Elucidation of the Mechanisms for the Underlying Depolarization and Reversibility by Photoactive Molecule

Light-induced control of the cell membrane potential has enabled important advances in the study of biological processes involving the nervous system and muscle activity. The use of these light-induced modifications is expected in various medical applications, including the control of physiological responses and the recovery of lost functions by regulating nerve activity. In particular, charge-separating linkage molecules (Charge-Separation (CS) molecules) can depolarize cells by photoexcitation without genetic processing. However, the molecular mechanisms underlying cell membrane depolarization are unknown and have hindered its application. Here, we show that CS molecules localized in the cell membrane of PC12 cells using a high-density lipoprotein (HDL)-based drug carrier can excite the cells through a novel membrane current regulation mechanism by light irradiation.

Membrane potential, channel activity, and membrane capacitance were measured by patch clamp method in rat adrenal gland pheochromocytoma (PC12) cells and K_V-overexpressing PC12 cells. CS molecules localized in the cell membrane of PC12 cells using HDL-based drug carrier. The localization of CS molecule was measured by a confocal microscopy. The mRNA expression was tested by RT-PCR.

Current clamp measurements revealed that the photo-activated CS molecule causes a sharp depolarization of about 15 mV. Furthermore, it was shown by voltage clamp measurement that this mechanism inactivates the voltage-dependent potassium current and simultaneously generates photo-activated CS molecule induced (PACS) current owing to the loss of the cell membrane capacitance. This activity continues the depolarization of the target cell, but is reversible via a regenerative mechanism such as endocytosis and exocytosis because the cell membrane is intact.

Thus, the mechanism of photo-induced depolarization concludes that photo-activated TC1 causes depolarization by generating PACS current in parallel with the suppression of the K⁺ current. Moreover, the depolarization slowly restores by internalization of TC1 from the membrane and insertion of new lipids into the cell membrane, resulting in the restoration of K_V to normal activity and eliminating PACS currents, without cell damage. These results suggest the possibility of medical application that can safely control membrane excitation.