Functional impact of transmembrane domain and turret on potassium channel gating

• Applicant: Professorin Dr. Indra Schröder
• Subject Area: Plant Biochemistry and Biophysics
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 265445268

This project utilizes the new potential of the viral Kcv channels for the investigation of structure/function correlates in potassium (K+) channels. Although the understanding of K+ channels has made breathtaking progress in the last decades with important implications for medical research, some basic issues are not yet fully understood on a structural level, e.g. how channels with the same selectivity filter can have different single-channel conductivities, or quasi identical voltage sensors (VS) can mediate activation by de- or hyperpolarization.Under the premise that the pore influences all gating properties of a channel, I will study fundamental structure/function correlates in the pore by using functional variants of the Kcv channels, which provide a model of the pore of all K+ channels. The power of this model system results from the fact that:- There are more than 60 Kcv orthologs with different functional properties in spite of minor sequence differences. This natural library provides a guideline to the functionally relevant amino acids (aa) and interactions in the protein.- Kcv channels are minimal channels (<100 aa per subunit). They are not yet functionally specialized, which prevents in more evolved channels many mutations and the creation of functional chimeras. This simplicity also favors structural studies by NMR or by molecular dynamics simulations.The project focuses on parts of the central channel pore, which have so far received little attention, but emerged in preliminary experiments as important: the extracellular turret and the outer transmembrane domain (TM1).One working hypothesis is that the turret mediates the coupling between TM1 and the selectivity filter. Kcv orthologs have two types of turrets which differ in length and charge. Chimeras and mutations which alter length and charge of the turret will be created and the impact on gating examined. Preliminary data show that such chimeras are functional and that the flavor of the turret affects channel gating.The second focus is on the role of TM1. While the role of TM2 is well established, TM1 has mostly been ignored even though it provides in Kv channels the mechanical coupling between VS and pore and is hence most likely relevant for gating. There are Kcv orthologs, which are >90% identical with minor differences in TM1 but with substantial differences in gating. Mutations and chimeras will reveal the key amino acids and interactions within the protein that affect gating.Both subprojects will merge in experiments investigating the interaction of turret and TM1. The general strategy is a combination of molecular biology (mutations, chimeras) and my special expertise in single-channel analysis and modeling of gating. The project will lay the grounds for a more detailed correlation of channel function and structure. I have started a cooperation with the department of chemistry to synthesize the small Kcv channels for solid state NMR spectroscopy.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Gerhard Thiel