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Proc Natl Acad Sci U S A. Aug 5, 2008; 105(31): 10641–10642.
Published online Jul 31, 2008. doi:  10.1073/pnas.0806110105
PMCID: PMC2504841

A Rosetta stone for analysis of human membrane protein function

Knowledge of the function of membrane proteins in normal and pathological human organs would be helpful in understanding a wide range of disorders, but general strategies for obtaining it are constrained by the availability of tissue for study. Procuring living samples can be difficult and postmortem tissues are often degraded or preserved by methods that alter or destroy membrane protein function. Even given the integrity of these components, studies of their function face additional hurdles. Their relatively low abundance and investment by the lipid bilayer render purification and characterization challenging. The genes that encode them are often not available to allow analysis of their function by expression in heterologous systems. In addition, one needs to be able to study the properties of tissue- and disorder-specific posttranslationally modified proteins. In this issue of PNAS, Limon, Reyes-Ruiz, and Miledi (1) use two methods to address these issues. The first involves classical mRNA expression. The second utilizes a more recently developed and powerful technique that takes advantage of membrane fusion to analyze the function of proteins without the mRNA for the genes that encode them. The authors apply these methods to study ion channels from frozen and archived human postmortem brain tissue of normal and autistic subjects. This approach should provide a Rosetta stone with which to decipher the normal functions and altered properties of membrane proteins from all organ systems of human subjects.

Frozen material for study was provided by several brain tissue banks and pairs of autistic and normal brains were matched for age (4 to 39 years old). Postmortem intervals to preservation (9–27 h) and durations of storage (2–5 years) were comparable. However, the substantial delays in tissue preservation and the considerable durations of storage raised the question of whether mRNA and protein integrity was maintained. The cerebellum and temporal cortex were selected for investigation because neuronal organization is abnormal in these regions in autistic brains (24).

Limon et al. (1) lead off by using the Xenopus oocyte expression system to address the integrity of transcripts from postmortem tissue. Gurdon and his colleagues (5) first reported the use of frog eggs and oocytes for the study of mRNA and its translation in living cells. These cells are protein synthesis machines, geared up for embryonic development, and efficiently translate transcripts into functional proteins. Miledi et al. (6, 7) pioneered this technique as an assay for the function of genes encoding receptors and ion channels, and the oocyte became a standard tool for neuroscientists, cell biologists, and biophysicists. Limon et al. homogenized 1–2 g of frozen tissue to isolate poly(A+) RNA for oocyte injection. Electrophysiological recordings revealed that oocytes expressing temporal lobe mRNA from normal or autistic brains generate similar current responses to a range of neurotransmitters, including acetylcholine, GABA, glutamate, kainate, and serotonin; both ionotropic and metabotropic receptors were expressed. These results identify a high level of integrity of transcripts in the banked specimens.

Limon et al. have demonstrated that human proteins can be assayed even after long intervals postmortem.

The authors then use an assay that relies on the propensity of membranes to form vesicles and the ease with which these vesicles fuse with the plasma membrane when they are injected intracellularly (810). The evolutionary origin of the cell membrane immediately created the need for a repair mechanism after damage. Electrophysiologists are familiar with the sealing of the cell membrane after withdrawal of intracellular or patch electrodes. The machinery for repair involves recruitment of intracellular membranes along with protein catalysts (11). Membrane fusion with the damaged plasma membrane is achieved through calcium-dependent exocytosis. The assay appears to rely on the endogenous machinery for the repair of the plasma membrane. Although this is a mechanism common to all cells, the large size of the Xenopus oocyte makes it particularly suited to this manipulation.

Limon et al. prepare membrane proteins by homogenization and centrifugation of 0.2–0.6 g of frozen tissue; 50 nl of 1 mg/ml protein are injected in oocytes. Within several hours the membrane vesicles have fused with the oocyte surface membrane, achieving microtransplantation of receptors. Previous work showed that patches of membrane expressing labeled receptors could be visualized directly in these chimeric oocytes (9). Electrophysiological recordings yielded current responses to GABA, glutamate, kainate, and acetylcholine, and again both ionotropic and metabotropic receptor activation was observed. No differences were noted in the currents elicited from oocytes expressing mRNA from normal or autistic brains. AMPA-class glutamate-induced currents showed rapid desensitization and were potentiated by cyclothiazide; GABAA-class GABA-induced currents were potentiated by pentobarbital. Interestingly, current amplitudes increased with time, probably as a result of continued fusion of membrane vesicles with the oocyte membrane, and were maximal at 1–3 days. Thus, these proteins appear to be well preserved and amenable to analysis after prolonged periods of storage.

Although this study does not reveal molecular differences between receptors from autistic and normal brains, mutations in ion channels and receptors have been associated with autism spectrum disorders (12). These disorders are thought to arise from defects in cell migration, neurite morphology, and synapse formation (13), all of which depend on electrical activity at early stages of development (1416). It will be useful to examine more components involved in these processes and to study tissues at earlier stages in their differentiation.

The techniques of mRNA expression and receptor and channel transplantation enable rapid functional characterization of many normal and abnormal membrane proteins for which the genes have not yet been cloned. The transplantation technique is particularly useful because posttranslationally modified proteins can be analyzed as well. These modifications may arise through complex cellular signaling events not easily recapitulated by simple mRNA injection. In addition, relatively small amounts of tissue are required. Limon et al. have demonstrated that human proteins can be assayed even after long intervals postmortem. This approach will be valuable for studies of channelopathies and defects in other membrane components. Microtransplantation of defective receptors should also provide a useful assay system for screening new drugs and refining their efficacy. The ability to test the actions of compounds on membrane proteins expressing modifications particular to different tissues or disorders is a great asset, and the opportunity to study receptors in large experimentally tractable cells may be a technical advantage. Much as the Rosetta stone led to deciphering of hieroglyphic writing, we can look forward to deciphering of the functions of the membrane proteome.

Footnotes

The author declares no conflict of interest.

See companion article on page 10973.

References

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