+ on I ex suggests inactivation of Cl− channels. Furthermore, ryanodine‐sensitive Ca2+ stores seem to be irrelevant for electrical excitation in Chara.

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.