Characterization of Membrane Proteins: From a gated plant aquaporin to animal ion channel receptors

Abstract: Membrane proteins play several important roles in a cell. Among these proteins are aquaporins (AQPs) and transient receptor potential (TRP) ion channels that mediate water transport, temperature and noxious chemical sensation, respectively. The function of some AQPs, for example the spinach isoform SoPIP2;1 is regulated by pH, phosphorylation and heavy metals such as mercury. However, the mechanisms by which mercury activate or inhibits AQPs are poorly understood. We suggest that mercury binds to SoPIP2;1 close to the C-terminal end and that the binding of mercury results in destabilization of the C-terminal region. This may affect its interaction with the residues forming the gate and therefore lead to an increase of the water permeability of SoPIP2;1 (Paper II). SoPIP2;1 is a highly selective water channel and can be produced as a functional protein in high yield in a heterologous system which suggest that SoPIP2;1 is a good choice for insertion in biomimetic membranes to be used for water purification. However, the stability of SoPIP2;1 in artificial membranes needed to be demonstrated. Thus we determined the stability of SoPIP2;1 in different lipids and identified E. coli polar lipids as the best system for reconstitution of SoPIP2;1. The results will contribute towards the effort to use SoPIP2;1 in biomimetic water filtration technology (Paper I). The animal TRP ion channel subtype A1 (TRPA1) from fruit fly, snake and mosquito has been implicated in warm temperature sensation. However, the threshold temperature which activates human TRPA1 (hTRPA1) is controversial. We addressed this issue by reconstituting the purified hTRPA1 in artificial lipid membranes. The purified hTRPA1 was found to be activated by cold temperatures and electrophilic chemicals. The results resolve the controversy surrounding the threshold temperature for the activation of hTRPA1 (Paper IV). The Anopheles gambiae TRPA1 (AgTRPA1) was found to be activated by heat and electrophilic compounds when reconstituted in artificial membranes after purification. The temperature activation as well as the binding of electrophilic ligands to AgTRPA1 resulted in the quenching of fluorescence suggesting that thermal and chemical activation brought about similar conformational changes of the protein and perhaps reflect the dynamic change in the conformation of residues involved in the gating process (Paper III). We also demonstrated that the N-terminal domain of both human and mosquito TRPA1 is not essential for thermal/chemical sensation (Paper III and Paper IV) as opposed to previous reports.