Biomedical subjects

Ricardo Miledi

Publications and source records attributed to Ricardo Miledi.

30 records · Page 2Linked to original sources

PubMed2004

Functional expression in frog oocytes of human rho 1 receptors produced in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was engineered to express the rho 1 subunit of the human gamma-aminobutyric acid rho 1 (GABA rho 1) receptor. RNA that was isolated from several transformed yeast strains produced fully functional GABA receptors in Xenopus oocytes. The GABA currents elicited in the oocytes were fast, nondesensitizing chloride currents; and the order of agonist potency was GABA > beta-alanine > glycine. Moreover, the receptors were resistant to bicuculline, strongly antagonized by (1,2,5,6 tetrahydropyridine-4-yl)methylphosphinic acid, and modulated by zinc and lanthanum. Thus, the GABA receptors expressed by the yeast mRNA retained all of the principal characteristics of receptors expressed by cRNA or native retina mRNAs. Western blot assays showed immunoreactivity in yeast plasma membrane preparations, and a rho 1-GFP fusion gene showed mostly intracellular distribution with a faint fluorescence toward the plasma membrane. In situ immunodetection of rho 1 in yeast demonstrated that some receptors reach the plasma membrane. Furthermore, microtransplantation of yeast plasma membranes to frog oocytes resulted in the incorporation of a small number of functional yeast rho 1 receptors into the oocyte plasma membrane. These results show that yeast may be useful to produce complete functional ionotropic receptors suitable for structural analysis.

Amino Acid Sequence↗

PubMed2003

Uptake of serotonin by adult rat corpus callosum is partially reduced by common antidepressants.

The corpus callosum (CC) is the main white matter tract involved in interhemispheric brain communication. We establish that uptake of [3H]5-hydroxytryptamine (5-HT) in CC is partially inhibited by some antidepressants. Slices of the adult rat CC had a high-affinity uptake of 5-HT. About 80% of this uptake was Na+ dependent, with a Michaelis-Menten constant, Km, of 420 +/- 80 nM and a rate of 5-HT uptake, Vmax, of 9.5 +/- 0.8 pmol/mg protein/min. The 5-HT uptake was reduced approximately 60% at pH 5 compared with that at pH 7. Fluoxetine (Prozac) inhibited only 43% of 5-HT uptake in a concentration-dependent manner, with an affinity constant, Ki, of 44.7 +/- 10.0 nM. We also studied the effects of other monoamine uptake inhibitors, all at 10 microM, and found that zimelidine, imipramine, and clomipramine inhibited 5-HT uptake in the CC by approximately 30-40%. The fluoxetine-insensitive 5-HT uptake was not altered by high concentrations of dopamine plus norepinephrine. The present data show that Na(+)-dependent 5-HT uptake occurs in the CC and optic nerve and that this uptake is partially sensitive to antidepressants and probably mediated by the serotonin transporter, which may be relevant during depression.

Animals↗

PubMed2003

e-Phys: a suite of intracellular neurophysiology programs integrating COM (component object model) technologies.

Current computer programs for intracellular recordings often lack advanced data management, are usually incompatible with other applications and are also difficult to adapt to new experiments. We have addressed these shortcomings in e-Phys, a suite of electrophysiology applications for intracellular recordings. The programs in e-Phys use Component Object Model (COM) technologies available in the Microsoft Windows operating system to provide enhanced data storage, increased interoperability between e-Phys and other COM-aware applications, and easy customization of data acquisition and analysis thanks to a script-based integrated programming environment. Data files are extensible, hierarchically organized and integrated in the Windows shell by using the Structured Storage technology. Data transfers to and from other programs are facilitated by implementing the ActiveX Automation standard and distributed COM (DCOM). ActiveX Scripting allows experimenters to write their own event-driven acquisition and analysis programs in the VBScript language from within e-Phys. Scripts can reuse components available from other programs on other machines to create distributed meta-applications. This paper describes the main features of e-Phys and how this package was used to determine the effect of the atypical antipsychotic drug clozapine on synaptic transmission at the neuromuscular junction.

Intracellular Fluid↗

PubMed2003

Microtransplantation of membranes from cultured cells to Xenopus oocytes: a method to study neurotransmitter receptors embedded in native lipids.

