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S Sala

Publications and source records attributed to S Sala.

At least 19 recordsLinked to original sources
PubMed1998

A residue in the middle of the M2-M3 loop of the beta4 subunit specifically affects gating of neuronal nicotinic receptors.

An aspartate residue in the M2-M3 loop of neuronal nicotinic receptor alpha7 subunits is a major determinant of the channel functional response. This residue is conserved in most beta4 subunits, e.g. human and rat, but not in others, e.g. bovine. We have used these differences to examine the mechanism by which this residue alters the functional properties of alpha3beta4 receptors. Having ruled out an effect on the macroscopic binding ability of the agonist, the level of receptor expression, or the single channel conductance, the results suggest that receptors lacking that residue have a deficient coupling between binding and gating.

Animals↗
PubMed1997

Role of the putative transmembrane segment M3 in gating of neuronal nicotinic receptors.

The involvement of some structural domains in the gating of the neuronal nicotinic acetylcholine receptor (AChR) was studied by expressing functional alpha7/alpha3 chimeric subunits in Xenopus oocytes. Substitution of the M3 transmembrane segment in the alpha7 subunit modifies the kinetic properties of the chimeric AChRs as follows: (a) a 6-fold reduction in the maximal current evoked by nicotinic agonists, (b) a 10-fold decrease in the macroscopic desensitization rate, (c) an increase of almost 1 order of magnitude in the apparent affinity for acetylcholine and nicotine, and (d) a decrease in the affinity for alpha-bungarotoxin. Computer simulations showed that the first three effects could be accounted for by a simple kinetic model in which chimeric AChRs presented a smaller ratio of the gating rates, beta/alpha, and a slightly slower desensitization rate. It is concluded that the M3 domain influences the gating of neuronal AChRs.

Amino Acid Sequence↗
PubMed1997

Neuronal nicotinic acetylcholine receptors on bovine chromaffin cells: cloning, expression, and genomic organization of receptor subunits.

Neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells play a primary role in triggering catecholamine secretion. In the present study, their constituent subunits were characterized. In addition to the alpha 3 subunit, which we have previously cloned, the presence of alpha 5 and beta 4 but not of beta 2 subunits was detected by reverse transcription-PCR analysis of mRNA from adrenal medulla. In situ hybridization indicated that alpha 3, alpha 5, and beta 4 subunits are coexpressed in all chromaffin cells. The primary structure of alpha 5 and beta 4 subunits was determined and functional receptors were obtained upon coinjection of subunit cRNAs into Xenopus oocytes. In contrast to other beta 4-containing nicotinic receptors, the ones formed by the bovine beta 4 subunit are insensitive to the agonist cytisine. Finally, we characterized the intergenic region of alpha 3 and alpha 5 subunits, which together with the beta 4 subunit, form a gene cluster in rats and chickens. RNase assays and the existence of overlapping cDNAs indicate that, in the bovine genome, the alpha 3 and alpha 5 genes overlap at their 3' ends. This fact is probably due to inefficient transcription termination, as a result of weak polyadenylation signals.

Animals↗
PubMed1997

[The absorbed dose to the gonads in adult patients undergoing defecographic study by digital or traditional radiographic imaging].

INTRODUCTION: Doses to the gonads and skin of adult people were evaluated during the study of functional anorectal disorders with defecography. MATERIALS AND METHODS: The radiologic procedure relies heavily on fluoroscopy with lateral and anteroposterior projections for a mean time of 2.4 minutes; the stages of interest are recorded on a mean of 6 films. Absorbed doses to the gonads and to the skin were measured with LiF thermoluminescence dosimeters placed inside and outside the anthropomorphic "Rando" phantom. The administered doses during the different examination phases (anteroposterior and lateral fluoroscopy and radiography) were measured. Digital and conventional radiography were compared. RESULTS: Gonadal absorbed doses in a standard DSI examination were: 5.0 +/- .1 mGy to the right ovary, 25.2 +/- .7 mGy to the left ovary and 2.7 +/- .1 mGy to testes when at the margin of the irradiation field. Doses to gonads and skin are about 15% higher when the examination is performed with conventional radiography. DISCUSSION: The considerable radiation dose to ovaries shows that defecography can cause radiation-induced genetic effects in women younger than 40 years with a probability of the order of one thousandth of the natural incidence genetic defects. CONCLUSIONS: Defecography must be correctly indicated in fertile women, because of the relatively high absorbed dose to ovaries. In fact, the ovaries absorb about twice as many radiations during defecography than radiologic examinations of the lower gastrointestinal tract.

