Structural and functional characterization of aquaporin 4 from the freshwater gastropod Pomacea canaliculata under temperature stress.

BACKGROUND

Aquaporins (AQPs) are key membrane channels involved in water transport and osmotic regulation, yet their structural and functional diversity in mollusks remains insufficiently understood.

METHODS AND

RESULTS

In this study, aquaporin 4 (PcAqp4L) was identified and characterized by the freshwater gastropod Pomacea canaliculata, with particular attention to its structural properties, transport activity, transcriptional response to temperature stress, and mass spectrometry. Sequence and structural analysis showed that PcAqp4L possesses noncanonical pore architecture, including an atypical NPA/NPI motif and a distinctive V-V-G-R aromatic/arginine (ar/R) constriction. Functional assays in Xenopus laevis oocytes demonstrated that PcAqp4L significantly increased osmotic water permeability, while no detectable transport was observed for glycerol or other tested solutes, indicating that PcAqp4L functions as a strictly water-selective aquaporin. Transcriptional responses of PcAqp4L to temperature stress showed a contrasting pattern-upregulated under cold and downregulated under heatAlonsoimplicating it in temperature-dependent osmoregulation. Furthermore, mass spectrometry identified endogenous PcAqp4L-specific peptides, providing direct evidence of protein-level expression and suggesting potential regulatory complexity.

CONCLUSIONS

Collectively, these results indicate that PcAqp4L is a structurally atypical but functionally conserved water channel that may contribute to temperature-responsive water balance in P. canaliculata. By clarifying this link between structure, regulation, and function, our work broadens the current understanding of AQP structural diversity in mollusks and establishes a crucial mechanistic foundation for comprehending the roles of aquaporins in osmotic adaptation and stress tolerance among freshwater invertebrates.