Genetic manipulation of ion channels: a new approach to structure and mechanism.

Christopher Miller Graduate Department of Biochemistry Brandeis University Waltham, Massachusetts 02254 Introduction The central position of ion channels in the nervous system-as catalysts mediating the ionic fluxes that give rise to electrical excitation-has for many years provided a neurobiological alias for those whose real interest is the molecular and structural basis of this catalytic func- tion. By what mechanisms do ion channels change con- formation rapidly in response to the binding of ligands or changes in transmembrane electric field? How do channels, once open, select so well among the many types of inorganic ions present in the aqueous phases on the two sides of the membrane? How do specific toxins and drugs modify the normal functions of channels? These have been the central questions driving research on the molecular mechanisms of channel function for nearly half a century. Until about a decade ago, these sorts of mechanistic questions were attacked largely by biophysically minded electrophysiologists, whose voltage clamps and single- channel recording techniques gave them exclusive ad- tnission to the study of channels. In the last 10 years, however, biochemical and molecular genetic techniques have burst onto the scene, and as a result, the character of mechanistic channel research has changed profoundly. Now, it is possible to carry out informative research on ion channels without ever impaling a cell or looking at an oscilloscope trace, but rather by microsequencing protein bands from SDS gels and cloning the appropri- ate genes. The ease and availability of molecular biologi- cal methods have elicited a breathtaking onrush of new types of ion channel studies-approaches that only 10 years ago would have been unthinkable. My purpose here is to review a small subset of this work: the use of molecular genetics to draw inferences about the molec- ular structures and basic catalytic mechansims of ion channels, Neither the biophysical nor the molecular genetic ap- proach alone can tell us very much in detail about the structures of channel proteins. But it is now becoming clear that the combination of these two approaches is exceedingly powerful in attacking questions of how ion channels are built and how they work. The molecular biologist’s surgical precision in manipulating channel subunit composition and primary sequence allows the routine production of mutant channel genes. These genes can then be expressed in an electrophysiologically accessible system, the Xenopus oocyte being the most widely used, and the altered functions of the mutant channels may be examined with the very high resolution method of single-channel recording. The basic point of thus approach is to identify specrfic regions or residues in the channel protein that directly affect opera- tion and to draw conclusions about the underlying structure of the channel. I will focus here on the new, understanding emerging about channel structure and mechanism as a result of the application of these “struc- ture-from-function” efforts. Before discussing these results, however, I should point out that when viewed in a biochemically broad context, it may seem premature to attack ion channel proteins by the mutation-structure-function route. There simply are no channels for which high resolution struc- tural information is available. In the past, site-directed mutagenesis techniques have been most productively applied to the mechanisms of water-soluble enzymes of known structures. There are two reasons why such en- zymes can be studred so well in this way. First, with a known macromolecular structure in hand, it is relatively easy to make guesses about which amino acid residues are involved directly in function, and so to design tight experiments in which detarled predictions of the func- tional consequences amino acid residue alterations can be made. Second, knowledge of the structure of both the natrve and the mutant proteins eliminates the great bugaboo of this approach-that an observed func- tional change may be the consequence not of the spe- cific local amino acid replacement, but rather of a global change in protein structure resulting from the mutation. With ion channels, the absence of atomic resolution structures would seem to throw enormous obstacles in the way of anyone wishing to pursue this line of attack. It would seem not only hopeless to guess where to mu- tate the channel, but also impossible to understand meaning of any functional changes observed in these mutants. But in fact these difficulties are not insuperable. Ion channels present two great advantages to the experi- menter that partially compensate for the lack of struc- tural information. First, the basic mechanism of ion transport through channels-passive diffusion through a water-filled pore-is a very simple one. In channel- mediated transport, the protein does not need to un- dergo a cycle of specific conformational changes for each ion transported, as is the case, for instance, with active transport pumps. Crudely stated, the channel, once open, just sits there, presenting a fixed diffusion pathway for ions to traverse. Moreover, the existence of an aqueous hole through a membrane-spanning protein places such severe constraints on the structure of channel that we can make reasonable guesses about the folding pattern of the polypeptide chain given its se- quence, a trick virtually impossible to pull off with water- soluble globular proteins. The second advantage unique to ion channels is that they can be observed one at a time. In site-directed mutagenesis experiments, the ca- pability of observing functional changes at single- channel level allows a much easier interpretation of