147
Oocytes and Receptors and Channels
is composed of eight members that are involved in diverse
biological functions such as temperature sensing, inf ammation, insulin secretion, and redox sensing (Gao and Liao 2019;
Huang et al. 2020). The TRPM4 channel is expressed in the
oocyte. This channel is widely expressed and regulates calcium oscillations after T cell activation and prevents cardiac
conduction and smooth muscle contraction (Wang et al. 2018).
TRPM4 is activated by intracellular calcium and voltage; thus,
the activation mechanisms converge with those of TMEM16A
(or T out in the oocyte; Miledi, 1982). Finally, TRPV4 has also
been found in the proteomic analysis of Xenopus oocytes.
This calcium-permeable non-selective channel performs
multiple physiological roles in diverse organs; the channel is also gated by osmotic pressure, mechanical pressure,
and biochemical signaling. Mutations in the gene encoding
TRPV4 induce skeletal dysplasia, osteoarthritis, and neurological motor disorders (McCray et al. 2014; Rosenbaum et al.
2020); however, the study of the endogenous TRPV4 has
escaped analysis in oocytes.
Follicle-enclosed oocytes present several ion currents,
including a barium-sensitive potassium current that is activated upon depolarization of the membrane. This current is
removed by collagenase treatment, indicating the need for
the intercommunication between the oocyte and surrounding cells (Parker and Miledi 1988). In the oocyte proteome,
two potassium channels have been identif ed: Kcnq1, which
has multiple functions such as the regulation of gastric
acid secretion, thyroid hormone synthesis, salt and glucose
homeostasis, and cell volume (Dixit et al. 2020), and Kcnn2,
a member of the calcium-activated potassium channel family. The latter channel is targeted by the ubiquitin-protein
ligase E3A, whose function is reduced in Angelman’s syndrome; thus, in the disease, Kcnn2 function increases, leading to changes in synaptic function (Sun et al. 2015, 2020).
Oocytes rarely express ligand-gated ion channels of the
Cys-loop family. One case is the α3 subunit of the glycine
receptor that is capable of forming homomeric receptors
with high affnity to glycine but generates small chloride currents upon exposure to glycine (Kuhse et al. 1990;
Nikolic et al. 1998). Thus, considering the limited number
of selective agonists and antagonists of the glycine receptors
( Zeilhofer et al. 2018), the oocyte may be valuable to screen
for new active molecules.
9.4. INSIGHT OF NATIVE ION
CURRENTS OF THE FOLLICLE
As mentioned, a poorly explored advantage of the oocyte as
an experimental model for understanding the role of receptors and ion channels is the follicle-enclosed oocyte. In
the ovary, cells that surround the oocyte include epithelial
and follicular cells that maintain a close physical interaction and exchange biochemical signals mediated by calcium
and cAMP, for example (Miledi and Woodward 1989a).
Follicle cells produce important modulators such as serotonin, dopamine and noradrenaline, gonadotropins (folliclestimulating hormone, luteinizing hormone and growth
hormone), prostaglandins, and neuropeptides (oxytocin, atrial
natriuretic peptide, corticotropin releasing factor, gonadotropin-releasing hormone, calcitonin gene-related peptide)
and express zinc-gated ion channels (Miledi et al. 1989).
Follicles also express acetylcholine and angiotensin receptors that couple to inositol-3-phosphate, which in turn
releases calcium and gates TMEM16A (Miledi and Parker
1984; Woodward and Miledi 1987; Parker and Miledi 1988;
Miledi and Woodward 1989a ; Arellano et al. 1995; Arellano
et al. 1996 ). Studies on these endogenous receptors and the
ion-currents elicited upon activation are very important for
understanding follicular physiology, inter- and intracellular
communication, and biochemical signaling and place the
follicular oocyte as an important model for studying diverse
processes which will help us understand the physiology of
diverse cellular systems.
