eNpHR30

eNpHR3.0, short for enhanced Natronomonas pharaonis halorhodopsin 3.0, is an engineered optogenetic inhibitor used to silence neurons. It is a modified version of the natural halorhodopsin (NpHR) that has been optimized for improved trafficking to the neuronal plasma membrane and higher expression levels in mammalian systems. In many constructs it is fused to a fluorescent reporter, such as EYFP, to verify expression and localization.

Mechanism and light sensitivity. eNpHR3.0 functions as a light-activated chloride pump. When illuminated with amber to yellow light, typically around 590 nm, it pumps chloride ions into the neuron, driving the membrane potential toward hyperpolarization and suppressing action potential generation. The result is inhibition of neuronal activity during illumination, with cessation of the effect when light is removed. Because it is a pump rather than a channel, the dynamics depend on pump turnover and light intensity, yielding rapid onset and offset on the millisecond to tens of milliseconds scale in many preparations.

Applications and usage. eNpHR3.0 is widely used in optogenetic experiments to selectively silence defined neuronal populations or circuits in vivo and in vitro. It is commonly delivered via viral vectors (such as AAV) under a suitable promoter or genetic system (e.g., Cre-dependent designs) to target specific cell types and brain regions. It is compatible with chronic experiments and dual-hemisphere or multi-parameter optogenetic setups, often paired with excitatory tools for bidirectional control of circuits.

Considerations and limitations. While eNpHR3.0 provides robust silencing, high light intensities are sometimes required for sustained inhibition, which can raise concerns about tissue heating and off-target effects. Prolonged or excessive expression may perturb chloride homeostasis or cellular physiology in some contexts. Researchers select specific variants and delivery strategies to balance expression, trafficking, and experimental goals.