280 7.4  Molecular Cloning

using the energy from the absorption of photons of visible light, as well as other membrane

proteins that act as ion and voltage sensors (Figure 7.2).

For example, bacteriorhodopsin, proteorhodopsin, and archaerhodopsin are all proton

pumps integrated in the cell membranes of either bacteria or archaea. Upon absorption of

blue-​green light (the activation wavelengths (λ) span the range ~390–​540 nm), they will

pump protons from the cytoplasm to the outside of the cell. Their biological role is to estab­

lish a proton motive force across the cell membrane, which is then used to energize the pro­

duction of ATP (see Chapter 2).

Similarly, halorhodopsin is a chloride ion pump found in a type of archaea known as

halobacteria that thrive in very salty conditions, whose biological role is to maintain the

osmotic balance of a cell by pumping chloride into their cytoplasm from the outside, energized

by absorption of yellow/​green light (typically 540 nm < λ < 590 nm). Channelrhodopsin

(ChR), which is found in the single-​celled model alga C. reinhardtii, acts as a pump for a

range of nonspecific positive ions including protons, Na^+​ and K^+​ as well as the divalent Ca^2+​

ion. However, here longer wavelength red light (λ > 590 nm) fuels a pumping action from the

outside of the cell to the cytoplasm inside.

In addition, light-​sensitive protein sensors are used, for example, chloride and calcium ion

sensors, as well as membrane voltage sensor protein complexes. Finally, another class of light-​

sensitive membrane integrated proteins are used, the most commonly used being the optoXR

type. These undergo conformational changes upon the absorption of light, which triggers

intracellular chemical signaling reactions.

The light-​sensitive pumps used in optogenetics have a typical “on time” constant of a

few milliseconds, though this is dependent on the local excitation of laser illumination. The

importance of this is that it is comparable to the electrical conduction time from one end of a

single nerve cell to the other and so in principle allows individual action potential pulses to be

probed. The nervous conduction speed varies with nerve cell type but is roughly in the range

FIGURE 7.2  Optogenetics techniques. Schematic of different classes of light-​sensitive opsin

proteins, or luminopsins, made naturally by various single-​celled organisms, which can be

introduced into the nerve cells of animals using molecule cloning techniques. These luminopsins

include proton pumps called archeorhodopsins, bacteriorhopsins, and proteorhodopsins that

pump protons across the cell membrane out of the cell due to absorption of typically blue

light (activation wavelength λ1 ~ 390–​540 nm), chloride negative ion (anion) pumps called

halorhodopsins that pump chloride ions out of the cell (green/​yellow activation wavelength

λ2 ~ 540–​590 nm), and nonspecific positive ion (cation) pumps called channelrhodopsins that

pump cations into the cell (red activation wavelength λ3 > 590 nm).