The capacity of Xenopus laevis
to translate exogenous RNAs was first demonstrated nearly 40 years ago when Gurden and coworkers detected substantial expression of rabbit hemoglobin in frogs’ oocytes that had been co-injected with hemin and a subfraction of rabbit reticulocyte RNA [1
]. Early evidence that oocytes could be used to express exogenous ion transporters include demonstrations that oocytes injected with Torpedo
electric organ mRNA [2
] and those injected with denervated cat-muscle-mRNA [4
] incorporate functional nicotinic acetylcholine-receptor-channels into their plasma membranes.
Once the cDNA encoding a transporter has been cloned, oocytes can be microinjected with the corresponding cRNA [5
], allowing: (a) the study of that transporter in relative isolation—barring contributions from endogenous proteins [9
]—forging a strong link between injected message and detected ion transport activity (e.g., ref. [8
]) and (b) the efficient and rapid study of transporter mutants expressed from engineered cDNAs (e.g., [6
]). The link between injected message and detected ion-transport activity has since been exploited by investigators aiming to elucidate the molecular identity of a particular ion-transporter activity. This process—expression cloning (reviewed in ref. [10
])—can follow the production of soluble proteins or the manifestation of transporter activity in oocytes microinjected with increasingly sub-fractionated pools of mRNA until a single, causal mRNA species is isolated (for early examples see refs [11
]). Oocytes are now routinely used for expressing and studying the function and regulation of wild-type and mutant ion transporters along with their associated protein-partners.
In this review, we describe a system in which we (a) microinject oocytes with cRNA that encodes a transporter fused to enhanced green fluorescent protein, (b) use fluorometry to assess the yield of the fusion protein in live oocytes, and (c) use ion-sensitive microelectrodesb
to assay the activity of expressed transporter fusion. The following two sections of the present review are written for the investigator who has ion-transporter cDNA in hand and who wishes to express that transporter in oocytes. Thus, section 2 brings together methods for producing cRNA, isolating oocytes and microinjecting cRNA into those oocytes and section 3 presents a series of experiments in which we assess the expression of an enhanced green fluorescence protein-tagged transporter in living oocytes using three different fluorometric methods. Section 4 of this review is written for the investigator who is already expressing ion transporters in oocytes and who wishes to study net transporter-mediated ion-movements using ion-sensitive microelectrodes (ISMs). Thus section 4 brings together methods for the fabrication, calibration, and utilization of intracellular and extracellular/surface ISMs. Section 5 of this review considers the utility of combining fluorometric and electrophysiological measurements.