Diane M. Papazian

Multiple potassium–channel components are produced by alternative splicing at the Shaker locus in Drosophila

At least four probable components of potassium channels are encoded at the Drosophila Shaker locus, by a family of alternatively spliced transcripts. Alternative splicing may provide one mechanism of generating the remarkable diversity of potassium channels.

Functional expression of Shaker K+ channels in a baculovirus-infected insect cell line

We constructed a recombinant baculovirus, A. californica nuclear polyhedrosis virus, containing the Drosophila Shaker H4 K+ channel cDNA under control of the polyhedrin promoter. When infected with this recombinant baculovirus, the cell line Sf9, derived from the army-worm caterpillar S. frugiperda, expresses fully functional Shaker transient K+ currents, as assayed by whole-cell recording. K+ currents begin to appear at about 15 hr after infection, and they continue to increase over the next 3 days. Over the same period of time, a 75 kd band appears on SDS gels stained with Coomassie blue. The identity of this band as a Shaker gene product is confirmed by Western blot analysis using an anti-Shaker antiserum. The 75 kd band accounts for a substantial fraction of the membrane protein in Shaker-infected Sf9 cells. These results give hope that the baculovirus system, which has been used successfully for high-level expression of soluble proteins from higher eukaryotes, may be appropriate for producing large amounts of cloned ion channel proteins as well.

Immunological characterization of K+ channel components from the Shaker locus and differential distribution of splicing variants in Drosophila.

Antibodies were raised to three portions of the predicted sequences of Shaker, a gene that encodes a family of K+ channel components that are produced by the alternative splicing of transcripts. On immunoblots, the protein products appear to be 65,000-85,000 daltons in size. No smaller products were detected. Immunocytochemistry has revealed a nonuniform distribution of Shaker products in the brain of the adult fly. By comparing antisera directed against regions shared by all the splicing variants to antisera that are directed against one particular group of splicing variants, we have determined that there is a differential distribution of that group of variants. Thus, the alternative splicing of Shaker transcripts appears to produce different subtypes of A-channels in different tissues.

Molecular Studies of Voltage-gated Potassium Channels

The cloning and characterization of the voltage-activated Shaker potassium channel gene in Drosophila have led to the identification of structural elements involved in potassium channel gating. As found for the voltage-activated sodium channel, the S4 segment, located in the conserved core of the protein, plays a central role in voltage-dependent activation. Potassium channels appear to be formed by the assembly of several polypeptides into multisubunit channels. This is directly analogous to the proposed folding of the four internally homologous pseudosubunits of sodium and calcium channels. The amino- and carboxy-terminal regions of Shaker channels are specialized for, and appear to interact in, inactivation gating. This interaction probably includes interaction between subunits, as may be said for the role in inactivation gating of the junction between the carboxyl terminus of the third domain and amino terminus of the fourth domain of sodium channel (Vassilev et al. 1988). The capacity for coassembly in potassium channels extends not only to the alternatively spliced products of the same gene, but also to the products of different genes. Heteromultimeric channels that are formed in this way have kinetic and pharmacological properties that differ from homomultimers of their constituents and, as such, broaden the functional diversity of channels that can be produced by any given number of compatible potassium channel genes.

Application of drosophila molecular genetics in the study of neural function — studies of the shaker locus for a potassium channel

Abstract One general approach to studying molecules that are important for neuronal function or development, even if the gene products are not defined biochemically, is to make use of classical and molecular genetics; these tools have been well developed for organisms such as Caenorhabditis elegans and Drosophila melanogaster . The cytogenetics made possible by the polytene chromosomes in Drosophila further facilitates gene cloning. If one can identify genes that are important for the nervous system, one should be able to clone them and then study these genes and their products in molecular terms.

A Family of Potassium Channels from the Shaker Locus of Drosophila

Potassium channels are found in nerve, muscle, epithelial, endocrine, exocrine and immune cells. Despite their importance in the control of excitation and ion transport, biochemical understanding of the channels has grown slowly because there are no high abundance sources of these channels and because high affinity ligands for them have only recently been found (Carbone et al., 1982; Miller et al, 19854; Seagar et al., 1986; Sternsfeld et al., 1987). An alternative route to studying these molecules has been provided by the fruit fly, Drosophila melanoqaster. Many neurological mutations of this organism have been found that define loci that may encode important neuronal proteins (Ganetzky and Wu, 1986). One of these, the Shaker locus, appeared likely from electrophysiological and genetic studies (Jan et al., 1977; Salkoff and Wyman, 1983; Timpe and Jan, 1987) to contain the structural gene for a type of potassium channel, called the A channel, that opens in response to depolarization and inactivates thereafter. The gene was mapped to band 16F on the X-chromosome (Tanouye et al., 1981). An entry point to cloning the region was provided by a cDNA, cloned for independent reasons (Wollfner, 1980); the cDNA is not related to Shaker but hybridizes to the same band on the X-chromosome.