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.

Abstract (Swedish)

Popular Abstract in English

Cells are the basic unit of life that exhibit dependence on water and ambient temperature to flourish and survive. So it is remarkable that the proteins responsible for the interaction of water and heat with the cell were not discovered until relatively recently. It was assumed for decades that water freely diffuse into and out of the cells without support. The cells are surrounded by a lipid (fat) membrane which forms a barrier to the passage of molecules that are not hydrophobic (fat soluble). Since lipid and water are immiscible, water does not readily pass through the membranes and require assistance in transport across the membranes. The identity of the proteins that form the channel in the membranes for the transport of water molecules was discovered in 1992. These proteins are now called as aquaporins (AQPs). In addition to water, some AQPs also transport other nutrient molecules such as glycerol, urea, ammonia, carbon dioxide and also signaling molecules such as hydrogen peroxide. The AQPs are vital to plant and animal function as exemplified by growth defects and diseases associated with AQP aberrant expression and function and therefore it is essential to study their function. The mechanism of water and nutrient conduction through AQPs is now well established but there are some aspects of the AQP function that are not very well understood. For example, the function of some of these AQPs is known to be inhibited by the mercury a toxic metal found in nature. However, some AQPs are activated by mercury despite sharing the similar structure with the AQPs inhibited by mercury. Although, the activation of AQP by mercury has been recognized for some time, the mechanism of activation is not known. In this thesis, we found out that mercury activate a spinach AQP. The structure of the spinach AQP is known which suggests that some part of the AQP protein act as a gate and open up the channel for water transport upon phosphorylation (a type of modification that is commonly known to regulate the function of large number of proteins). We speculate that mercury activated the spinach AQP by binding close to the gate of the channel and disrupting interactions in the closed form and thereby open it in a similar way as phosphorylation. Toxic heavy metals such as mercury and nickel produced by industrial waste are increasingly incorporated in the food chain and known to modulate the function of AQPs. Our finding shed new light into the mechanism of activation by mercury and will contribute towards a better understanding of AQP function. The spinach AQP is a highly selective water channel and it can be produced in abundance thus making it attractive to use in biotechnological application such as biomimetic water purification systems. However, this application requires the demonstration of stability of spinach AQP in different biomimetic membranes. We have conducted these studies and we hope that it will contribute towards the efforts to use the spinach AQP in biotechnological application. As mentioned earlier, the heat responsive proteins present in the cell membranes were discovered very recently. These proteins are called transient receptor potential (TRP) ion channels and have several different subtypes with a distinct threshold for temperature activation. Some of these TRPs also lined our taste buds and responsible for the strong taste sensation when activated by chemicals found in food such as wasabi, pepper, chili, oregano, thyme, garlic, onion and compound found in mustard oil, eucalyptus oil, lemon grass and menthol etc. The fruit fly, snake and mosquito TRP type A1 (TRPA1) mediate warm temperature sensation. However, the temperature activation of human TRPA1 is mired in controversy with different researchers reporting disparate threshold temperatures. Moreover, there is no demonstration of TRP activation by temperature in a pure system without presence of accessory proteins and small molecules. We demonstrated the activation of human and mosquito TRPA1 by cold and heat, respectively, in a purified system, thus proving for the first time that temperature sensation is indeed intrinsic to TRPA1, which will eventually resolve the controversy regarding the cold activation of human TRPA1. A large part of the TRPA1 protein is formed by a repetitive unit known as ankyrin repeats. Some researchers have proposed that these repeats are essential for thermal and chemical sensation. We demonstrate in this thesis that TRPA1 still retain the temperature and chemical sensation even after the removal of these repeats. These results will contribute towards a better understanding of TRPA1 function which has been recognized as a target for pain management. Inhibitors of human TRPA1 will have an application as painkillers. Similarly, mosquitos use TRPA1 to identify hosts for sucking blood, thus compounds that modulate the function of TRPA1 may function as mosquito repellants. However, these pursuits will require a better understanding of TRPA1 function. The results of this study may contribute towards these efforts by providing the evidence that ankyrin repeats are not responsible for temperature and chemical sensation and therefore future drug design should target other parts of TRPA1 proteins for selective inhibition of chemical and temperature sensing domain.

• author: Survery, Sabeen
• supervisor: Urban Johanson; Per Kjellbom
• opponent: Prof Beitz, Eric, Pharmaceutical and Medicinal Chemistry University of Kiel, Germany
• organization: Biochemistry and Structural Biology
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: Biological Sciences
• keywords: MIPs, AQPs, TRP ion channels, water transport, themo sensor, Electrophiles
• pages: 156 pages
• defense location: Hall B Kemicentrum
• defense date: 2015-06-12 10:15:00
• ISBN: 978-91-7422-403-0
• language: English
• LU publication?: yes
• id: 2738a10e-24fe-41aa-b5a9-bdff7fbdec75 (old id 5415972)
• date added to LUP: 2016-04-04 11:39:18
• date last changed: 2018-11-21 21:06:16