Gene arrangement within concatemeric constructs governs the functional divergence of human and rat ASIC3 concatemers

Acid-sensing ion channels (ASICs) are proton-gated sodium channels encoded by four genes in mammals. The ASIC3 isoform is widely expressed and plays a critical role in pain signaling and inflammatory responses. Despite a high degree of sequence homology between rat and human ASIC3 orthologs, they exhibit pronounced functional differences. Notably, rat ASIC3 undergoes steady-state desensitization (SSD) at extracellular pH values of 7.1-7.0, whereas human ASIC3 is largely desensitized at physiological pH (7.4-7.3), underscoring substantial interspecies divergence with important translational implications. In this study, we systematically investigated the functional properties of rat and human ASIC3 channels using concatemeric constructs that enable precise control over subunit stoichiometry and gene order within engineered trimeric assemblies. Using whole-cell electrophysiology, we show that homomeric concatemeric channels (r-r-r and h-h-h) closely recapitulate the biophysical properties of their respective wild-type channels. Substitution of a single subunit at the N-terminal position of the concatemer with an ASIC3 ortholog produced minimal functional perturbations and yielded channel properties most similar to those of the corresponding homomeric channel. In contrast, replacement of the C-terminal subunit resulted in the most pronounced shifts in pH₅₀SSD values and significant alterations in activation kinetics and current characteristics, indicating that functional properties of ASIC3 concatemers are strongly influenced by subunit position within the construct. Together, these findings demonstrate that both subunit composition and positional arrangement contribute to the functional behavior of engineered ASIC3 concatemers and provide a useful experimental framework for analyzing interspecies differences between rat and human ASIC3 channels. The results also highlight important limitations of concatemeric approaches, including the potential emergence of non-native channel properties in certain construct configurations.