The Use of Xenopus Oocytes
9 to Study the Biophysics and
Pharmacological Properties
of Receptors and Channels
Ataúlfo Martínez-Torres and Elizabeth Pereida-Jaramillo
CONTENTS
9.1. Historical Background ............................................................................................................................................... 143
9.2. Past Observations ....................................................................................................................................................... 143
9.3. Present Status ............................................................................................................................................................. 144
9.4. Insight of Native Ion Currents of the Follicle ............................................................................................................ 147
9.5. Future Directions ....................................................................................................................................................... 147
Acknowledgments ................................................................................................................................................................ 148
References ............................................................................................................................................................................ 148
9.1. HISTORICAL BACKGROUND
Oocytes of the frog Xenopus laevis have been widely used as an
experimental system for expressing ion channels and receptors
cloned from diverse sources. Relatively simple electrophysiological techniques, such as the two-electrode voltage-clamp
confguration, allow the study of biophysical and pharmacological characteristics of voltage- and neurotransmitter-gated
ion channels. Development of the oocyte expression system
began by serendipity in the mid-70s as Ricardo Miledi and
co-workers explored the early development of acetylcholine
responses at the frog neuromuscular junction. At some point,
the biological preparation was so diffcult to perform due to the
small size of the animals that impaling electrodes in tadpoles
became impractical to determine at what age the responses
to the neurotransmitter emerged; that is when the researchers
decided to explore if frog oocytes responded to acetylcholine.
To their surprise, many oocytes elicited an electric response
when exposed to acetylcholine (Kusano et al. 1977), and many
other endogenous ion currents were subsequently discovered
(Arellano et al. 1995, 1996; Miledi et al. 1989; Parker and
Miledi, 1987, 1988). Other studies disclosed the presence of
receptors in follicle-enclosed oocytes, such as noradrenaline,
angiotensin, vasointestinal peptide, and follicle stimulating
hormone (Woodward and Miledi 1987; Miledi and Woodward
1989b; Hershey et al. 1991; Woodward and Miledi 1991).
These studies paved the way to “transplant” ion channels by
injecting mRNA isolated from the brain and later from cloned
cDNAs isolated from diverse tissues and species (Miledi et al.
1982, 1983; Barnard et al. 1982). Another application, also
developed by Miledi, showed that plasma membranes isolated
from brain tissue can be “transplanted” into oocytes, retaining
fully functional receptor and channel activity that can be studied more than ten years after the samples are obtained (Eusebi
et al. 2009; Miledi et al. 2002). This has allowed researchers to determine the pharmacological and functional changes
of receptors in neurological disorders such as epilepsy,
Alzheimer’s disease, and autism and opens the possibility for
applying a combination of proteomics and electrophysiology
to establish a relationship between the pathological state and
molecular imbalances.
There are nearly 10,000 citations in PubMed using the
keywords Xenopus oocyte and ion channel. This gives an idea
of the impact that this methodology has on the feld and the
diffculty to offer a thorough review on the subject. Nonetheless,
here we provide an account of past observations and the present
status of the feld, offering new perspectives to be explored.
9.2. PAST OBSERVATIONS
The earliest use of the Xenopus oocyte as an expression system was demonstrated by Sir John Gurdon (Gurdon et al.
1971), who published a series of papers that f rmly established that oocytes can synthesize a protein from a foreign
mRNA. (Lane et al. 1971) conclude that
when injected into a living cell, the 9 s RNA is fairly stable
and has the properties of a hemoglobin messenger. The messenger requires no reticulocyte-specifc factors for translation, and the translational machinery of the oocyte will
accept the messenger RNA from a totally different cell type,
from another species.
DOI: 10.1201/9781003050230-10
143
9 to Study the Biophysics and
Pharmacological Properties
of Receptors and Channels
Ataúlfo Martínez-Torres and Elizabeth Pereida-Jaramillo
CONTENTS
9.1. Historical Background ............................................................................................................................................... 143
9.2. Past Observations ....................................................................................................................................................... 143
9.3. Present Status ............................................................................................................................................................. 144
9.4. Insight of Native Ion Currents of the Follicle ............................................................................................................ 147
9.5. Future Directions ....................................................................................................................................................... 147
Acknowledgments ................................................................................................................................................................ 148
References ............................................................................................................................................................................ 148
9.1. HISTORICAL BACKGROUND
Oocytes of the frog Xenopus laevis have been widely used as an
experimental system for expressing ion channels and receptors
cloned from diverse sources. Relatively simple electrophysiological techniques, such as the two-electrode voltage-clamp
confguration, allow the study of biophysical and pharmacological characteristics of voltage- and neurotransmitter-gated
ion channels. Development of the oocyte expression system
began by serendipity in the mid-70s as Ricardo Miledi and
co-workers explored the early development of acetylcholine
responses at the frog neuromuscular junction. At some point,
the biological preparation was so diffcult to perform due to the
small size of the animals that impaling electrodes in tadpoles
became impractical to determine at what age the responses
to the neurotransmitter emerged; that is when the researchers
decided to explore if frog oocytes responded to acetylcholine.
To their surprise, many oocytes elicited an electric response
when exposed to acetylcholine (Kusano et al. 1977), and many
other endogenous ion currents were subsequently discovered
(Arellano et al. 1995, 1996; Miledi et al. 1989; Parker and
Miledi, 1987, 1988). Other studies disclosed the presence of
receptors in follicle-enclosed oocytes, such as noradrenaline,
angiotensin, vasointestinal peptide, and follicle stimulating
hormone (Woodward and Miledi 1987; Miledi and Woodward
1989b; Hershey et al. 1991; Woodward and Miledi 1991).
These studies paved the way to “transplant” ion channels by
injecting mRNA isolated from the brain and later from cloned
cDNAs isolated from diverse tissues and species (Miledi et al.
1982, 1983; Barnard et al. 1982). Another application, also
developed by Miledi, showed that plasma membranes isolated
from brain tissue can be “transplanted” into oocytes, retaining
fully functional receptor and channel activity that can be studied more than ten years after the samples are obtained (Eusebi
et al. 2009; Miledi et al. 2002). This has allowed researchers to determine the pharmacological and functional changes
of receptors in neurological disorders such as epilepsy,
Alzheimer’s disease, and autism and opens the possibility for
applying a combination of proteomics and electrophysiology
to establish a relationship between the pathological state and
molecular imbalances.
There are nearly 10,000 citations in PubMed using the
keywords Xenopus oocyte and ion channel. This gives an idea
of the impact that this methodology has on the feld and the
diffculty to offer a thorough review on the subject. Nonetheless,
here we provide an account of past observations and the present
status of the feld, offering new perspectives to be explored.
9.2. PAST OBSERVATIONS
The earliest use of the Xenopus oocyte as an expression system was demonstrated by Sir John Gurdon (Gurdon et al.
1971), who published a series of papers that f rmly established that oocytes can synthesize a protein from a foreign
mRNA. (Lane et al. 1971) conclude that
when injected into a living cell, the 9 s RNA is fairly stable
and has the properties of a hemoglobin messenger. The messenger requires no reticulocyte-specifc factors for translation, and the translational machinery of the oocyte will
accept the messenger RNA from a totally different cell type,
from another species.
DOI: 10.1201/9781003050230-10
143