Dissecting the mechanism of ligand regulation in cyclic nucleotide gated channels at the single molecule level.

The ability of a cell to transport ions across the cell membrane is one of the most fundamental biological processes and allows a cell to interact with its environment. Transport of ions is achieved by proteins called ion channels, that provide pathways for ions to cross the otherwise impermeable cell membrane. The opening and closing (gating) of ion channels in response to the binding of other small molecules (ligands) is a critical step in many signal transduction pathways. The cyclic-nucleotide gated (CNG) ion channel governs the signalling mechanisms that permit both sight and smell. Like other members of this family of ion channels, CNG channels are formed by the assembly of four individual subunits, with a portion of each subunit contributing to the channel structure. Each subunit contains a ligand-binding site, and binding to these portions of the protein is thought to cause an structural change that is transduced into opening/closure of the ion channel. This process of ligand binding, transduction, and channel gating is central to understanding the dynamic molecular mechanism of ligand gating in ion channels. A better understanding of ligand regulation in CNG would help us in designing methods to manipulate these important signalling mechaims. We will develop apparatus capable of simultaneous measurements of ligand binding and ion channel gating at the level of individual molecules. By observing ligand binding and transduction at the single-molecule level we will be able to study each step in this signal transduction process; observing both the ligand binding event and the subsequent response of the ion channel. We will use this approach to address the molecular mechanism of ligand regulation in a chimeric cyclic-nucleotide gated potassium channel, KCNG.

The ability of a cell to facilitate the selective movement of ions and small molecules across the plasma membrane is one of the most fundamental biological processes and allows a cell to interact with its environment. Ion channels are integral membrane proteins that provide ion-selective pathways across the otherwise impermeable cell membrane. The gating of ion channels in response to the binding of chemical ligands is a critical step in many signal transduction pathways. Cyclic-nucleotide gated (CNG) ion channels govern the signalling mechanisms that permit both sight and smell. Like other members of this family of ion channels, CNG channels are formed by the assembly of four individual subunits into a tetramer, with a portion of each subunit contributing to the channel structure. Each subunit contains a ligand-binding site, and binding to these domains is thought to cause an structural change that is transduced into opening/closure of the central pore. This process of ligand binding, transduction, and channel gating is central to understanding the dynamic molecular mechanism of ligand gated ion channels. We will develop apparatus capable of simultaneous measurements of ligand binding and ion channel gating at the single molecule level. This apparatus will combine single-molecule fluorescence measurements of fluorogenic ligands with electrical recording of ion current through the channel. This will enable us to directly relate a single ligand binding event to the electrical response of the channel. By observing ligand binding and transduction in one molecule we will be able to study each step in this signal transduction process. We will then use this approach to address the molecular mechanism of ligand regulation in a chimeric cNTP-gated potassium channel. These measurements will enable us to study in detail the relationship between binding, transduction and gating in a ligand gated ion channel.