144
Xenopus
Using this information while working at University College
London, Miledi and co-workers tried to express ion channels and receptors involved in synaptic transmission; after
using diverse strategies, they realized that the critical step
was the preparation of high-quality mRNA. In the early
days of molecular cloning, that methodology was a tedious
procedure involving ultracentrifugation of Cesium chloride
gradients and other biochemical tricks that made the effort
a heroic act. Selection of mRNA by Oligo dT chromatography to enrich the poly-A + mRNA fraction was crucial to
prove that acetylcholine receptors from denervated muscle
injected in oocytes indeed have the same electrophysiological and pharmacological characteristics as the receptors
embedded in the muscle membrane (Miledi et al. 1982).
After those initial steps, many brain ion channels and receptors were expressed after mRNA injection (Gundersen et al.
1983, 1984; Sumikawa et al. 1984). Ultracentrifugation of
sucrose density gradients of mRNA allowed the identif cation of specifc fractions containing mRNAs with different
coding potential for functional receptors; this strategy was
used to separate different functional fractions and became an
expression cloning approach to synthesize cDNA clones for
proteins essential for synaptic transmission (e.g. GABA-A
and nicotinic receptors, as well as K + , Na + , and Cl-channels).
Today, these studies are rarely found in the recent literature
because of the current availability of gene clones, synthetic
genes, and other resources that permit a straightforward
experimental approach to assess protein function. But they
led to major advances in the f eld.
For many years, studies aiming to understand the function and pharmacology of ion channels and receptors were
potentiated by using Xenopus oocytes. One of the most
effective approaches to studying the inner workings of
these proteins consisted of a combination of site-directed
mutagenesis and functional analysis in the oocyte. This
approach revealed important structural characteristics,
including voltage sensors, neurotransmitters, and pharmacologically relevant binding sites, and allowed researchers
to evaluate functional effects such as gating, desensitization, and inactivation.
9.3. PRESENT STATUS
From the mid-80s, researchers in the feld primarily focused on
the Xenopus oocyte to understand how ion channels and neurotransmitter receptors work. Key biophysical and structural
traits of these molecular entities that have been approached
experimentally are voltage sensing, gating, neurotransmitter binding sites, and permeability (see subsequently). When
oocytes began to be used as an experimental tool for expressing ion channels, they were ready to be exploited during the
molecular cloning era.
Voltage-gated ion channels constitute a diverse class
of proteins that are crucial for neuronal excitability and
plasticity. They gate an ion channel upon sensing changes
in the plasma membrane voltage. “Gating” is an intrinsic
electrophysiological characteristic of ion channels that refers
to the opening (by activation) or closing (by deactivation or
inactivation) of the protein complex that forms the conducting pore of the ions. Gating involves changes in conformation in response to changes in membrane voltage or to an
agonist. This biophysical characteristic of ion channels has
been widely studied using Xenopus oocytes, from the f rst
reports of their molecular cloning (Mackinnon et al. 1988;
Takumi et al. 1988; Murai et al. 1989) to recent approaches
that combine site-directed mutagenesis and structural models
of crystallized proteins (Carvalho-de-Souza and Bezanilla
2019; Hou et al. 2019; Rinné et al. 2019).
Another fundamental structural component of both
sodium- and potassium-selective channels is the so-called
“voltage sensor.” Studies in frog oocytes were critical for
deciphering the molecular structure and function of voltage sensors that were found to be highly conserved among
channel families and from different species (Noda et al.
1986; Stühmer et al. 1988; Tempel et al. 1988; Timpe et al.
1988; Stühmer et al. 1989, 1989), which currently remains an
intensive feld of study (Catterall et al. 2017; Ori et al. 2020).
Mapping the binding sites of the agonist to ligand-gated
ion channels was essential to understand their molecular
structure. Xenopus oocytes played a central role in determining fne pharmacology and specifc interaction sites for
nicotinic, GABA, serotonin, and glycine receptors (Blair
et al. 1988; Bertrand et al. 1990; Grenningloh et al. 1990;
Kuhse et al. 1990; Maricq et al. 1991; Amin et al. 1994), as
well as for the family of glutamate receptors AMPA/kainite
and NMDA (Bettler et al. 1990; Boulter et al. 1990; Egebjerg
et al. 1991). High-resolution structures of these receptor
complexes have confrmed many of the observations f rst
determined by electrophysiology in oocytes (Kesters et al.
2013; Hassaine et al. 2014; Miller and Aricescu 2014).
Detailed electrophysiological characterization of Xenopus
follicle-enclosed oocytes was prompted when it was realized that they could be very useful to study ion channels and
receptors. Many studies have shown that electrical responses
to neurotransmitters and hormones generated by ovarian
follicular cells require maintenance of the electrical communication between the oocyte and its surrounding cells.
Therefore, the responses originate in the membrane of the
follicular cells and these cells express membrane receptors
and ion channels. Several “native” ion currents and receptors
that are fundamental for neural transmission and other physiological processes are endogenously expressed by Xenopus
oocytes. Here we provide some examples of ion channels in
the oocyte membrane whose currents were identif ed years
ago. The presence of these currents is supported by new proteomic (Table 9.1) or other analyses and is associated with
human diseases (Session et al. 2016; Peshkin et al. 2019).
