iC++

Stages

1. 1.
   Structure-informed pore selectivity modeling(library_design)

   This stage exists to identify rational pore modifications expected to invert or improve ion selectivity before experimental engineering.

   Selection: Development of a structure-informed electrostatic model for pore selectivity to guide residue changes in the ion conduction pathway.

2. 2.
   Crystal structure-guided engineering of next-generation chloride channels(library_build)

   This engineering stage converts model predictions into specific channel variants and seeks to overcome the small photocurrents of first-generation chloride-conducting channels.

   Selection: Introduce positively charged side chains into the ion conduction pathway and remove residues hypothesized to support negatively charged binding sites for cations, then further develop next-generation variants iC++ and SwiChR++.

3. 3.
   Functional characterization under physiological conditions(functional_characterization)

   This stage tests whether engineered channels improve the electrophysiological properties that limited first-generation chloride-conducting channels.

   Selection: Assess net photocurrents, reversal potential, and inhibition of spiking under physiological conditions.

4. 4.
   In vivo and behavioral validation(in_vivo_validation)

   This stage validates that the engineered channels function beyond physiological recordings and can support behavioral control in living animals.

   Selection: Demonstrate strong expression in vivo and control of freely moving behavior.

Steps

1. 1.
   Develop a structure-informed electrostatic model for pore selectivity

   Guide engineering decisions about which pore residues to modify to alter ion selectivity.

   The abstract states that first-generation engineering was guided in part by this model, so modeling precedes residue-level design.

2. 2.
   Introduce positively charged pore residues and remove putative cation-binding residues

   Reconfigure the ion conduction pathway to favor chloride selectivity.

   These residue changes operationalize the pore selectivity model before functional testing.

3. 3.
   Engineer next-generation variants iC++ and bistable SwiChR++engineered constructs

   Further develop and validate the pore model with improved chloride-conducting channelrhodopsins.

   After establishing the design logic, the campaign advances to named next-generation constructs intended to overcome first-generation limitations.

4. 4.
   Measure photocurrent and reversal potential under physiological conditionstested constructs

   Quantify whether next-generation variants improve electrophysiological performance over first-generation chloride-conducting channels.

   Electrophysiological characterization is needed before in vivo use to confirm that the engineered channels have stronger and more favorable inhibitory properties.

5. 5.
   Test inhibition of spiking relative to chloride gradients and intrinsic cell propertiestested constructs

   Determine whether inhibitory function tracks expected cellular determinants.

   After basic electrophysiological improvement is established, the next step is to confirm functional spike inhibition in relevant physiological contexts.

6. 6.
   Validate in vivo expression and behavioral controlvalidated constructs

   Establish practical in vivo utility and demonstrate control of freely moving behavior.

   In vivo expression and behavior are downstream validations that follow successful design and physiological functional characterization.