9.5. FUTURE DIRECTIONS
Miledi and co-workers developed a novel, powerful assay
that relies upon the ability of cell membranes to spontaneously form vesicles and the ease with which these vesicles
fuse with the plasma membrane when they are injected in
the oocyte (Eusebi et al. 2009). This assay offers the possibility of studying the characteristics of ion channels and
neurotransmitter receptors embedded in their original lipid
environment from samples that were frozen years before or
from freshly resected brain tissue (Palma et al. 2005, 2006,
2007). This assay has shed some light on the characteristics
of important components of the synaptic function and how
they are altered in human disorders such as autism, epilepsy,
and Alzheimer’s disease (Miledi et al. 2004; Limon et al.
2008; Roseti et al. 2008).
A combination of proteomic, transcriptomic, and functional assays of glutamate receptors “transplanted” from
postmortem samples of schizophrenic brain showed the
electrophysiological and pharmacological characteristics of
AMPA receptors from the dorsolateral prefrontal cortex and
its impairment in the disease ( Zeppillo et al. 2020). Another
example of the power of this assay is the microtransplantation of brain samples from Rett syndrome patients. This
disease is caused by mutations within the methylcytosinebinding protein 2 (MECP2) gene, although evidence has
shown linkage to the DKL5 and FOXG1 genes. Rett syndrome is associated with the X chromosome, and patients
develop language and communication problems and learning and coordination defcits (Brunetti and Lumsden 2020;
Sandweiss et al. 2020). In a study by Ruffolo et al. (2020),
membranes from the prefrontal cortex of Rett syndrome
patients showed an imbalance in the excitatory/inhibitory
ratio given by AMPA and GABA responses, a modif cation of GABA currents towards a more depolarizing value,
and differences in the AMPA/GABA ratio. Interestingly, a
transgenic mouse model of Rett syndrome exhibited similar
functional impairments (Ruffolo et al. 2020).
Because the human neurotransmitter receptors are
“microtransplanted” in their native cell lipid environment,
Oocytes and Receptors and Channels
is composed of eight members that are involved in diverse
biological functions such as temperature sensing, inf ammation, insulin secretion, and redox sensing (Gao and Liao 2019;
Huang et al. 2020). The TRPM4 channel is expressed in the
oocyte. This channel is widely expressed and regulates calcium oscillations after T cell activation and prevents cardiac
conduction and smooth muscle contraction (Wang et al. 2018).
TRPM4 is activated by intracellular calcium and voltage; thus,
the activation mechanisms converge with those of TMEM16A
(or T out in the oocyte; Miledi, 1982). Finally, TRPV4 has also
been found in the proteomic analysis of Xenopus oocytes.
This calcium-permeable non-selective channel performs
multiple physiological roles in diverse organs; the channel is also gated by osmotic pressure, mechanical pressure,
and biochemical signaling. Mutations in the gene encoding
TRPV4 induce skeletal dysplasia, osteoarthritis, and neurological motor disorders (McCray et al. 2014; Rosenbaum et al.
2020); however, the study of the endogenous TRPV4 has
escaped analysis in oocytes.
Follicle-enclosed oocytes present several ion currents,
including a barium-sensitive potassium current that is activated upon depolarization of the membrane. This current is
removed by collagenase treatment, indicating the need for
the intercommunication between the oocyte and surrounding cells (Parker and Miledi 1988). In the oocyte proteome,
two potassium channels have been identif ed: Kcnq1, which
has multiple functions such as the regulation of gastric
acid secretion, thyroid hormone synthesis, salt and glucose
homeostasis, and cell volume (Dixit et al. 2020), and Kcnn2,
a member of the calcium-activated potassium channel family. The latter channel is targeted by the ubiquitin-protein
ligase E3A, whose function is reduced in Angelman’s syndrome; thus, in the disease, Kcnn2 function increases, leading to changes in synaptic function (Sun et al. 2015, 2020).
