Date of Award
Spring 5-6-2025
Document Type
Thesis
Degree Name
Bachelor of Arts
Department
Biology
First Advisor
Chris Gillen
Abstract
Aedes aegypti cation chloride cotransporter 2 (aeCCC2) belongs to a family of Na+-dependent cation chloride cotransporters which includes Na+-K+-2Cl- (NKCC) cotransporters (Duong et al. 2022). Despite high sequence similarity to the electroneutral NKCCs, aeCCC2 transports ions electrogenically, suggesting a transport mechanism that has differentiated from the NKCCs (Kalsi et al. 2019). In this thesis, we use site-directed mutagenesis and a Xenopus laevis expression system to explore residue-specific contributions to the transport properties of aeCCC2 in order to further investigate its transport mechanism. We found that aeCCC2-mediated transport saturated at low extracellular ion concentrations. Furthermore, single mutations at ion-binding residues L155 or W158 decreased ion affinity three to four-fold, while constructs carrying mutations at both L155 and W158 had a ten-fold decrease in ion affinity. aeCCC2 mutants had decreased Li+ uptake rates and increased Rb+ uptake rates in comparison to aeCCC2 WT. In fact, the Li+:Rb+ uptake rate ratio was roughly two to four-fold lower than aeCCC2 WT in aeCCC2 mutants. To explore the effects of hypotonic activation on aeCCC2-mediated transport, we measured the ion affinity of aeCCC2 in response to hypotonic stimulation, as well as the Li+ transport rate of aeCCC2 constructs carrying mutations at the predicted SPS/Ste20 proline-alanine-rich kinase (SPAK)-binding site. Hypotonic activation does not alter the ion affinity of aeCCC2, and aeCCC2-mediated Li+ uptake is abolished in kinase-binding mutants. Altogether, these results suggest a carrier-like mechanism of aeCCC2-mediated transport that is dependent on basal activation by the with no lysine (WNK)/SPAK kinase complex, where L155 and W158 both contribute to ion binding and may contribute to ion selectivity.
Recommended Citation
Yarcusko, Ryan, "Characterization of functionally important residues in the transport mechanism of Aedes aegypti cation chloride cotransporter 2 (aeCCC2)" (2025). Honors Theses. 980.
https://digital.kenyon.edu/honorstheses/980
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