One case is the calcium-dependent chloride channel
TMEM16A (Anoctamin 1), originally discovered in the
oocyte (Miledi 1982; Miledi and Parker 1984) but known to
play a crucial role in regulating anxiety-related behaviors, for
it is expressed in cholinergic neurons of the medial habenula
Xenopus
Using this information while working at University College
London, Miledi and co-workers tried to express ion channels and receptors involved in synaptic transmission; after
using diverse strategies, they realized that the critical step
was the preparation of high-quality mRNA. In the early
days of molecular cloning, that methodology was a tedious
procedure involving ultracentrifugation of Cesium chloride
gradients and other biochemical tricks that made the effort
a heroic act. Selection of mRNA by Oligo dT chromatography to enrich the poly-A + mRNA fraction was crucial to
prove that acetylcholine receptors from denervated muscle
injected in oocytes indeed have the same electrophysiological and pharmacological characteristics as the receptors
embedded in the muscle membrane (Miledi et al. 1982).
After those initial steps, many brain ion channels and receptors were expressed after mRNA injection (Gundersen et al.
1983, 1984; Sumikawa et al. 1984). Ultracentrifugation of
sucrose density gradients of mRNA allowed the identif cation of specifc fractions containing mRNAs with different
coding potential for functional receptors; this strategy was
used to separate different functional fractions and became an
expression cloning approach to synthesize cDNA clones for
proteins essential for synaptic transmission (e.g. GABA-A
and nicotinic receptors, as well as K + , Na + , and Cl-channels).
Today, these studies are rarely found in the recent literature
because of the current availability of gene clones, synthetic
genes, and other resources that permit a straightforward
experimental approach to assess protein function. But they
led to major advances in the f eld.
For many years, studies aiming to understand the function and pharmacology of ion channels and receptors were
potentiated by using Xenopus oocytes. One of the most
effective approaches to studying the inner workings of
these proteins consisted of a combination of site-directed
mutagenesis and functional analysis in the oocyte. This
approach revealed important structural characteristics,
including voltage sensors, neurotransmitters, and pharmacologically relevant binding sites, and allowed researchers
to evaluate functional effects such as gating, desensitization, and inactivation.
9.3. PRESENT STATUS
From the mid-80s, researchers in the feld primarily focused on
the Xenopus oocyte to understand how ion channels and neurotransmitter receptors work. Key biophysical and structural
traits of these molecular entities that have been approached
experimentally are voltage sensing, gating, neurotransmitter binding sites, and permeability (see subsequently). When
oocytes began to be used as an experimental tool for expressing ion channels, they were ready to be exploited during the
molecular cloning era.
Voltage-gated ion channels constitute a diverse class
of proteins that are crucial for neuronal excitability and
plasticity. They gate an ion channel upon sensing changes
in the plasma membrane voltage. “Gating” is an intrinsic
electrophysiological characteristic of ion channels that refers
to the opening (by activation) or closing (by deactivation or
inactivation) of the protein complex that forms the conducting pore of the ions. Gating involves changes in conformation in response to changes in membrane voltage or to an
agonist. This biophysical characteristic of ion channels has
been widely studied using Xenopus oocytes, from the f rst
reports of their molecular cloning (Mackinnon et al. 1988;
Takumi et al. 1988; Murai et al. 1989) to recent approaches
that combine site-directed mutagenesis and structural models
of crystallized proteins (Carvalho-de-Souza and Bezanilla
2019; Hou et al. 2019; Rinné et al. 2019).
Another fundamental structural component of both
sodium- and potassium-selective channels is the so-called
“voltage sensor.” Studies in frog oocytes were critical for
deciphering the molecular structure and function of voltage sensors that were found to be highly conserved among
channel families and from different species (Noda et al.
1986; Stühmer et al. 1988; Tempel et al. 1988; Timpe et al.
1988; Stühmer et al. 1989, 1989), which currently remains an
intensive feld of study (Catterall et al. 2017; Ori et al. 2020).
Mapping the binding sites of the agonist to ligand-gated
ion channels was essential to understand their molecular
structure. Xenopus oocytes played a central role in determining fne pharmacology and specifc interaction sites for
nicotinic, GABA, serotonin, and glycine receptors (Blair
et al. 1988; Bertrand et al. 1990; Grenningloh et al. 1990;
Kuhse et al. 1990; Maricq et al. 1991; Amin et al. 1994), as
well as for the family of glutamate receptors AMPA/kainite
and NMDA (Bettler et al. 1990; Boulter et al. 1990; Egebjerg
et al. 1991). High-resolution structures of these receptor
complexes have confrmed many of the observations f rst
determined by electrophysiology in oocytes (Kesters et al.
2013; Hassaine et al. 2014; Miller and Aricescu 2014).
Detailed electrophysiological characterization of Xenopus
follicle-enclosed oocytes was prompted when it was realized that they could be very useful to study ion channels and
receptors. Many studies have shown that electrical responses
to neurotransmitters and hormones generated by ovarian
follicular cells require maintenance of the electrical communication between the oocyte and its surrounding cells.
Therefore, the responses originate in the membrane of the
follicular cells and these cells express membrane receptors
and ion channels. Several “native” ion currents and receptors
that are fundamental for neural transmission and other physiological processes are endogenously expressed by Xenopus
oocytes. Here we provide some examples of ion channels in
the oocyte membrane whose currents were identif ed years
ago. The presence of these currents is supported by new proteomic (Table 9.1) or other analyses and is associated with
human diseases (Session et al. 2016; Peshkin et al. 2019).
One case is the calcium-dependent chloride channel
TMEM16A (Anoctamin 1), originally discovered in the
oocyte (Miledi 1982; Miledi and Parker 1984) but known to
play a crucial role in regulating anxiety-related behaviors, for
it is expressed in cholinergic neurons of the medial habenula