What determines the folding and therefore the function of membrane proteins? Our laboratory addresses this question in studies on ion-conducting channels: channels formed by antibiotic molecules, such as the linear gramicidins; and channels formed by integral membrane proteins, such as voltage-dependent sodium channels and CFTR (cystic fibrosis transmembrane conductance regulator) chloride channels. The structure of gramicidin channels is known at near-atomic resolution, permitting detailed structure-function studies based on a combination of sequence substitutions, spectroscopy, conformational energy calculations, and electrophysiological (single-channel) measurements. The experiments address the following questions:
- What are the determinants of channel folding and membrane insertion? Gramicidin channels are right-handed beta-helices; the helix preference is determined by side chain-side cahin and side chain-water interactions. These interactions are probed through amino acid substitutions and determinations of the helix preference.
- How does the surrounding bilayer modulate channel function? The modulation results, at least in part, from alterations in the bilayer's mechanical properties, which provides a novel mechanism for the control of membrane protein function. We have developed methods to measure the changes in bilayer mechanics, which will be used to relate changes in membrane structure to changes in channel function.
- What determines the microphysics of channel-mediated ion movement? The permeability characteristics of channels having different amino acid sequences are investigated to define the energetic and kinetic consequences of sequence substitutions on ion permeation.
The three-dimensional structures of voltage-dependent Na+ channels or CFTR channels are unknown, but electrophysiology provides detailed insights into channel function. These experiments address further questions:
- What are the roles of charged amino acid residues for channel function? These charges establish local electrostatic potential in the vicinity of the channel entrance and ligand binding sites; ion permeation and ligand binding are affected by changes in these electrostatic potentials, which therefore are important for channel function.
- Why do single voltage-dependent sodium channels gate in several distinct modes? This observation offers unique insights into the dynamics and functional heterogeneity of single molecules. Moreover, alterations in membrane environment, as occur during coronary occlusion and other pathophysiological states, alter the pattern of channel gating, but the mechanisms are presently not understood. We are testing whether alterations in the gating of bilayer-incorporated channels can be related to alterations in the surrounding bilayer's mechanical properties.