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

Abstract

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.

Figure 1

Definitions, recapitulation, and reaction schemes of the working hypothesis. (A) Typical time course of ChR2 photocurrent upon a bright, rectangular light pulse: after an early peak (I_P), the current relaxes to a stationary level, I_∞; extrapolation of the steepest slope to t = 0 yields the initial current, I₀. (B) Simplified photocycle of ChR (6,7) with early and late conducting (open) states O₁ and O₂, respectively, plus two nonconducting (closed) states, C₁ and C₂. Asterisks indicate photoisomerization steps. Due to the irreversible step from C₂ to C₁, the steady-state occupation probabilities, p, of the four states are p_C1 = 1, and p_O1 = p_O2 = p_C2 = 0 in the dark; hence, I₀ can be assigned to state O₁ exclusively. (C) Reaction scheme for experiments with concentration changes of external ions; bold line indicates rapid equilibria. Derivation of three-state model from standard four-state model (gray) for uniport of one substrate i through enzyme E when no internal concentration changes take place. Subscripts c and e represent cytoplasmic and external measures, respectively. In the global model, n ion species, i, with their specific valencies, z_i, compete for the empty binding site with charge z_E, resulting in n three-state cycles sharing reorientation of the empty binding site between the cytoplasmic and external sides. (D) Current-voltage relationships for current amplitudes of I₀ and I_∞ versus holding voltage, recorded at pH_e 9 and 6. I_S is the reference current at pH_e 7.5 with 100 mM external Na⁺, for standardizing results from various preparations. Arrows indicate the estimated reversal voltages, and insets show individual current tracings at the indicated holding voltages.

Figure 2

Typical experiment for determination of reversal voltages E_r0 and E_r∞ of ChR2 in Xenopus oocytes under various ionic conditions (in mM): internal, ∼120 Na⁺, ∼40 Cl⁻, pH_c 7.3; external, 0.1 CaCl₂, 2 MgCl₂, and 100 mM NMGCl (A) or 100 mM NaCl (B). Voltage-clamp recordings of initial and stationary currents, I, were carried out at holding voltages, E_C, in the vicinity of reversal voltages. Readings of E_r are from intersections of regression lines with the zero-current line in the absence of external Na⁺ (A), and in the presence of 100 mM external Na⁺ (B) (for statistical evidence, see Fig. 4D; for quantitative analysis, see Table 1). (C) Original photocurrent records, measured at the indicated holding voltages. Note change of current sign in middle tracing (−34 mV), indicating change of reversal voltage and selectivity during a light pulse. Since the experiments in A–C were from different oocytes, the current scale in C was adjusted to match A and B.

Figure 3

Typical experiment for determination of reversal voltages E_r0 and E_r∞ of ChR2 in HEK cells under various ionic conditions (in mM): internal, 120 NaCl, 10 Hepes, 2 MgCl₂, 10 EGTA, and 2 CaCl₂, pH_c 7.3; and external, 2 CaCl₂, 2 MgCl₂, and 100 mM NMGCl (A) or 100 mM NaCl (B). Voltage-clamp records of initial and stationary currents, I, are shown at holding voltages, E_C, in the vicinity of reversal voltages. Readings of E_r are from intersections of regression lines with the zero-current line. in the absence of external Na⁺ (A), and with 100 mM added external Na⁺ (B). The predominant result was E_r∞ < E_r0 throughout, indicating increased H⁺ selectivity in the stationary state compared to the initial state (for statistical evidence, see Fig. 4A, and for quantitative analysis, see Table 1). (C) Original photocurrent records. Note change of current sign in the middle tracing (−25 mV), indicating change of reversal voltage and selectivity during a light pulse. The current scale was adjusted to match A and B.

Figure 4

Initial (red) and stationary (blue) reversal voltages, E_r0 and E_r∞, of ChR2 as a function of pH_e, recorded with and without 100 mM external Na⁺ in HEK cells and Xenopus oocytes. Values are means of n ≥ 3 independent recordings. (A) Absolute E_r values in HEK cells (internal ion concentrations, in mM: 120 NaCl, 10 Hepes, 2 MgCl₂, 2 CaCl₂, and 10 EGTA, pH_c 7.3); small SEs of 0.5–5.7 mV (n = 3) not illustrated. dotted line: theoretical Nernstian E_H(pH_e) with slope m = 1 (here, rounded 60 mV/pH unit); solid lines: theoretical relationships according to Eq. 10 with mean conductance ratios α and β from Table 1 at pH_e 9.0, with and without 100 mM Na⁺_e. (B) Same as A, but for Xenopus oocytes (internal ion concentrations, in mM: 120 Na⁺, 40 Cl⁻, pH_c 7.4; data taken from Zhang and Prigge (4); small SEs of 1.0–5.0 mV (n = 3–7) are not shown. (C and D) Statistically supported differences, E_r∞ − E_r0, at pH_e 9, for HEK cells and Xenopus oocytes, respectively.

Figure 5

Effect of divalent cations, D²⁺ (Mg²⁺ + Ca²⁺), on E_r. (A) Theoretical relations: impact of 10-fold increase and decrease of conductance ratios α (k_cH⁰/k_cM⁰), β (k_cD⁰/k_cM⁰), and γ (k_ce⁰/k_cM⁰) on E_r([D²⁺]_e) relationships compared to reference configuration (inset). Note that increase of γ and decrease of β are almost equivalent in this configuration ([M⁺]_c = 120 mM (100 mM K⁺ and 20 mM Na²⁺), [D²⁺]_c = 2 mM (2 mM Mg²⁺ and 0.25 μM Ca²⁺), pH_c 7.3, pH_e [M⁺]_e = 0, [D²⁺]_c as marked by pCa_e on abscissa). To circumvent corresponding fluctuations in fits, γ was arbitrarily fixed at γ = 1 for numerical analysis. (B) Experimental results (symbols, means ± SE) and fits (solid lines) of E_r at various external [Ca²⁺]; cytoplasmic ions, γ = 1, [M⁺]_e = 0, pH_e 9.0, as in A. To obtain fair fits, an ad hoc correction, δ_D, was introduced to account for the impact of external Mg²⁺: [D²⁺]_e = δ_D + [Ca²⁺]_e (see numerical results in Table 2). (Inset) Example of original current-voltage data for ChR2 in HEK cells (pH_e 9, [M⁺]_e = 0, internal solution, as in main figure) showing [Ca²⁺]_e dependency of E_r.