Recent work has indicated that there is a very substantial redistribution of ions between intracellular and extracellular ion levels throughout the day^1,2. Furthermore, ionic redistribution plays a major role in different episodic neural pathology such as seizures and cortical spreading depressions³. Particular attention has focused upon K⁺ and Cl^– levels where even small changes may affect the likelihood of pathological discharges such as seizures or cortical spreading depression⁴. Optogenetics allows for the control of ionic levels using light-controlled ion channels and pumps (opsins). While optogenetics is a well-established field, there is relatively untapped potential for pairing opsins in order to manipulate ionic movement.  The only example of this is Cl-out, which couples an Arch outward proton pump with an inhibitory channelrhodopsin⁵. The outward proton pump hyperpolarises the cell, thereby creating an electrochemical gradient for extruding chloride anions when the chloride channel is opened.

We have now developed and tested five new co-opsins for the manipulation of ionic movement.  We used PCR and overlapping PCR to create the constructs, inserted the vectors into HEK293 cells. The constructs contain a fluorescent tag for easy identification of expressing cells. Whole cell patch clamp was used to demonstrate light activated currents indicative of targeted membrane expression, and functionality.

1. Cl-out4KCR contains GtACR2, a general anion (chloride) channel, and KCR, a potassium channel. This couples chloride and potassium movement so that chloride will be extruded against its concentration gradient due to the outward movement of potassium. (n=10 cells)
2. K-in has KCR, a potassium channel, and ArchT3.0, an outward proton pump. ArchT3.0 moves positive charge out of the cell to manipulate the electrochemical gradient of potassium, thereby causing potassium to move into the cell through the open KCR channels. (n=5)
3. K-out combines KCR and ChR2, a general cation channel, with a reversal potential close to zero.  ChR therefore creates the electrochemical gradient for moving potassium out of cells when KCR2 is opened. (n=11)
4. HaloKCR combines KCR and NpHR, an inward chloride pump. When NpHR is activated it will change the electrical gradient of potassium so that when KCR1 is activated, potassium will enter the cell. (n=6)
5. HaloReverseKCR changes the order of NpHR and the inflexible linker βHK in order to flip NpHR. With NpHR flipped, chloride will be pumped out of the cell. This will change the electrochemical gradient of potassium so that when KCR is activated potassium will leave the cell in order to create a net outward ionic redistribution. (n=5)

We will illustrate these findings, and discuss these in relation to models of the constructs within the membrane, generated using Alphafold. We will discuss how these might be used for exploring novel pathophysiological mechanisms involving ionic redistribution.