Genetics of ion channels

Genetic and molecular genetic techniques have now been added to the traditional tools for studying ion-channel biology. For more than two decades after the pioneering work of Hodgkin and Huxley, nearly all understanding of what ion channels are and how they work was obtained by biophysical investigations employing the voltage-clamp technique. Investigations with the techniques of protein biochemistry followed these initial studies. However, the difficulties of working with sparsely distributed integral membrane proteins, where only a few molecules may be present per square micron of membrane, have limited the rate of progress in characterizing these proteins. Progress has been slow even for the two channel types where available tissues contain relatively greater quantities of the protein; both the AChR and the Na⁺ channel have had to wait for the introduction of molecular genetic techniques to reveal much of their structure. All of these techniques will probably go hand in hand, and a thorough characterization of the structure and function of at least these two proteins should be forthcoming. For the characterization of other channel molecules, e.g., the various K⁺ and Ca²⁺ channels, genetic approaches may be essential. These channel molecules are sparsely distributed in tissues and have no specific high-affinity binding ligands. Thus traditional genetic approaches may be useful for generating and selecting mutants that have alterations in the structure or regulation of these ion channels that confer an identifiable behavioral phenotype. Cloning techniques, at least in Drosophila, have advanced to the point where virtually any identified mutant locus can be cloned. Ultimately, molecular genetic techniques can contribute to a comprehensive understanding of the structure-function relationships of channel proteins. The relationship of structure to function can be investigated in a systematic way by exploiting such new techniques as site-directed mutagenesis, which can engineer specific amino acid substitutions or small deletions that leave the codon reading frame in register. With available transformation techniques, such as those that are already highly developed in Drosophila, or by injection of mutant mRNA into Xenopus oocytes, systematically altered molecules can be tested for alterations in their biophysical properties. This approach is underway to dissect the functional properties of the AChR. With these techniques, evidence has been obtained that supports the hypothesis that the ACh-binding site is located at the NH₂-terminal area of the α-subunit and suggests that an amphipathic region may form the pore. Altered channel molecules may also be produced by expression systems that can yield sufficient amounts of the proteins for X-ray crystallography comparison studies. A possible area of difficulty in working with voltage-sensitive channels may be their large size. Substantial difficulties are involved in getting full-length cDNAs or RNA molecules equal in size to the Na⁺ channel, but the Na⁺ channel may be at the large end of the spectrum for channel molecules. Finally, molecular genetic techniques applied across species can reveal the evolutionary history of these molecules. General themes than can be learned about these proteins, like the tendency to evolve repetitive homology units, may reveal the chosen functional design of nature for channel proteins required to do certain tasks.