Absorption↗
PubMed1996

Acetylcholine receptor subunit homomer formation requires compatibility between amino acid residues of the M1 and M2 transmembrane segments.

The neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha3 and alpha7 have different assembly behavior when expressed in heterologous expression systems: alpha3 subunits require other subunits to assemble functional nAChRs, whereas alpha7 subunits can produce homomeric nAChRs. A previous analysis of alpha7/alpha3 chimeric constructs identified a domain comprising the first putative membrane-spanning segment, M1, as essential to homomeric assembly. The present study dissected further this domain, identifying three amino acid residues, which are located at the most intracellular third of the M1 transmembrane segment, as important in the assembly of homomers. Moreover, formation of homooligomeric complexes seems to require a compatible accommodation between this region and certain residues of the second transmembrane segment, M2. Thus, compatibility between defined domains of the M1 and M2 transmembrane segments appears as a determinant factor governing homomer association of nAChR subunits.

Amino Acid Sequence↗
PubMed1996

A single residue in the M2-M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors.

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of changes that activate a cation-selective conductance. By measuring electrophysiological responses in chimeric alpha7/alpha3 receptors expressed in Xenopus oocytes, we have showed the involvement of the M2-M3 loop in coupling agonist binding to the channel gate. An aspartate residue therein, Asp-266 in the alpha7 subunit, was identified by site-directed mutagenesis as crucial, since mutants at this position exhibited very poor functional responses to three different nicotinic agonists. We have extended this investigation to another neuronal nicotinic receptor (alpha3/beta4), and found that a homologous residue in the beta4 subunit, Asp-268, played a similar role in coupling. These findings are consistent with a hypothesis that the aspartate residue in the M2-M3 loop, which is conserved in all homomer-forming alpha-type subunits and all neuronal beta-type subunits that combine to form functional receptors, is a major determinant of information transmission from binding site to channel gate in all neuronal nicotinic receptors.

Amino Acid Sequence↗
PubMed1995

alpha-Bungarotoxin-sensitive nicotinic receptors on bovine chromaffin cells: molecular cloning, functional expression and alternative splicing of the alpha 7 subunit.

Chromaffin cells from the bovine adrenal medulla express alpha-bungarotoxin-sensitive acetylcholine receptors whose subunit composition is unknown. Northern blot analysis showed that the alpha 7 subunit, a main component of these alpha-bungarotoxin-sensitive acetylcholine receptors in avian and rat brain, is expressed in chromaffin cells. The cDNA of this bovine alpha 7 subunit was cloned by polymerase chain reaction amplification of adrenal medulla RNA for detailed characterization of structure and function. The protein-coding region revealed 92% amino acid sequence identity to rat alpha 7 and 89% to chicken alpha 7 subunits. The alpha-bungarotoxin affinity of alpha 7 homomers expressed in Xenopus oocytes was similar to that observed previously with native chromaffin alpha-bungarotoxin-sensitive acetylcholine receptors. Cross-linking and sucrose gradient experiments suggested that, like the muscular and neuronal acetylcholine receptors; the alpha 7 receptor has a pentameric structure. Upon activation with nicotinic agonists the alpha 7 receptor exhibited rapidly desensitizing cation currents that were blocked by nicotinic antagonists and showed inward rectification. The amplification of adrenal medulla RNA by reverse transcription-polymerase chain reaction methods revealed an alternatively spliced isoform of the bovine alpha 7 subunit, where the exon that codes for the M2 transmembrane segment was skipped during mRNA processing. Oocyte expression of this isoform does not yield functional channels. However, this alternative mRNA exhibits dose-dependent inhibition of alpha 7 homomer expression when coinjected with the undeleted isoform.

Alternative Splicing↗
PubMed1995

Conformation and interactions of all-D-, retro-all-D- and retro-bombolitin III analogues in aqueous solution and in the presence of detergent micelles.

