Date of Award

Spring 5-12-2021

Document Type

Thesis

Degree Name

Bachelor of Arts

Department

Biochemistry & Molecular Biology

First Advisor

Prof. Chris Gillen

Abstract

The orthologs Drosophila melanogaster NCC83 and Aedes aegypti aeCCC2 and aeCCC3 share high sequence identity with related NKCC1 proteins of Drosophila melanogaster (NCC69) and Aedes aegypti (aeNKCC1). However, there are functional differences between NKCC1 and CCC2. While the well-characterized NKCC1 is electroneutral, chloride dependent and sensitive to loop diuretics, aeCCC2 is an electrogenic sodium transporter that is chloride-independent and insensitive to loop diuretics. Mutagenesis and structural studies of mammalian NKCC1 proteins have identified regions of the TMDs that participate in ion and inhibitor binding or are otherwise crucial to transport activity. We hypothesize that the functional differences between CCC2 and NKCC1 arise from structural differences in protein sequence at regions in the transmembrane domains (TMDs), both the residues that remain undiscovered and those previously identified by cryo-EM and mutagenesis. We wrote R programs to evaluate the conservation of amino acid identity between CCC2 and NKCC1 sequences from mosquitos and fruit flies. We observed that CCC2 is better conserved in fruit flies while NKCC1 is better conserved in mosquitos. Of 55 residues with known functional importance in the first ten TMDs of CCC2 and NKCC1, 36 residues were fully conserved in all mosquito and fruit fly CCC2 and NKCC1 sequences. Around one third of these 36 fully conserved and previously identified residues were different amino acids in the CCC2 and NKCC1 paralogs, indicating that conserved differences between paralogs may be connected to functional differences. In addition to these known residues, we discovered 16 new residues that meet the same criteria of conservation between CCC2 and NKCC1 and therefore are good candidates for future studies that aim to elucidate CCC2’s role in the secretory system.

Rights Statement

All rights reserved. This copy is provided to the Kenyon Community solely for individual academic use. For any other use, please contact the copyright holder for permission.

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