Channelrhodopsin-2 is a leaky proton pump.

Since its discovery, the light-gated cation channel Channelrhodopsin-2 (ChR2) has proven to be a long-sought tool for the noninvasive, light-activated control of neural cells in culture and in living animals. Although ChR2 is widely used in neurobiological applications, little is known about its molecular mechanism. In this work, the unitary conductance of ChR2 was determined for different cations, for example 40 fS at 200 mM NaCl and -60 mV, using noise analysis. The kinetics of the ion channel obtained by noise analysis is in excellent agreement with the photocurrent kinetics obtained by voltage-clamp and time-resolved spectroscopy. The inward rectification of the channel could be explained by the single channel parameters. ChR2 represents an ion channel with a 7 transmembrane helix motif, even though the sequence homology of its essential amino acids to those of the light-driven H(+) pump bacteriorhodopsin (bR) is high. Here, we also show that when ChR2 is expressed in electrofused giant HEK293 cells or reconstituted on planar lipid membranes, it can indeed act as an outwardly driven H(+) pump, demonstrating that ChR2 is bifunctional, and in-line with other microbial rhodopsins, a H(+) pump but with a leak that shows ion channel properties.

Time resolved kinetics of the guinea pig Na-Ca exchanger (NCX1) expressed in Xenopus oocytes: voltage and Ca(2+) dependence of pre-steady-state current investigated by photolytic Ca (2+)concentration jumps.

Kinetic properties of the Na-Ca exchanger (guinea pig NCX1) expressed in Xenopus oocytes were investigated by patch clamp techniques and photolytic Ca(2+) concentration jumps. Current measured in oocyte membranes expressing NCX1 is almost indistinguishable from current measured in patches derived from cardiac myocytes. In the Ca-Ca exchange mode, a transient inward current is observed, whereas in the Na-Ca exchange mode, current either rises to a plateau, or at higher Ca(2+) concentration jumps, an initial transient is followed by a plateau. No comparable current was observed in membrane patches not expressing NCX1, indicating that photolytic Ca(2+) concentrations jumps activate Na-Ca exchange current. Electrical currents generated by NCX1 expressed in Xenopus oocytes are about four times larger than those obtained from cardiac myocyte membranes enabling current recording with smaller concentration jumps and/or higher time resolution. The apparent affinity for Ca(2+) of nonstationary exchange currents (0.1 mM) is much lower than that of stationary currents (6 muM). Measurement of the Ca(2+) dependence of the rising phase provides direct evidence that the association rate constant for Ca(2+) is about 5 x 10(8) M(-1) s(-1) and voltage independent. In both transport modes, the transient current decays with a voltage independent but Ca(2+)-dependent rate constant, which is about 9,000 s(-1) at saturating Ca(2+) concentrations. The voltage independence of this relaxation is maintained for Ca(2+) concentrations far below saturation. In the Ca-Ca exchange mode, the amount of charge translocated after a concentration jump is independent of the magnitude of the jump but voltage dependent, increasing at negative voltages. The slope of the charge-voltage relation is independent of the Ca(2+) concentration. Major conclusions are: (1) Photolytic Ca(2+) concentration jumps generate current related to NCX1. (2) The dissociation constant for Ca(2+) at the cytoplasmic transport binding site is about 0.1 mM. (3) The association rate constant of Ca(2+) at the cytoplasmic transport sites is high (5 x 10(-8) M(-1)s(-1)) and voltage independent. (4) The minimal five-state model (voltage independent binding reactions, one voltage independent conformational transition and one very fast voltage dependent conformational transition) used before to describe Ca(2+) translocation at saturating Ca(2+) concentrations is valid for Ca(2+) concentrations far below saturation.