Bombolitins are five structurally related heptadecapeptides which lyse erythrocytes and liposomes and enhance the activity of phospholipase A2. The conformational properties of the all-D-, retro-all-D, and retro-L-analogues of bombolitin III were investigated by circular dichroism (CD) and two-dimensional 1H-nuclear magnetic resonance (NMR) techniques. In water, all three sequences are in a random conformation and aggregate to various extents depending on pH and concentration. The two enantiomeric retro-sequences exhibit a higher propensity to form beta-aggregates, while the D-analogue of the native sequence tends to form aggregates of alpha-helices. In the presence of an excess of sodium dodecyl sulfate (SDS), the peptides fold into an amphiphilic alpha-helical conformation (left-handed in the case of the all-D sequences). NMR studies on the L-retro-analogue indicate that the helical segment is localized in the central part of the sequence. Combined CD and surface tension measurements indicate that the critical micellar concentration of SDS is raised in the presence of peptide and that helical folding occurs before micelle formation. These results are interpreted in terms of formation of peptide-detergent complexes. It is estimated that the helical structure forms when 40-50 molecules of detergent are bound to each peptide molecule. These data and our previous results in vitro biological tests confirm that the ability to form amphiphilic helices is the major determinant of biological activity of bombolitins.

Amino Acid Sequence↗
PubMed1994

Role of two acetylcholine receptor subunit domains in homomer formation and intersubunit recognition, as revealed by alpha 3 and alpha 7 subunit chimeras.

Differential expression of subunit genes from the nicotinic acetylcholine receptor (AChR) superfamily yields distinct receptor subtypes. As each AChR subtype has a specific subunit composition and many subunit combinations appear not to be expressed, each subunit must contain some information leading to proper assembly. The neuronal AChR subunits alpha 3 and alpha 7 are expressed in bovine chromaffin cells, probably as constituents of two different AChR subtypes. These subunits have different assembly behavior when expressed in heterologous expression systems: alpha 7 subunits are able to produce homomeric AChRs, whereas alpha 3 subunits require other "structural" subunits for functional expression of AChRs. This feature allows the dissection of the requirements for subunit interactions during AChR formation. Analysis of alpha 7/alpha 3 chimeric constructs identified two regions essential to homomeric assembly and intersubunit recognition: an N-terminal extracellular region, controlling the initial association between subunits, and a second domain within a region comprising the first putative transmembrane segment, M1, and the cytoplasmic loop coupling it to the pore-forming segment, M2, involved in the subsequent interaction and stabilization of the oligomeric complex.

Amino Acid Sequence↗
PubMed1994

A delayed rectifier potassium channel cloned from bovine adrenal medulla. Functional analysis after expression in Xenopus oocytes and in a neuroblastoma cell line.

Using a cDNA library from bovine adrenal medulla, and, subsequently, a bovine genomic library, we have isolated the gene coding for a non inactivating potassium channel. This gene encodes a 597-amino acid protein which we have called BAK5 as its sequence is very similar to members of Kv1.5 potassium channel family. Neuroblastoma cells (Neuro-2a cell line) were stably transfected with BAK5 DNA. Protein expression was under the control of a heat-shock promoter. Transfected cells showed a current highly selective for potassium, insensitive to tetraethylammonium but reversibly blocked by 4-aminopyridine. Oocytes injected with BAK5 mRNA also expressed a potassium current with the same characteristics.

4-Aminopyridine↗
PubMed1994

Sources of errors in different single-electrode voltage-clamp techniques: a computer simulation study.

The use of voltage clamp with a single electrode has been useful in estimating kinetic parameters for a number of ionic whole-cell currents. There are two main types of such a technique: discontinuous voltage clamp (dSEVC) (Brennecke and Lindemann, 1974), and continuous voltage clamp (cSEVC) (Hamill et al., 1981). We have studied, by means of computer simulations, the performance of both types of clamp on estimating activation kinetics parameters of a typical neuronal Ca2+ current. Deviations from the theoretical values are shown to be sensitive on both set-up and cell properties. Both types of clamp are shown to lose voltage control when either access resistance or absolute membrane conductance are increased. In contrast, changes in membrane capacitance affect differently to the estimates obtained by the two types of clamp. Cell size is also shown to affect cSEVC performance but not that of dSEVC. The nature and magnitude of errors obtained by using both types of clamp in different situations are discussed.

