Background

Research into the processing and regulation of information in nerve cells is a core task of modern neuroscience. Optogenetics, a technique that expresses light-sensitive channelrhodopsins (ChRs) derived from microalgae to open and close ion channels with light, is widely used to control brain cell activity. In particular, techniques for suppressing overexcited neurons attract significant attention as they may provide clues for developing treatments for brain disorders. To this end, attempts have continued to precisely block specific neural activity using potassium-selective channelrhodopsins (KCRs) such as HcKCR1 and WiChR1. When potassium ions move out of the cell, the membrane potential decreases, naturally inhibiting the release of neurotransmitters. However, for a long time, the actual amount of charge these channels conduct at the single-molecule level and the physical mechanism by which the ion channels open in response to light remained unclear. The single-channel currents were extremely weak, on the order of hundreds of femtoamperes (fA), making it impossible to distinguish signals from noise using conventional equipment. To optimize the intensity and frequency of optical stimulation, it is essential to clarify the microscopic operation of the ion channels, and the academic community has been waiting for the measurement techniques and analytical models to achieve this.

Key Findings

A joint research team led by Professor Klaus Benndorf from Friedrich Schiller University Jena and Professor Peter Hegemann from Humboldt University Berlin precisely measured the single-ion channel currents of HcKCR1 and WiChR1 using a single-channel recording device with femtosiemens (fS) resolution. The analysis revealed that both potassium channelrhodopsins do not fully open their ion channels immediately upon activation by light but instead pass through diverse intermediate conductance states. Both single-ion channels exhibited burst phenomena, where current was released in pulse-like patterns over a certain period. During these bursts, HcKCR1 recorded a dominant maximum conductance of 1.6 picosiemens (pS), and WiChR1 recorded 2.4 pS. At the same time, heterogeneous and continuous micro-conductances ranging from 0.3 pS to 3.0 pS were also observed. Among these, the burst duration of WiChR1 was approximately four times longer than that of HcKCR1. The research team clarified that the chemical cycle in response to light, known as the photocycle, and the physical opening and closing of the ion channel (closed-open gating) are independent, orthogonal processes. This single-molecule current data fully supports the behavior of whole-cell currents measured at the cellular level.

Significance and Outlook

This discovery is of great significance in revealing the operational principles of optogenetic tools at an extreme resolution at the single-molecule level. Contrary to the conventional belief that channelrhodopsins maintain a single fixed open state, the finding that the ion channel fluctuates through various microstructures within the open complex to allow ion passage suggests a new direction for the design of optogenetic proteins. By leveraging these physicochemical properties, it becomes easier to design customized optogenetic inhibitory tools that can enhance potassium ion conductivity or adjust the duration of the open state. Applications such as artificially modulating the conductance of the channel to selectively suppress target neurons while minimizing unnecessary ion leakage are also feasible. However, further research is required to examine additional variables, such as how the lipid composition of the neuronal membrane or changes in membrane potential affect channel structural fluctuations. It is also essential to verify whether the artificial experimental conditions can be replicated in the complex environment of brain tissue.