Are cysteine repeat modular proteins ion channels? Initial characterisation of a conserved Apicomplexan protein family of putative calcium channels

Abstract

The Cysteine Repeat Modular Proteins (CRMPs) are a protein family conserved in multiple Apicomplexan species including the malaria parasites, Toxoplasma, Neospora, Eimeria, Theileria, Babesia and Cryptosporidium. CRMPs have been predicted to be membrane proteins with nine transmembrane domains, a poly-cysteine repeat modular domain and two conserved multi-transmembrane domains. Furthermore, these CRMP proteins have been reported to be essential for host cell invasion in Toxoplasma gondii, a process mediated by calcium signaling. Interestingly, bioinformatic analysis show similarities with a protein family of putative fungal calcium channels, called Flavin carrier proteins (FLCs). Therefore, I hypothesize that CRMPs are calcium channels that trigger the molecular mechanisms involved in cell invasion. To investigate this, I have generated constructs to achieve heterologous expression and membrane trafficking of TgCRMPet and ScFLC-2 in HEK293 cells and have performed patch clamp electrophysiology to generate a protocol to assess their ion channel activity. These results provide initial characterization of a previously undescribed Apicomplexan family protein, updates their phylogeny, and set the foundations to further investigate the ion channel activity of these proteins.