Amplifiers, Electronic↗
PubMed1994

Quantitative analysis of T wave abnormalities and their prognostic implications in the idiopathic long QT syndrome.

OBJECTIVES: We evaluated the diagnostic and prognostic value of morphologic abnormalities of the T wave (mainly notched or biphasic T waves) in patients affected by the idiopathic long QT syndrome. BACKGROUND: In the long QT syndrome, these abnormalities in T wave morphology are often observed and are of uncertain significance. METHODS: The T wave abnormalities in the electrocardiogram (ECG) of 53 patients with the long QT syndrome and 53 control subjects of similar age and gender were analyzed, and their association with major cardiac events was defined. RESULTS: Notched or biphasic T waves were defined according to morphologic criteria. They were present in 33 (62%) of 53 patients with the long QT syndrome and in 8 (15%) of 53 control subjects (p < 0.001). Moreover, among patients with the long QT syndrome they were much more frequent in symptomatic (history of syncope or cardiac arrest) than in asymptomatic subjects (30 [81%] of 37 vs. 3 [19%] of 16, p < 0.001). The same distribution was observed within families with the long QT syndrome, in which symptomatic members had more pronounced T wave abnormalities than did their asymptomatic siblings or parents. In symptomatic patients, the occurrence of T wave abnormalities was independent of the length of repolarization (corrected QT). These T wave abnormalities were associated with the presence of a specific pattern of abnormal left ventricular wall motion. CONCLUSIONS: This study has quantified an ECG pattern typical of the long QT syndrome and provides the first evidence that morphologic analysis of T wave abnormalities may contribute to the diagnosis of the long QT syndrome and the identification of patients at higher risk for syncope or cardiac arrest.

Adrenergic beta-Antagonists↗
PubMed1994

Effect of calcium channel block on the wall motion abnormality of the idiopathic long QT syndrome.

BACKGROUND: We recently showed the frequent occurrence of an unusual ventricular wall motion abnormality, assessed by echocardiography, in patients with the idiopathic long QT syndrome (LQTS). Two new quantitative indexes were developed: Th1/2 (time needed to reach half of the maximal systolic thickening), which was smaller in LQTS patients than in controls; and TSTh (time spent at a very low thickening rate before rapid relaxation), which was much greater in LQTS patients, indicating the presence of a slow contraction in the late thickening phase. This marked late systolic "plateau," either rectilinear or with a peculiar double peak pattern, was significantly more frequent in patients with a history of syncope or cardiac arrest. The mechanism underlying this puzzling phenomenon remained unexplained. METHODS AND RESULTS: The present study assessed the effects of the calcium channel blocker verapamil on the contraction pattern in 10 LQTS patients (9 females and 1 male; mean age, 19 +/- 7 years) with a marked plateau pattern and in 6 healthy controls (4 females and 2 males; mean age, 28 +/- 5 years). Either verapamil (0.1 mg/kg) or saline was randomly injected over 2 minutes. Saline had no effect. In LQTS patients, verapamil increased Th1/2 by 27%, from 16.9 +/- 3.2% to 21.4 +/- 3.9% of the cardiac cycle (P = .005), and dramatically reduced TSTh by 92%, from 13.7 +/- 5.3% to 1.08 +/- 0.6% of the cardiac cycle (P < .00001). At the peak effect of verapamil, the contraction pattern of all patients was normal. In healthy control subjects, verapamil did not significantly change either Th1/2 (from 17.6 +/- 2.5% to 18.5 +/- 3.5% of the cardiac cycle) or TSTh (from 0.92 +/- 0.47% to 1.17 +/- 0.74%). CONCLUSIONS: This study demonstrates that the wall motion abnormality of LQTS is completely abolished by verapamil. These results suggest that symptomatic LQTS patients may have an abnormal increase in the intracellular calcium concentration before relaxation has completed, possibly linked to an early afterdepolarization, and that the contraction abnormality may be the mechanical equivalent of an early afterdepolarization.

Adult↗