Voltage-gated ion channels (VGC) are proteins found in the membranes of practically all cells, that through opening and closing (gating) events let ions flow through between the internal and external milieu of the cells acting as very fast signaling entities. The most characteristic and intriguing aspect of VGC is that their function is modulated by voltage. That means that the protein senses changes in the electrical field and responds by opening, possibly through a sequence of conformational changes. With the advent of high resolution electrical recording techniques combined with the molecular cloning and engineering of ion channel proteins, it has been possible to identify parts of VGC that would serve as voltage-sensors, which has led to proposal of several mechanistic models on how the voltage-sensing event is translated into channel opening. Yet, the molecular and physical natures of the events that take place during voltage-gating are not resolved. It is the long-term goal of this proposal to contribute a physical molecular model of how VGC gate by studying intramolecular distances at rest and while channels are open, using optical tools along with functional recordings. The recent cloning of a bacterial sodium channel, NaChBac, which can be produced in large quantities, purified and reconstituted into lipid membranes, provides a unique opportunity to address these questions in great molecular detail.
The specific aims are: 1) Search for regions and residues that undergo distances changes associated with the voltage sensor and between the sensor and the gate region using lanthanide-based resonance energy transfer (LRET) in the reconstituted protein in different conformational states induced by voltage changes in proteoliposomes; 2) Measurement of distances in tandem proteins, purified and reconstituted, bearing a single donor acceptor pair using the same technique as in aim 1; and 3) Functional analysis of voltage sensing and gating using electrophysiology and site directed fluorescence and its correlation to structure and structural changes studied in aims 1 and 2. To measure distances, cysteines are introduced in different parts of the protein and a special sequence, an EF-hand motif that binds lanthanides, is introduced in another part of the same protein. Fluorescent probes are then used to label the cysteine group and are prompted to emit upon excitation of the lanthanide with light. Because groups will be placed in areas suspected to participate in voltage gating, these measurements are expected to contribute real molecular distances and information on molecular rearrangements occurring during voltage gating. VGC are particularly important in nerve and muscle cells because they determine cell excitability and participate in cell-to-cell communication. The results from this work should help in our understanding of a large number of VGC that are crucial in health and in drawing strategies to 1'ameliorate or perhaps eventually cure some illnesses that involve the dysfunction of this important family of channels.
