[Frontiers in Bioscience 1, d214-233, September 1, 1996]


Martin Davies1, Alan N. Bateson1,2 and Susan M. J. Dunn1,2

1 Department of Pharmacology

2Division of Neuroscience, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Received 07/16/96; Accepted 07/22/96; On-line 09/01/96


The common approach in mutagenesis studies is to express wildtype or mutant receptors in suitable cell lines or in Xenopus oocytes and to characterize receptor properties by radiolabelled ligand binding or electrophysiological techniques. In our experience, a frequently encountered problem is that the introduction of mutations into one or more subunits of the receptor oligomer leads to dramatic changes in the level of receptor expression. Even very conservative amino acid substitutions may greatly reduce, or even eliminate, the expression of receptor at the cell surface with protein being accumulated instead in the endoplasmic reticulum (e.g. 38). Such a profound effect of what are presumed to be small changes in protein structure is perhaps not surprising given the complexity of folding and assembly of large multisubunit transmembrane proteins. This intrinsic complexity has been illustrated in studies of nAChR expression which have shown that only a small fraction of the total protein that is synthesized is correctly assembled as functional receptors (for review, see 39). Thus, until a complete understanding of the mechanisms which govern expression and assembly of complex proteins is obtained, the approach of mutagenesis remains essentially a "hit or miss" procedure.

A related problem in some published mutational studies is a failure to determine if an absence of detectable current or measurable radioligand binding is due to a lack of receptor expression or to a much reduced affinity for the ligand. To distinguish between these possibilities, it is necessary to determine levels of expression using an appropriate technique such as Western blotting carried out in conjunction with detailed studies of receptor-ligand interactions.

Another difficulty associated with mutational analysis of LGICs is the determination of the precise receptor property that is altered. Although radiolabelled ligand binding and electrophysiological experiments are routinely used to study mutant receptor properties, most mutational studies are restricted to the use of only one or other of theses types of analysis. Radioligand binding has the advantage that no specialized equipment is needed and information can be obtained relatively quickly. Although this technique can reveal changes in the apparent Kd or Ki values for specific ligands resulting from the introduction of mutations, it has the limitation that normally only high affinity sites that are present at equilibrium can be measured. Frequently, receptors are characterized by radiolabelled antagonist binding which provides information only on the inactive, blocked state of the receptor. Furthermore, GABAA receptors, like other members of this ion channel family, become desensitised upon prolonged exposure to agonists. Thus, equilibrium studies of radiolabelled agonist binding provide information only on the desensitised state of the receptor. Usually, therefore, equilibrium radioligand binding studies alone are unable to reveal the functional impact of a mutation.

In order to investigate the effects of mutation on receptor function, a whole cell patch clamp study is often the method of choice. Normally, this technique is used to determine EC50 values for various ligands, with a shift in EC50 being interpreted as a change in the properties of the mutant receptor. However, the EC50 value is a complex function of several variables, including the rate constants for agonist association and dissociation, and the kinetics of channel opening and closing. Since a change in EC50 may result from an alteration in any one of these properties, it is not possible to identify the precise molecular event that has been affected by the mutation. Single channel analysis overcomes some of these limitations by permitting detailed analysis of gating mechanisms. However, this technique also has the shortcoming that the interpretation of the mutation's effect is, to a large extent, model dependent. Thus, the accuracy of the model used to describe the various states of the receptor is critical in obtaining meaningful data.

The points listed above dictate that caution must be exercized when interpreting the effects of mutations on receptor properties. Because electrophysiological and radioligand binding studies generally examine two different states of the receptor (the resting "active" state and the equilibrium ligand-bound state respectively), crucial information might be missed if a mutant is analyzed by only one of the two techniques. To obtain a more complete view of the impact of a mutation, it is obviously preferable to use both approaches. Even then, an important caveat remains with mutational analysis of all such complex membrane-bound proteins. Since none of these receptors has, thus far, proved amenable to crystallization, no high resolution structural information is available. Specific mutations may thus have unpredictable consequences and experimental data are subject to serious misinterpretation. At the present time, we can, however, hope for consistency of results and a merging of information from different approaches. We can then begin to develop models of receptor structure and function which will undoubtedly need refinement as better structural data become available.

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