Legier V. Rojas

Functional expression of Kir 6.1/SUR1‐KATP channels in frog retinal Müller glial cells

The retinae and brains of larval and adult amphibians survive long‐lasting anoxia; this finding suggests the presence of functional KATP channels. We have previously shown with immunocytochemistry studies that retinal glial (Müller) cells in adult frogs express the KATP channel and receptor proteins, Kir6.1 and SUR1, while retinal neurons display Kir6.2 and SUR2A/B (Skatchkov et al., 2001a : NeuroReport 12:1437–1441; Eaton et al., in press : NeuroReport). Using both immunocytochemistry and electrophysiology, we demonstrate the expression of Kir6.1/SUR1 (KATP) channels in adult frog and tadpole Müller cells. Using conditions favoring the activation of KATP channels (i.e., ATP‐ and spermine‐free cytoplasm‐dialyzing solution containing gluconate) in Müller cells isolated from both adult frogs and tadpoles, we demonstrate the following. First, using the patch‐clamp technique in whole‐cell recordings, tolbutamide, a blocker of KATP channels, blocks nearly 100% of the transient and about 30% of the steady‐state inward currents and depolarizes the cell membrane by 5–12 mV. Second, inside‐out membrane patches display a single‐channel inward current induced by gluconate (40 mM) and blocked by ATP (200 μM) at the cytoplasmic side. The channels apparently show two sublevels (each of ∼27–32 pS) with a total of 85‐pS maximal conductance at −80 mV; the open probability follows a two‐exponential mechanism. Thus, functional KATP channels, composed of Kir6.1/SUR1, are present in frog Müller cells and contribute a significant part to the whole‐cell K+ inward currents in the absence of ATP. Other inwardly rectifying channels, such as Kir4.1 or Kir2.1, may mediate the remaining currents. KATP channels may help maintain glial cell functions during ATP deficiency. GLIA 38:256–267, 2002.

Tryptophan Substitutions at Lipid-exposed Positions of the Gamma M3 Transmembrane Domain Increase the Macroscopic Ionic Current Response of the Torpedo californica Nicotinic Acetylcholine Receptor

Abstract. Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (γM3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltage-clamped oocytes. For all the substitutions the concentration for half-maximal activation, EC50, is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2- to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized αC418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the γM3/αM4 double mutants, the EC50s were similar to those measured for the αC418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs.

K+ channel density increases selectively in the endfoot of retinal glial cells during development of Rana catesbiana

The radial glial cells that span the retina, described by Müller in 1851, have a remarkable distribution of ion channels in adult amphibia that mediate extracellular K+ spatial buffering. 94% of the total membrane conductance of these cells resides in inward rectifier K+ channels in the endfoot processes apposed to the vitreous humour. We now report that this regional specialization is found in Müller cells isolated from adult (>120 day old) bullfrogs but to a far less extent in those from 10–20 day old tadpoles (stages 34–36). Using the cell attached configuration of the patch‐clamp technique, we found, in agreement with previous studies in salamanders, that the endfoot of adult cells had 19.2 ± 2.4 (mean ± S.E., n = 81) channels/patch, whereas the soma had 1.81 ± 0.28 (n = 21) channels/patch. In the tadpole, the respective values were 4.29 ± 0.26 (n = 79) for the endfoot and 2.26 ± 0.24 (n = 27) for the soma. The slope conductance of the inward rectifier K+ channel in 115 mM K+, 19.2 ± 0.25 pS (n = 205), channel kinetics and the resting membrane potential (−69 ± 2.7 mV, n = 224) were similar at both the endfoot and soma of both adults and embryos. We conclude that during development, the K+ conductance of the Müller cell endfoot, but not of the soma, increases due to a selective clustering of inwardly rectifyiing K+ channels in that specific region of the cell membrane. The properties of the channels change little during the transformation from tadpole to adult bullfrog. GLIA 25:199–203, 1999.

