Using hierarchical thermodynamic linkage analysis to study ion channel gating

Ion channels are multi-subunit, multi-modal proteins that exhibit hierarchical organization, whereby the different components interact in a cooperative manner to realize a tunable function of the channel as a whole. Synergy is thus a fundamental aspect of ion channels threaded across all three complex system characteristics, namely, hierarchy, dynamism, and regulation. A full understanding of a system thus requires a formalism to assess the synergy between any two or more system components and across all system hierarchies. Thermody-namic linkage analysis (Wyman, 1964) offers such unified formalism, with the principle behind this approach being context dependence (Carter et al., 1984; Ackers and Smith, 1986). Two Perspectives addressing conforma-tional coupling in voltage-gated ion channels (Chowdhury and Chanda, 2012) and in voltage-and ligand-activated BK channels (Horrigan, 2012) published in the Decem-ber 2012 issue of the JGP highlighted the importance of domain-level linkage analysis for understanding how the coupling between gating and pore domains could lead to channel opening. Both papers demonstrated that when it comes to explaining the molecular details of such coupling, residue-level information, in particular for those residues found at domain interfaces, is required. Furthermore, knowing the magnitude and state dependence of residue interactions across such domain interfaces is essential for discriminating between possible gating mechanics scenarios. An accepted method for attaining such residue-level information is double mutant cycle coupling analysis (Horovitz and Ferst, 1990). Here, we point out that double mutant cycle analysis is, in fact, a context-dependent linkage analysis at a residue level and that, when properly applied, such linkage analysis is powerful and can reveal changes in residue interactions along the gating pathway of the channel. We further highlight that when combined, domain-and residue-level linkage analyses can reveal the molecular details of domain coupling leading to channel opening. The conceptual analysis method of thermodynamic linkage (Wyman, 1964) offers a unified formalism for assessing interactions, as exemplified for the case of li-gand-linked conformational changes found at the heart Correspondence to Ofer Yifrach: o f e r y @ b g u. a c. i l of the MWC classical allosteric model (Monod et al., 1965). In such formalism, the strength of nonadditive interactions and their contributions to protein function can be evaluated using the thermodynamic square presented in Fig. 1 A (Carter et al., 1984; Ackers and Smith, 1986), analogous, in essence, to Wyman's linkage cycle. In brief, at any level of channel protein (P) hierarchy, two components, X …