Central Targeting of Channelrhodopsin2 by the Motif of Potassium Channel Kv2.1 Can be Altered Due to Overexpression of the Construct

Subcellular targeting of opsins in optogenetics provides new possibilities for studying the function of nerve cells. One of the widely used motifs for central targeting of opsins is the motif of the potential-dependent potassium channel Kv2.1. In our study, ChR2-Venus-Kv2.1 construct was expressed in the layer 2/3 pyramidal neurons of murine cerebral cortex by means of in utero electroporation. We found that, although the majority of neurons expressing ChR2-Venus-Kv2.1 demonstrated mainly central localization of fluorescence, limited to the soma, proximal dendrites, and axon, a considerable quantity of neurons remarkably exhibited disrupted targeting, i.e., fluorescent protein distributed far beyond the soma and proximal parts of all processes. We hypothesized that the observed mislocalization might have been caused by overexpression of the construct. It was found that a decrease in the plasmid concentration during the in utero electroporation from 1 to 0.35 μg/μl resulted in almost complete disappearance of the neurons that had altered targeting. Moreover, we found a pronounced correlation between the absolute brightness of the somatic fluorescence and the visible length of the apical dendrite, which also provides evidence that indeed overexpression might be the reason for the mislocalization of ChR2-Venus-Kv2.1 construct. Thus, the possibility of disruption of central targeting of ChR2 by the potassium channel Kv2.1 motif should be taken into account when using this construct in optogenetic experiments when somatic localization of the opsin is necessary. Lowering plasmid concentration during transfection procedure could be a solution for the above problem.