Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physiological processes. While excessive ROS damages cells, small fluctuations in ROS levels represent physiological signals important for vital functions. Despite the physiological importance of ROS, many fundamental questions remain unanswered, such as which types of ROS occur in cells, how they distribute inside cells, and how long they remain in an active form. The current study presents a ratiometric sensor of intracellular ROS levels based on genetically engineered voltage-gated sodium channels (roNaV). roNaV can be used for detecting oxidative modification that occurs near the plasma membrane with a sensitivity similar to existing fluorescence-based ROS sensors. Moreover, roNaV has several advantages over traditional sensors because it does not need excitation light for sensing, and thus, can be used to detect phototoxic cellular modifications. In addition, the ROS dynamic range of roNaV is easily manipulated in real time by means of the endogenous channel inactivation mechanism. Measurements on ROS liberated from intracellular Lucifer Yellow and genetically encoded KillerRed have revealed an assessment of ROS lifetime in individual mammalian cells. Flashlight-induced ROS concentration decayed with two major time constants of about 10 and 1000 ms.

The voltage-dependent block of AMPA receptor (AMPAR) channels by a series of dicationic compounds was studied on native GluR2-lacking receptors of striatal giant interneurons isolated from rat brain slices. The dicationic derivatives of adamantane, dimethyladamantane, diphenyl, and phenylcyclohexyl were used. The voltage dependence of the blockade and of the unblocking rate constant suggests that the compounds permeate the open AMPAR channels. The permeation of adamantane derivatives was demonstrated previously. However, for derivatives of phenylcyclohexyl this finding is surprising because of the large dimensions of the phenylcyclohexyl moiety. All these compounds were found to get trapped in the closed state of the channel. However, time-dependent decrease of trapping was found. This effect is accelerated by hyperpolarization, suggesting that blockers can escape from trapping into the cytoplasm channel. Importantly, there is a correlation between permeation through the open channel and escape from the closed channel. Dicationic compounds were demonstrated to block open and closed AMPAR channels from inside of the cell Thus, trapping of AMPAR channel blockers after agonist removal does not prevent escape of blockers into the cytoplasm It is concluded that closure of the AMPAR channel gates at the extracellular vestibule is not coupled with plugging of the pathway between the selectivity filter and cytoplasm. Possible physiological importance of this blocking mechanism is discussed.
Neuropharmacology 54(4): 653-64 (2008)