The Xenopus oocyte is used as a convenient cell expression system to study the structure and function of heterogenic transmitter receptors and ion channels. Recently, we introduced a method to microtransplant already assembled neurotransmitter receptors from the human brain to the plasma membrane of Xenopus oocytes. The same approach was used here to transplant neurotransmitter receptors expressed from cultured cells to the oocytes. Membrane vesicles prepared from a human embryonic kidney cell line (HEK293) stably expressing the rat glutamate receptor 1 were injected into oocytes, and, within a few hours, the oocyte plasma membrane acquired alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors, which had the same properties as those expressed in the original HEK cells. Analogously, oocytes injected with membranes prepared from rat pituitary GH(4)C1 cells, stably expressing homomeric human neuronal alpha 7 nicotinic acetylcholine receptors (alpha 7-AcChoRs), incorporated in their plasma membrane AcChoRs that behaved as those expressed in GH(4)C1 cells. Similar results were obtained with HEK cells stably expressing heteromeric human neuronal alpha 4 beta 2-AcChoRs. All this makes the Xenopus oocyte a powerful tool for detailed investigations of receptors and other proteins expressed in the membrane of cultured cells.

Animals↗

PubMed2002

Expression of human epileptic temporal lobe neurotransmitter receptors in Xenopus oocytes: An innovative approach to study epilepsy.

Poly(A(+)) RNA was extracted from the temporal lobe (TL) of medically intractable epileptic patients which underwent surgical TL resection. Injection of this mRNA into Xenopus oocytes led to the expression of ionotropic receptors for gamma-aminobutyric acid (GABA), kainate (KAI) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Membrane currents elicited by GABA inverted polarity at -15 mV, close to the oocyte's chloride equilibrium potential, were inhibited by bicuculline, and were potentiated by pentobarbital and flunitrazepam. These basic characteristics were also displayed by GABA currents elicited in oocytes injected with mRNAs isolated from human TL glioma (TLG) or from mouse TL. However, the GABA receptors expressed by the epileptic TL mRNA exhibited some unusual properties, consisting in a rapid current run-down after repetitive GABA applications and a large EC(50) (125 microM). AMPA alone evoked very small or nil currents, whereas KAI induced larger currents. Nevertheless, upon cyclothiazide treatment, AMPA elicited substantial currents that, like the KAI currents, were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Furthermore, the glutamate receptor 5 (GluR5) agonist, ATPA, failed to evoke an obvious current although both RT-PCR and Western blot analyses showed GluR5 expression in the epileptic TL. Oocytes injected with mouse TL or human TLG mRNAs generated KAI and AMPA currents similar to those evoked in oocytes injected with epileptic TL mRNA but, in contrast to these, the mouse TL and human TLG oocytes were also responsive to ATPA. Our findings are in accord with the concept that both a depression of GABA inhibition and a dysfunction of the KAI-receptor system maintain a high neuronal excitability that results in epileptic seizures.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2002

Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms.

Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT(2) receptor subtypes increase IP(3) levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT(2A)-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT(2A) receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT(2A) receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT(2A) receptors internalize and return to the surface after both serotonin- and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT(2A) receptors are recycled.

Animals↗

PubMed2002

Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes.

The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl(-) channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to gamma-aminobutyric acid, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders.

Animals↗

PubMed2002

Effects of clomipramine on neuronal nicotinic acetylcholine receptors.

The action of the tricyclic antidepressant clomipramine on membrane currents elicited by acetylcholine was studied in Xenopus oocytes expressing neuronal alpha2beta4 nicotinic acetylcholine receptors. Clomipramine inhibited the acetylcholine responses rapidly and reversibly, with a similar IC(50) when the oocytes were preincubated with clomipramine (1.3+/-0.2 microM) or when they were exposed simultaneously with acetylcholine and clomipramine (1.5+/-0.3 microM). The EC(50) was 39.9+/-2.1 microM for acetylcholine alone and 65.7+/-3.6 microM for acetylcholine in the presence of 2 microM clomipramine. The inhibitory effect of clomipramine was weakly voltage-dependent, with an electric distance of approximately 0.14. Moreover, clomipramine increased the rate of decay of currents elicited by acetylcholine. From all of these, we conclude that clomipramine reversibly and noncompetitively regulates neuronal alpha2beta4 nicotinic acetylcholine receptors by blocking the open receptor-channel complex at a site close to the extracellular vestibule of the channel. The actions of clomipramine on neuronal nicotinic acetylcholine receptors may play an important role in the treatment of mental depression and other mood disorders.

Acetylcholine↗

PubMed2002

Inhibition of neuronal nicotinic acetylcholine receptors by La(3+).

A study was made of the effects of La(3+) on neuronal alpha 2 beta 4 nicotinic acetylcholine receptors expressed in Xenopus oocytes. La(3+) by itself (up to 10 microM) did not elicit significant membrane currents. However, La(3+) reversibly inhibited the ionic currents induced by acetylcholine (IC(50)=13.5+/-4.3 microM). When La(3+) and acetylcholine were simultaneously applied onto an oocyte, the level of inhibition of the acetylcholine response was the same as when the oocyte was first preincubated with La(3+) and then exposed to acetylcholine plus La(3+). In the presence of La(3+), the EC(50) decreased from 43.8+/-6.4 to 26.5+/-5.1 microM, suggesting a small increase in the affinity of acetylcholine for the receptors through a noncompetitive mechanism. The inhibition of acetylcholine response was independent of the membrane potential. From these results we conclude that La(3+) regulates nicotinic receptors, reversibly and noncompetitively, presumably by inhibiting allosterically the receptor through interactions at an external domain of the receptor complex.

