Structural energetics of voltage- and ligand-dependent gating in ion channels

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Abstract Ion channels are exquisite molecular machines that regulate the flow of ions across cell membranes in response to stimuli such as voltage and small molecule ligands (e.g. second messengers, and neurotransmitters). They underlie all electrical excitability in the brain and heart, and defects in ion channels are responsible for many human disorders. Despite decades of experiments and many high-resolution molecular structures, we still do not know, for any channel, the mechanisms for voltage- or ligand- dependent gating. The missing ingredient seems to be conformational energetics. The energetics of the different channel conformations governs the time course, voltage- dependence, and ligand-dependence of opening of the channel pore, and ultimately electrical excitability of the cell. In this proposal we will determine the mechanisms of voltage-dependent gating and ligand-dependent gating and fill important gaps in our understanding of ion channel biology. We will focus on the cyclic nucleotide-binding domain (CNBD) family of ion channels, which are structurally related, but functionally diverse. Whereas some CNBD channels are activated by depolarization, others are activated by hyperpolarization, and some members are activated by cAMP yet others are activated by cGMP. We will leverage breakthrough FRET methods we developed for measuring intramolecular distance distributions and conformational energetics using fluorescence lifetime imaging microscopy (FLIM), simultaneous with recordings of channel function using patch-clamp fluorometry (PCF). The data from multiple donor- acceptor sites throughout the channels will be compiled into a four-dimensional map (X, Y, Z, and energy) of the conformational rearrangements associated with ligand- dependent and voltage-dependent activation of CNBD channels. Our long-term vision is to understand the general themes that underlie allosteric regulation of ion channels, and these experiments promise rapid progress toward this goal. Ultimately, the methods and principles we discover will be of broad utility for elucidating mechanisms for all allosteric proteins.