Thaddeus A. Bargiello, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): There is considerable controversy surrounding current models of the structure of channels formed by the connexin gene family. A recent model of the Cx32 channel (Fleishman et al. 2004) that is based on the structure of Cx43, obtained by image processing of frozen hydrate 2D crystals (Linger et al. 1999) uses the third transmembrane segment, M3, to form the majority of the aqueous channel pore. This view is supported by results of SCAM (substituted cysteine accessibility method) studies of Cx32 intercellular channels (Skerrett et al., 2002) but not by the SCAM studies reported by Zhou et al. (1997) and Kronengold et al. (2003). These authors indicate that M1 and a portion of the first extracellular loop E1 form the pore of Cx32*43E1 and Cx46 functional hemichannels. We propose to use disulphide-trapping methods to test the helical contact points predicted by these disparate models. Our preliminary studies strongly support the view that the pore of connexin channels is formed primarily by M1 and demonstrate that the Cx32*43E1 hemichannel can be locked in a state dependent conformation by the formation of Cd2+bridges between substituted cysteine residues in adjacent M1/E1 helices. Our results suggest, that the closure of connexin channels by loop-gating results from a rotation of the M1/E1 segment. We propose to continue studies of disulphide bond formation between substituted cysteines in the M1/E1 region to determine the proximity relations of residues located deeper in the hemichannel pore and to establish their functional correlates. The ability to lock channel channels in open and closed conformations provides a means to explore the nature of conformational changes that underlie voltage gating. We propose to use state-dependent lock to establish the relation between Vj and loop-gating, two forms of voltage gating that are common to all connexins. We will continue to use NMR to solve the structure of wild type and mutant N-termini. Our past studies have suggested that the structure of N-terminus is determined largely by hydrophobic interactions among conserved non-polar residues and by the presence of highly flexible turn in the vicinity of the 12th residue. We propose solve the structure of mutant peptides to test these hypotheses. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Time is requested to perform a 10.5 [unreadable]s all atom simulation of the 43E1 hemichannel embedded in an explicit POPC membrane and solvent system with a large applied electric field to explore the conformational path made by the channel as it transits from the open to closed state in response to voltage. The basic strategy of our simulation is to increase the applied potential and/or system temperature in 1 [unreadable]s increments while monitoring the trajectories of voltage-dependent (charge interactions) and voltage-independent (hydrophobic interactions) properties that are directly related to stability of the open channel and must change with channel closure. The conformations of the channel pore in the open and closed states have been determined experimentally and provide a means of validating the end point of the simulation.

DESCRIPTION (provided by applicant): The N-terminal domain (NT, residues 1-22) is an important determinant of perm-selectivity and voltage-dependent gating of connexin channels and a sensitive mutational target underlying two common inherited diseases: X-linked Charcot-Marie-Tooth (Cx32) and nonsyndromic and syndromic deafness (Cx26). This proposal will determine how disease causing mutations in the NT of Cx32 and Cx26 alter channel function and channel biosynthesis by applying synergistic computational and experimental approaches. Differences in function between wild type and disease causing NT mutations are hypothesized to arise from specific changes in channel structure. The study will examine 9 NT loci comprising mutations in both Cx32 and Cx26. In several cases, mutations of the same locus alter Cx26 and Cx32 channel function differently, suggesting that identical or homologous amino acid substitutions cause different structural defects in the two connexins. Studies will be guided by the crystal structure of a Cx26 hemichannel and a Cx32 homology model, both refined by all-atom molecular dynamics (MD) simulation and shown to closely correspond to the structure of the biological open channel. The study will solve the structure of mutant NT peptides by 2D NMR. Structural solutions of longer wild-type and mutant peptides (NT-CL domain, residues 1-114) in a membrane environment by 3D NMR, and assembled channels by x-ray crystallography have been initiated. Resulting atomic models of connexin channels will be refined by all-atom MD simulations, the permeabilities to ions and second messengers determined computationally and compared to experimental. This experimental strategy provides a sensitive test of the accuracy of atomic models, insights into molecular mechanisms of perm-selectivity and how these are changed by mutation, as well as testable hypotheses of structure-function relations. The study will investigate the role of the NT in channel biogenesis by determining the position and stability of the NT of connexin subunits inserted into canine microsomal membranes, the role of the NT in subunit oligomerization, and when and how the NT assumes its final position deep within the pore of assembled hemichannels prior to plasma membrane insertion. Parallel computational studies will provide a rigorous mechanistic framework that will guide these experimental studies. This new, fundamental knowledge will provide a framework for understanding the molecular defects of the class of disease causing NT mutations that are not plasma membrane inserted but trapped in cytosolic compartments and targeted for degradation. The project is highly collaborative, bringing together investigators with proven expertise in structural determination, computational methods and biophysical characterization of connexin channels. The results will provide new information fundamental to the elucidation of connexin disease etiology and to the development of effective treatments.