Acetylcholine↗

PubMed2002

Some properties of human neuronal alpha 7 nicotinic acetylcholine receptors fused to the green fluorescent protein.

The functional properties and cellular localization of the human neuronal alpha7 nicotinic acetylcholine (AcCho) receptor (alpha7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutalpha7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtalpha7 receptors decay much faster than those elicited by the mutalpha7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated alpha7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable "run-down" of the AcCho currents generated by mutalpha7-GFP receptors, whereas those of the wtalpha7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutalpha7-GFP oocytes was accompanied by a marked decrease of alpha-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtalpha7 and mutalpha7 receptors provides powerful tools to study the distribution and function of alpha7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins.

Animals↗

PubMed2002

The single-channel properties of human acetylcholine alpha 7 receptors are altered by fusing alpha 7 to the green fluorescent protein.

Neuronal nicotinic acetylcholine (AcCho) receptors composed of alpha7-subunits (alpha7-AcChoRs) are involved in many physiological activities. Nevertheless, very little is known about their single-channel characteristics. By using outside-out patch-clamp recordings from Xenopus oocytes expressing wild-type (wt) alpha7-AcChoRs, we identified two classes of channel conductance: a low conductance (gamma(L)) of 72 pS and a high one (gamma(H)) of 87 pS, with mean open-times (tau(op)) of 0.6 ms. The same classes of conductances, but longer tau(op) (3 ms), were seen in experiments with chimeric alpha7 receptors in which the wtalpha7 extracellular C terminus was fused to the green fluorescent protein (wtalpha7-GFP AcChoRs). In contrast, channels with three different conductances were gated by AcCho in oocytes expressing alpha7 receptors carrying a Leu-to-Thr 248 mutation (mutalpha7) or oocytes expressing chimeric mutalpha7-GFP receptors. These conductance levels were significantly smaller, and their mean open-times were larger, than those of wtalpha7-AcChoRs. Interestingly, in the absence of AcCho, these oocytes showed single-channel openings of the same conductances, but shorter tau(op), than those activated by AcCho. Accordingly, human homomeric wtalpha7 receptors open channels of high conductance and brief lifetime, and fusion to GFP lengthens their lifetime. In contrast, mutalpha7 receptors open channels of lower conductance and longer lifetime than those gated by wtalpha7-AcChoRs, and these parameters are not greatly altered by fusing the mutalpha7 to GFP. All this evidence shows that GFP-tagging can alter importantly receptor kinetics, a fact that has to be taken into account whenever tagged proteins are used to study their function.

Animals↗

PubMedDate not supplied

A single amino acid change within the ion-channel domain of the gamma-aminobutyric acid rho1 receptor accelerates desensitization and increases taurine agonism.

BACKGROUND: GABAC receptors are part of the ligand-gated ion channel family of receptors that share some functional and structural features: e.g., they have four putative transmembrane domains (TM1-TM4) and the TM2-segment is presumed to form the ion-channel. GABAC receptors open chloride channels and do not desensitize even after long exposures to GABA. These receptors are highly expressed in vertebrate retina, where they may play a unique role due to their unusual biophysical and pharmacologic characteristics. METHODS: To determine whether the TM2 domain plays a role in the process of desensitization of GABAC receptors, we used site-directed mutagenesis to produce several permutations within the leucine (L9') residue of the TM2 domain of the human GABArho1 subunit. Recombinant receptors were expressed in Xenopus laevis oocytes and their functional and pharmacologic properties were studied by using a two-microelectrode, voltage-clamp. RESULTS: Several amino acid changes led to receptors that did not generate GABA-currents, whereas an Asp for Leu mutation in the well-conserved L9' position of the rho1 subunit (L301D-rho1) generated a fast-desensitizing, bicuculline-resistant receptor that was antagonized by TPMPA, a specific GABAC receptor antagonist. Moreover, in contrast with wild-type rho1 receptors, which are practically not gated by taurine, L301D-rho1 mutant receptors generated substantial taurine-currents. CONCLUSIONS: Substitution of L9' residue in the TM2 region of GABArho1 receptor for an amino acid residue with an acidic lateral chain greatly accelerates its desensitization rate and increases taurine-agonism. This mutant will be useful to study mechanisms involved in gating and desensitization of GABAC receptors in particular, and of neurotransmitter receptors in general.

Animals↗