Alpha-1 adrenoreceptors modulate GABA release onto ventral tegmental area dopamine neurons

The ventral tegmental area (VTA) plays an important role in reward and motivational processes involved in drug addiction. Previous studies have shown that alpha1-adrenoreceptors (α1-AR) are primarily found pre-synaptically at this area. We hypothesized that GABA released onto VTA-dopamine (DA) cells is modulated by pre-synaptic α1-AR. Recordings were obtained from putative VTA-DA cells of male Sprague-Dawley rats (28-50 days postnatal) using whole-cell voltage clamp technique. Phenylephrine (10 μM; α1-AR agonist) decreased the amplitude of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) evoked by electrical stimulation of afferent fibers (n = 7; p < 0.05). Prazosin (1 μM, α1-AR antagonist), blocked this effect. Paired-pulse ratios were increased by phenylephrine application (n = 13; p < 0.05) indicating a presynaptic site of action. Spontaneous IPSCs frequency but not amplitude, were decreased in the presence of phenylephrine (n = 7; p < 0.05). However, frequency or amplitude of miniature IPSCs were not changed (n = 9; p > 0.05). Phenylephrine in low Ca(2+) (1 mM) medium decreased IPSC amplitude (n = 7; p < 0.05). Chelerythrine (a protein kinase C inhibitor) blocked the α1-AR action on IPSC amplitude (n = 6; p < 0.05). Phenylephrine failed to decrease IPSCs amplitude in the presence of paxilline, a BK channel blocker (n = 7; p < 0.05). Taken together, these results demonstrate that α1-ARs at presynaptic terminals can modulate GABA release onto VTA-DA cells. Drug-induced changes in α1-AR could contribute to the modifications occurring in the VTA during the addiction process.

The Polarity of Lipid-Exposed Residues Contributes to the Functional Differences between Torpedo and Muscle-Type Nicotinic Receptors

A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues αS287, αC412, βY441, γM299, γS460, δM293, δS297 and δN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [125I]-α-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC50), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC50. Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.

Novel β subunit mutation causes a slow-channel syndrome by enhancing activation and decreasing the rate of agonist dissociation

We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V --> F substitution in the M1 domain of the beta subunit (betaV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation betaV229F expressed normal amounts of AChRs and decreased the ACh EC50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the betaV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the betaV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the betaV229F mutation on the miniature endplate currents and thus are direct evidence that the betaV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.

Thyroid hormones regulate the frequency of miniature end‐plate currents in pre‐ and prometamorphic stages of the tadpole tail

Thyroid hormones (THs), primarily 3,3′,5‐triiode‐L‐thyronine (T3), have been clearly established as natural inducers of apoptosis during metamorphosis of anuran embryos. We decided to use this phenomenon to test the hypothesis that, prior to genomic activation, T3 has acute actions in the neuromuscular junction (NMJ) of the tail of amphibian embryos. We detected a dramatic increase in the production of miniature end‐plate currents (MEPCs) 2–5 min after continuous application of T3 (250 nM) using focal recordings under voltage clamp. Furthermore, this increase in the spontaneous release of neurotransmitter, evaluated by the MEPC frequency, was maintained for several hours. Reverse‐T3, the “inhibitory” form of THs, prevented this increase in MEPC frequency, suggesting that this is probably a highly specific action of T3. In addition, the elevation in MEPC frequency induced by T3 was unchanged in the presence or absence of extracellular calcium. The T3‐mediated increase in MEPC frequency was blocked by niflumic acid, a nonsteroidal antinflammatory fenamate used to prevent the apoptotic volume decrease observed in many systems. The present study demonstrated that T3 induces a remarkable nongenomic action in the NMJ of the tadpole tail at pre‐ and promatamorphic stages.

Aβ Peptide Originated from Platelets Promises New Strategy in Anti-Alzheimer’s Drug Development

The amyloid beta (Aβ) peptide and its deposits in the brain are known to be implicated in the neurodegeneration that occurs during Alzheimers disease (AD). Recently, alternative theories views concerning both the source of this peptide and its functions have been developed. It has been shown that, as in all other known types of amyloidosis, the production of Aβ originates in blood cells or cells related to blood plasma, from which it can then spread from the blood to inside the brain, with the greatest concentration around brain blood vessels. In this review, we summarize research progress in this new area and outline some future perspectives. While it is still unclear whether the main source of Aβ deposits in AD is the blood, the possibility of blocking the chain of reactions that lead to constant Aβ release from the blood to the brain may be exploited in an attempt to reduce the amyloid burden in AD. Solving the problem of Aβ accumulation in this way may provide an alternative strategy for developing anti-AD drugs.