Oocytes rarely express ligand-gated ion channels of the
Cys-loop family. One case is the α3 subunit of the glycine
receptor that is capable of forming homomeric receptors
with high affnity to glycine but generates small chloride currents upon exposure to glycine (Kuhse et al. 1990;
Nikolic et al. 1998). Thus, considering the limited number
of selective agonists and antagonists of the glycine receptors
( Zeilhofer et al. 2018), the oocyte may be valuable to screen
for new active molecules.
9.4. INSIGHT OF NATIVE ION
CURRENTS OF THE FOLLICLE
As mentioned, a poorly explored advantage of the oocyte as
an experimental model for understanding the role of receptors and ion channels is the follicle-enclosed oocyte. In
the ovary, cells that surround the oocyte include epithelial
and follicular cells that maintain a close physical interaction and exchange biochemical signals mediated by calcium
and cAMP, for example (Miledi and Woodward 1989a).
Follicle cells produce important modulators such as serotonin, dopamine and noradrenaline, gonadotropins (folliclestimulating hormone, luteinizing hormone and growth
hormone), prostaglandins, and neuropeptides (oxytocin, atrial
natriuretic peptide, corticotropin releasing factor, gonadotropin-releasing hormone, calcitonin gene-related peptide)
and express zinc-gated ion channels (Miledi et al. 1989).
Follicles also express acetylcholine and angiotensin receptors that couple to inositol-3-phosphate, which in turn
releases calcium and gates TMEM16A (Miledi and Parker
1984; Woodward and Miledi 1987; Parker and Miledi 1988;
Miledi and Woodward 1989a ; Arellano et al. 1995; Arellano
et al. 1996 ). Studies on these endogenous receptors and the
ion-currents elicited upon activation are very important for
understanding follicular physiology, inter- and intracellular
communication, and biochemical signaling and place the
follicular oocyte as an important model for studying diverse
processes which will help us understand the physiology of
diverse cellular systems.
9.5. FUTURE DIRECTIONS
Miledi and co-workers developed a novel, powerful assay
that relies upon the ability of cell membranes to spontaneously form vesicles and the ease with which these vesicles
fuse with the plasma membrane when they are injected in
the oocyte (Eusebi et al. 2009). This assay offers the possibility of studying the characteristics of ion channels and
neurotransmitter receptors embedded in their original lipid
environment from samples that were frozen years before or
from freshly resected brain tissue (Palma et al. 2005, 2006,
2007). This assay has shed some light on the characteristics
of important components of the synaptic function and how
they are altered in human disorders such as autism, epilepsy,
and Alzheimer’s disease (Miledi et al. 2004; Limon et al.
2008; Roseti et al. 2008).
A combination of proteomic, transcriptomic, and functional assays of glutamate receptors “transplanted” from
postmortem samples of schizophrenic brain showed the
electrophysiological and pharmacological characteristics of
AMPA receptors from the dorsolateral prefrontal cortex and
its impairment in the disease ( Zeppillo et al. 2020). Another
example of the power of this assay is the microtransplantation of brain samples from Rett syndrome patients. This
disease is caused by mutations within the methylcytosinebinding protein 2 (MECP2) gene, although evidence has
shown linkage to the DKL5 and FOXG1 genes. Rett syndrome is associated with the X chromosome, and patients
develop language and communication problems and learning and coordination defcits (Brunetti and Lumsden 2020;
Sandweiss et al. 2020). In a study by Ruffolo et al. (2020),
membranes from the prefrontal cortex of Rett syndrome
patients showed an imbalance in the excitatory/inhibitory
ratio given by AMPA and GABA responses, a modif cation of GABA currents towards a more depolarizing value,
and differences in the AMPA/GABA ratio. Interestingly, a
transgenic mouse model of Rett syndrome exhibited similar
functional impairments (Ruffolo et al. 2020).
Because the human neurotransmitter receptors are
“microtransplanted” in their native cell lipid environment,