Light-Driven Chloride Transport Kinetics of Halorhodopsin

Abstract

Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl^- transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl^-/protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle.

Figure 1

Kinetic model of the NpHR photocycle. The photocycle model is adapted from Chizhov et al. (25), where P₀ represents the ground state of NpHR and P₁–P₆ are six kinetically distinguishable intermediates. Corresponding half-lives for each transition are listed between the corresponding intermediates. A photon of light initiates the photocycle by promoting the transition between P₀ and P₁. The last and the longest step in this process, the transition from P₆ to the ground state (P₀) with a half-life of 20 ms, is currently accepted to be the rate-limiting step. The “bypass” hypothesis suggests that P₆ can absorb a photon of light and directly transition to P₁, bypassing P₀, under continuous illumination.

Figure 2

Purification and characterization of NpHR. Silver-stained SDS-PAGE (left) and anti-histidine tag Western blot (center) show a single band around 30 kDa consistent with the NpHR monomeric molecular weight. The absorbance spectrum of purified NpHR with an absorbance maximum at 580 nm (right).

Figure 3

Determination of the per-protein transport rate of NpHR expressed in oocytes from NpHR-induced photocurrents in a voltage-clamp set-up and NpHR quantification using Coomassie and Western blot analysis. (A) The determination of concentration of detergent-solubilized Myc-tagged NpHR from Coomassie gel analysis of NpHR-anti-Myc-antibody complex is shown. The gel can be found in Fig. S1A. Densitometry analysis was conducted on the gel band corresponding to the 50 kDa heavy chain of the antibody after denaturation of the NpHR-antibody complex and compared against a Coomassie calibration curve obtained with different known concentrations of 26 kDa glutathione S-transferase. (B) The determination of Myc-tagged NpHR expression in oocyte membranes using Western blot analysis is shown. The gel can be found in Fig. S1B. A calibration curve of a range of known concentrations of denatured NpHR-antibody complex was used to determine the concentration of NpHR per oocyte. The curve was obtained from a calibration curve of the band intensity from a Western blot analysis on independently purified Myc-tagged NpHR proteins. The band intensity was observed to reach saturation at the higher concentrations of purified NpHR, and only concentrations for which the calibration curve was linear were used to obtain the concentration of NpHR expressed in oocytes. Two different cRNA concentrations were used per batch of oocytes to induce different levels of NpHR expression. Each cRNA concentration was treated as a replicate. Two different batches of oocytes were thus tested, resulting in four replicates. (C) The current induced in NpHR-expressing oocytes was determined using two electrode voltage clamp (TEVC) on oocytes during illumination by a 589 nm laser. Normalizing the current at zero potential from (C) with corresponding NpHR per oocyte from (B) results in an average transport rate of 0.35 (± 0.16) ions/protein/photon/s. Current recordings were obtained from seven to nine oocytes per cRNA injection per oocyte batch.

Figure 4

High-repetition consecutive singe-turnover flash experiments on detergent-solubilized NpHR micelles. Detergent-solubilized NpHR micelles purified from E. coli were illuminated with consecutive actinic flashes at repetition rates of 20 Hz (50 ms between flashes, dash-dotted line), 50 Hz (20 ms between flashes, dashed line), 100 Hz (10 ms between flashes, dotted line), and 200 Hz (5 ms between flashes, solid line). The resulting light-induced changes in absorbance at 570 nm, representative of the chloride uptake step, were recorded as a function of time. Each trace is the difference between a single data collection with and without actinic pulses at the specified repetition rate. Arrows indicate actinic flash events at an intensity of 4 mJ/pulse at 532 nm. The amplitudes of the consecutive absorbance change events at 570 nm are comparable at all excitation rates tested, including the 100 and the 200 Hz repetition rates, which are more frequent than the P₆–P₀ half-life duration (t_1/2 = 20 ms).

Figure 5

NpHR transport rates obtained from voltage-clamped current measurements on NpHR expressing oocytes (this study) compared with indirectly measured or inferred transport rates and quantum yields from different techniques reported in literature. The normalized per-photon oocyte-based transport rate of NpHR of 0.35 (± 0.16) Cl⁻/photon obtained for an NpHR transport rate of 219 (± 98) Cl⁻/protein/s for a photon flux of 630 photons/protein/s is in agreement with the reported quantum yield of NpHR of 0.3. The measured value is also in agreement with the electrophysiology-based per-photon estimates on tandem fusion constructs of NpHR with ChR2, which resulted in a transport rate of 0.33 Cl⁻/photon or 1245 ions/protein/s at a photon flux of 3600 photons/protein/s (24).