Sep. 1, 2010
Potassium channels are dynamic proteins that undergo large conformational changes to regulate the flow of K+ ions across the cell membrane. Understanding the gating mechanism of these channels therefore requires methods for probing channel structure in both their open and closed conformations. Radiolytic footprinting is used to study the gating mechanism of the inwardly-rectifying potassium channel KirBac3.1. The purified protein stabilized in either open or closed conformations was exposed to focused synchrotron X-ray beams on millisecond timescales to modify solvent accessible amino acid side chains. These modifications were identified and quantified using high-resolution mass spectrometry. The differences observed between the closed and open states were then used to reveal local conformational changes that occur during channel gating. The results provide support for a proposed gating mechanism of the Kir channel and demonstrate a method of probing the dynamic gating mechanism of other integral membrane proteins and ion channels. This study also demonstrates that radiolytic footprinting coupled with mass spectrometry can provide a powerful probe of ion channel gating dynamics, and has the potential to be applied to the gating mechanism of many other classes of ion channels and membrane proteins. Furthermore, future studies that combine such recent advances in radiolytic labeling coupled to rapid O18 exchange mean this technique could also be used to probe channel gating processes in real time with millisecond resolution.
Figure 1. Radiolytic Labeling Identifies Residues in KirBac3.1 with Increased Modification Rates in the Open State. (A) Experimental scheme. (B) Dose response plot. A representative dose response plot for peptide, 120-GML*GL*AVAASLI-131 (*indicates modification identified by MS/MS). The closed and open states of KirBac3.1 were prepared in buffer containing 50 mM Mg2+ and 1 mM EDTA respectively. EDTA is an ,OH radical scavenger and reduces the X-ray dose by a factor of _2.5 (Figure S3). The observed rate constant kopen is corrected and kopen-corrected is compared with that of kclosed (Table 1). The solvent accessibility of L122 and L124 at the cavity of membrane pore are increased by a factor of _31 from close to open state of KirBac3.1. Rates were calculated for all the modified peptides and compared between the closed and open states to obtain information on conformation dynamics. Error bars represent standard deviation between experimental repeats. (C) Mapping the solvent accessibility changes. Modified residues are represented by sticks on the X-ray crystal structure of KirBac3.1 in the closed state (PDB 1XL4). The color codes indicate the changes in rates of modification or solvent accessibility on transition from the closed to open state. Only the residues with >2-fold increase are highlighted.
Results from: Gupta, S., Bavro, V.N., D.Mello, R., Tucker, S.J., Venien-Bryan, C., Chance, M.R. Structure, 18(7): 839-46, 2010.