Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients

https://science.sciencemag.org/content/sci/245/4922/1066.full.pdf

Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.

Voltage-sensitive ion channels mediate action potentials in electrically excitable cells and play important roles in signal transduction in other cell types. In the past several years, their protein components have been identified, isolated, and restored to functional form in the purified state. Na+ and Ca2+ channels consist of a principal transmembrane subunit, which forms the ion-conducting pore and is expressed with a variable number of associated subunits in different cell types. The principal subunits of voltage-sensitive Na+, Ca2+, and K+ channels are homologous members of a gene family. Models relating the primary structures of these principal subunits to their functional properties have been proposed, and experimental results have begun to define a functional map of these proteins. Coordinated application of biochemical, biophysical, and molecular genetic methods should lead to a clear understanding of the molecular basis of electrical excitability.

http://science.sciencemag.org/content/sci/242/4875/50.full.pdf

Structure and function of voltage-sensitive ion channels

A complementary DNA encoding an ATP-regulated potassium channel has been isolated by expression cloning from rat kidney. The predicted 45K protein, which features two potential membrane-spanning helices and a proposed ATP-binding domain, represents a major departure from the basic structural design characteristic of voltage-gated and second messenger-gated ion channels. But the presence of an H5 region, which is likely to form the ion conduction pathway, indicates that the protein may share a common origin with voltage-gated potassium channel proteins.

Cloning and expression of an inwardly rectifying ATP-regulated potassium channel

Autosomal recessive generalized myotonia (Becker's disease) (GM) and autosomal dominant myotonia congenita (Thomsen's disease) (MC) are characterized by skeletal muscle stiffness that is a result of muscle membrane hyperexcitability. For both diseases, alterations in muscle chloride or sodium currents or both have been observed. A complementary DNA for a human skeletal muscle chloride channel (CLC-1) was cloned, physically localized on chromosome 7, and linked to the T cell receptor beta (TCRB) locus. Tight linkage of these two loci to GM and MC was found in German families. An unusual restriction site in the CLC-1 locus in two GM families identified a mutation associated with that disease, a phenylalanine-to-cysteine substitution in putative transmembrane domain D8. This suggests that different mutations in CLC-1 may cause dominant or recessive myotonia.

https://science.sciencemag.org/content/sci/257/5071/797.full.pdf

The skeletal muscle chloride channel in dominant and recessive human myotonia.

Voltage-dependent potassium, sodium and calcium ion channels may share a common mechanism of activation, in which the conserved S4 sequence acts as the primary voltage sensor. Site-directed mutagenesis of the S4 sequence of the Shaker potassium channel and electrophysiological analysis suggest that voltage-dependent activation involves the S4 sequence but is not solely due to electrostatic interactions.

Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence.

We have cloned 215-kb DNA containing the maternal effect region (ME) of the Shaker gene complex (shC) at 16F of the Drosophila X chromosome. Five translocation and deletion breakpoints have been mapped on the cloned DNA allowing a correlation of the genetic map to transcription units. The ME region spans ˜100 kb. The genetic behavior of this region correlates with the occurrence of maternal RNAs in this part of the ShC. Two transcripts have been identified in the vicinity of chromosomal rearrangements which cause a Sh phenotype. These are a 4.5-kb transcript interrupted by T(x;2)B27 and a 2-kb transcript interrupted by T(X;3)ShLC and T(X;Y)W32. The latter transcript is derived from a primary transcript which spans >65 kb genomic DNA. The cDNA-sequencing data show that this Shaker (IAchannel) gene can encode a protein of ˜35 kd with three α-helical membrane-spanning sequences near its carboxyl terminus. These have a striking homology with membrane-spanning sequences of the vertabrate Na+ channel.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1987.tb02665.x

Molecular organization of the maternal effect region of the Shaker complex of Drosophila: characterization of an IA channel transcript with homology to vertebrate Na+ channel

The Shaker locus of Drosophila melanogaster encodes a family of A-type potassium channel subunits. Shaker mutants behave as antimorphs in gene dosage tests. This behaviour is due to the production of truncated A-channel subunits. We propose that they interfere with the function of their normal counterpart by forming multimeric A-channel structures. This hypothesis was tested by constructing transgenic flies carrying a heat-inducible gene encoding a truncated A-type potassium channel subunit together with a normal wild type doses of A-type potassium channel subunits. The altered subunit leads at larval, pupal or adult stages to the transformation of wild type into Shaker flies. The transformed flies exhibited a heat-inducible abnormal leg shaking behaviour and a heat-inducible facilitated neurotransmitter release at larval neuromuscular junctions. By the overexpression of an aberrant A-channel subunit the normal behaviour of transgenic D. melanogaster can be altered in a predictable way.

The interference of truncated with normal potassium channel subunits leads to abnormal behaviour in transgenic Drosophila melanogaster.

Potassium channels contribute to the most ubiquitous group of membrane ion channels and seem to be present in almost every eukaryotic cell. Voltage-sensitive potassium channels in excitable membranes are involved (Hille, 1984) in the regulation of action potential duration, in neuron bursting and in cardiac pacemaking. Recent studies indicated that the Shaker gene complex encodes a family of potassium channel proteins in the nervous system of Drosophila (Schwarz et al., 1988, Pongs et. al., 1988).

F. Müller-Holtkamp

Papers

Molecular organization of the maternal effect region of the Shaker complex of Drosophila: characterization of an IA channel transcript with homology to vertebrate Na+ channel

The interference of truncated with normal potassium channel subunits leads to abnormal behaviour in transgenic Drosophila melanogaster.

Molecular Basis of Biological Diversity at the Shaker Locus of Drosophila