Voltage-dependent ion channels mediate electrical signals in the nervous system; many sodium (Na+), calcium (Ca++) and potassium (K+) selective channels are structurally related, and thus represent a family. These proteins undergo interesting conformational changes in response to alterations in transmembrane potential. However, the functional determinants involved in these transitions are not well understood. Chapters 2A and 2B describe the identification and characterization of an amino acid sequence motif (a leucine-heptad repeat) that is evolutionarily conserved among this family of voltage-dependent ion channels. Conservative, single amino-acid substitutions within this region of Drosophila Shaker (Sh) proteins have substantial effects on the voltage-dependence of activation. The observed alterations suggest that the heptad-repeat region is an important determinant in the conformational transitions leading to channel opening.
Na+ and Ca++ channels are composed of four homologous domains, each of which is equivalent to a single K+ channel subunit. Thus, K+ channels are thought to be functional multimers. Furthermore, there are a large number of different voltage-dependent K+ genes and alternatively spliced products that potentially can be expressed in the same cell. Therefore, the potential number of different K+ channel multimers could be quite extensive. Chapter 3 describes the physiological characteristics of combinations of K+ channels belonging to the Sh family that have been coexpressed in Xenopus oocytes. Members of the same molecular class of Sh channel form heteromultimers with novel functional properties, adding to the diversity of K+ channel function. Members of different molecular classes do not form heteromultimeric channels, suggesting that there are distinct K+ channel systems. The Appendix describes an alternative exon in the “constant” region of the Drosophila Sh gene, the existence of which suggests, that the molecular diversity of this gene is greater than previously determined.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Tanouye, Mark}, } @phdthesis{10.7907/ycfq-1k97, author = {Ramaswami, Mani}, title = {Molecular genetic studies on voltage-gated ion channels}, school = {California Institute of Technology}, year = {1990}, doi = {10.7907/ycfq-1k97}, url = {https://resolver.caltech.edu/CaltechTHESIS:07282014-102712405}, abstract = {Several different methods have been employed in the study of voltage-gated ion channels. Electrophysiological studies on excitable cells in vertebrates and molluscs have shown that many different voltage-gated potassium (K+) channels and sodium channels may coexist in the same organism. Parallel genetic studies in Drosophila have identified mutations in several genes that alter the properties of specific subsets of physiologically identified ion channels. Chapter 2 describes molecular studies that identify two Drosophila homologs of vertebrate sodium-channel genes. Mutations in one of these Drosophila sodium-channel genes are shown to be responsible for the temperature-dependent paralysis of a behavioural mutant parats. Evolutionary arguments, based on the partial sequences of the two Drosophila genes, suggest that subfamilies of voltage-gated sodium channels in vertebrates remain to be identified.
In Drosophila, diverse voltage-gated K+ channels arise from alternatively spliced mRNAs generated at the Shaker locus. Chapter 3 and the Appendices describe the isolation and characterization of several human K+-channel genes, similar in sequence to Shaker. Each of these human genes has a highly conserved homolog in rodents; thus, this K+-channel gene family probably diversified prior to the mammalian radiation. Functional K+ channels encoded by these genes have been expressed in Xenopus oocytes and their properties have been analyzed by electrophysiological methods. These studies demonstrate that both transient and noninactivating voltage-gated K+ channels may be encoded by mammalian genes closely related to Shaker. In addition, results presented in Appendix 3 clearly demonstrate that independent gene products from two K+-channel genes may efficiently co-assemble into heterooligomeric K+ channels with properties distinct from either homomultimeric channel. This finding suggests yet another molecular mechanism for the generation of K+-channel diversity.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Tanouye, Mark}, } @phdthesis{10.7907/a32z-ye28, author = {Kamb, Alexander}, title = {Molecular Biology of Shaker, a Drosophila Gene that Encodes Multiple Potassium Channel Components}, school = {California Institute of Technology}, year = {1988}, doi = {10.7907/a32z-ye28}, url = {https://resolver.caltech.edu/CaltechTHESIS:01222013-094834563}, abstract = {Shaker (Sh) mutants of Drosophila suffer from a characteristic leg-shaking behavioral defect. Previous genetic and physiological experiments suggested that Sh encodes at least one component of a fast, transient, or A-type K⁺ channel. To address questions pertaining to the structure, function and heterogeneity of K⁺ channels, we have undertaken a molecular analysis of Sh. We have isolated molecular clones for the genomic region encompassing Sh as part of a 350 kb chromosomal walk. Using a combination of classical and molecular genetics, we have mapped several Sh mutations within this region, and localized the Sh gene. Sh mutations scatter over at least 65 kb of genomic DNA, and the Sh gene itself is large, spanning at least 95 kb. Comparative studies on a collection of Sh cDNA clones show that Sh encodes a diverse array of gene products. The basis for this diversity is a mechanism that generates a limited number of different 5’ and 3’ end segments, and splices these segments onto a central constant region. This differential splicing mechanism produces at least 10, and possibly 28 or more, predicted Sh proteins that differ at the carboxyl and/or amino terminus. The primary structures of Sh proteins deduced from the cDNAs reveal two general types of polypeptide: a protein that contains seven potential membrane-spanning domains, including a positively charged segment that is similar to a sequence called S4 in Na⁺ channels, and a smaller protein that lacks S4 and contains only three potential membrane-spanning regions. Variants of the flrst protein type range in size from 493 a.a. to 656 a.a., whereas variants of the second type range from 303 a.a. to 337 a.a. These polypeptides may assemble as homomultimers and/or as heteromultimers to produce K⁺ channels with different features.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Tanouye, Mark}, }