Campus Address: Science And Math Complex 310
I use molecular mutagenesis, combined with the Xenopus oocyte expression system to better understand the structure and function of membrane proteins. Current research projects focus on structure-function analyses of gap junction proteins. In animal cells, direct communication between cells occurs through gap junction channels which allow the passage of ions and metabolites with molecular weights lower than about 1 kilodalton.
Transmembrane Domain Interactions in Gap Junction Channels.
Connexins are the protein constituents of vertebrate gap junctions and there are at least 20 different mammalian connexins, each of which imparts unique characteristics to the gap junction channels it forms. Each gap junction is composed of 12 connexin proteins, each connexin subunit spans the membrane four times and the packing of the transmembrane helices plays an important role in the structure and function of gap junction channels. In order to identify critical regions of protein packing a tryptophan-scanning approach, which involves the systematic substitution of tryptophan for one amino acid at a time, is being applied to several connexins. Tryptophan mutants are functionally analyzed using the Xenopus oocyte expression system. This project is funded by an American Heart Association National Affiliate Scientist Development Grant to IMS.
Functional Analysis of Cx31 Mutations Associated with the Human Skin Disease EKV.
Mutations in specific connexin proteins lead to several diseases in humans. Connexin31 (Cx31) mutations cause a hereditary form of skin disease known as Erythrokeratadermia variablis (EKV). In order to better understand the link between mutations and disease, we are currently developing a method to optimize the expression of Cx31 in Xenopus ooctyes. Disease-related mutations will then be created using site-directed mutagenesis and their effects on channel function, particularly interactions with other connexins will be assessed.
Invertebrate Gap Junctions
The innexin family of proteins make up gap junctions in invertebrates. Innexins are functionally analogous to vertebrate connexins but were identified only recently as the molecular component of gap junctions in invertebrates. Relatively little is known about the structure and function of this diverse family of proteins. Our aim is to express innexins from Drosophila in Xenopus oocytes to determine aspects of protein structure and function.
BIO100- Principles of Biology
BIO211- Introduction to Cell Biology and Genetics
BIO314- Advanced Cell Biology
BIO301- Cell Physiology
BIO616- Topics in Cell Biology
BIO616- Topics in Cell Physiology
Toloue M.M., Woolwine, Y, Karcz, J.A., Kasperek, E.M., Nicholson, B.J., and Skerrett I.M. (2008). Site-Directed Mutagenesis Reveals Putative Regions of Protein Interaction within the Transmembrane Domains of Connexins. Cell Communication and Adhesion (in press)
Li, H-L., Qu, Y-J., Chun, L., Bondarenko, V.E., Wang, S., Skerrett, I.M., and Morales, M.J. (2006). DPP10 is an inactivation modulator of Kv4.1 and Kv4.3. Am. J. Cell Physiol. 291: C966-C976.
Skerrett, I.M., Di, W.-L., Kasperek, E.M., Kelsell, D.P., and Nicholson, B.J. (2004). Aberrant Gating, but a Normal Expression Pattern, Underlies the Recessive Phenotype of the Deafness Mutant Connexin26M34T. FASEB J. 10.1096/fj.03-0763fje.
Skerrett, I.M., Aronowitz, J.A., Shin, J., Cymes, G., Kasperek, E., F.L. Cao and Nicholson, B.J. (2002). Identification of amino acid residues lining the pore of a gap junction channel. Journal of Cell Biology 159: 349-359
Zhang, W.H., Skerrett, M., Walker, N.A., Patrick, J.W., and Tyerman, S.D. (2002) Nonselective Currents and Channels in Plasma Membranes of Protoplasts from Coats of Developing Seeds of Bean. Plant Physiol. 128: 388-399
Skerrett, I.M., Kasperek, E., Cao, F.-L., Shin, J.H., Aronowitz, J., Ahmed, S., and Nicholson, B.J. (2000). Application of SCAM to (Substituted Cysteine Accessibility Method) to Gap Junction Intercellular Channels. Cell Communication and Adhesion 8: 179-186.
Dermietzel, R. Kremer, M., Paputsoglu, G., Stang, A., Skerrett, I.M., Gomes, D., Srinivas, M., janssen-Bienhold, U., weiler, R., Nicholson, B., Bruzzone, R., and Spray, D.C. (2000). Molecular and functional diversity of neural connexins in the retina. J. Neurosci 20: 8331-8342.
Skerrett, I.M., Merritt, M., Zhou, L., Zhu, H., Cao, F.L., Smith, J.F., and Nicholson, B.J. (2000). Applying the Xenopus Oocyte Expression System to the Analysis of Gap Junction Proteins. In: Methods in Molecular Biology: Connexin Channels. R. Bruzzone and C. Giaume Eds. Humana Press, NJ, USA. pp 225-249.
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