Michael C. Puljung, Ph.D. - US grants

Gap junction channels, dodecamers of connexin proteins, provide direct coupling between the cytoplasm of adjacent cells, each cell contributing a hexameric connexon, or hemichannel. Mutations in connexin genes are associated with diseases including X-linked Charcot-Marie-Tooth disease (CMTX), a peripheral neuropathy caused by defects in connexin32. The applicant aims to characterize the pharmacology of conducting hemichannels formed by human connexin37 (hCx37) and bovine connexin44 (Cx44), which have diverse properties with respect to gating. Experiments are proposed to test the hypothesis that block of these hemichannels by heptanol, halothane, and divalent cations should be the same in hemichannels as in intact intercellular channels, indicating that gap junctional uncoupling is due to hemichannel block. Voltage effects on divalent block will also be assessed. Single channel currents for hCx37 will determine whether the hemichannel currents have a role under physiological conditions. Native preparations will be used to further explore this possibility. Finally, chimeric channels, containing domains of the hCx37 and U44, will be made to determine the molecular basis of gating. Chemical gating properties will be assessed in chimeric hemichannels that exchange the cytoplasmic loop and carboxy-terminal domains, regions implicated in chemical gating of connexins. Voltage gating properties will be examined in hemichannels and intercellular channels formed by chimeras that exchange the extracellular loop domains. Site-directed mutants can also be made in the domains involved in gating. Once the molecular basis of gating is identified, specific residues may be used as targets for rationale drug design to treat illnesses like CMTX.

[unreadable] DESCRIPTION (provided by applicant): Cyclic nucleotide-gated (CNG) ion channels are allosteric proteins regulated by the cooperative binding of cytoplasmic cyclic nucleotides to a conserved cyclic nucleotide binding domain (CNBD). CNG channels generate the primary electrical signal responsible for transduction of visual and olfactory information to the brain. Human mutations in CNG channels cause various diseases including complete achromatopsia or colorblindness and retinitis pigmentosa, which leads to retinal degeneration and blindness. The ligand-bound structure of the C-terminal domain of HCN2, a closely related ion channel, which includes a CNBD, has been determined by x-ray crystallography. This domain is formed by an eight-stranded [unreadable] roll followed by two helices (B and C) with cyclic nucleotide binding in a pocket between the C helix and the [unreadable] roll. The structure of the unliganded CNBD and the details of the movements accompanying ligand binding and channel gating have not been determined. The goal of the proposed study is to monitor this conformational change using fluorescence measurements of isolated CNBD's in solution and intact channels. Distance-dependent quenching between fluorophores and quenchers attached to various positions on the channel will address specific questions about the nature of the CNBD conformational change. How does the C helix reorient relative to the [unreadable] roll upon agonist binding and channel gating? Does the C helix maintain its secondary structure in the absence of agonist? Fluorescence measurements can be performed simultaneously with current measurements from CNG channels, which allows for structural changes to be correlated directly with a functional protein state, a resolution not possible for other types of proteins. Therefore, not only will the proposed studies be informative with respect to ion channel gating mechanisms, but they will also provide insight into the movements of structurally-related molecules, including certain kinases, transcription factors and guanine nucleotide exchange factors. PUBLIC HEALTH RELEVANCE The goal of this study is to understand the dynamic structural rearrangements that occur in a family of proteins known as cyclic nucleotide-gated ion channels. These proteins are necessary to relay visual information from the eye to the brain, and mutations in the genes encoding these channels result in blindness. Using a combination of structural and functional assays, the normal, molecular motions of these channels will be determined, which will be instrumental in understanding their regulation by cellular signals in healthy individuals, and their misregulation in disease. [unreadable] [unreadable] [unreadable]