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A P Moreno

Publications and source records attributed to A P Moreno.

At least 19 recordsLinked to original sources
PubMed2003

LacSwitch II regulation of connexin43 cDNA expression enables gap-junction single-channel analysis.

Metabolic and electrical coupling through gap junction channels is implicated in cell differentiation, tissue homeostasis, and electrotonic propagation of signals in excitable tissues. The characterization of gating properties of these channels requires electrophysiological recordings at both single- and multiple-channel levels. Hence, a system that is able to control connexin expression by external means would provide a useful tool. To regulate the expression of connexins in cells, plasmids encoding a transactivator and/or a lac-operon IPTG response-dependent Cx43 target gene were transfected into communication-deficient N2a neuroblastoma cells. Immunoblotting, dye coupling, and electrophysiological methods revealed that expression of Cx43 in selected clones could be tightly regulated. After 15-20 h of acute induction with IPTG, cell-to-cell communication reached its peak with junctional conductances of 15-30 nS. Chronic induction at specific doses of IPTG produced constant, controlled levels of Cx43 expression, which were reflected by predictable junctional coupling levels. These conditions allowed prolonged recordings from either lowly or highly coupled cells, making lac operon an ideal regulatory system for channel gating studies at a single-channel level.

Animals↗
PubMed2001

The carboxyl terminal domain regulates the unitary conductance and voltage dependence of connexin40 gap junction channels.

Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain model, in which the carboxyl terminal (CT) domain acts as a gating particle that binds to a receptor affiliated with the pore. Moreover, Cx40 channels can be closed by a heterodomain interaction with the CT domain of Cx43 and vice versa. Here, we report similar interactions in the establishment of the unitary conductance and voltage-dependent profile of Cx40 in N2A cells. Two mean unitary conductance values ("lower conductance" and "main") were detected in wild-type Cx40. Truncation of the CT domain at amino acid 248 (Cx40tr248) caused the disappearance of the lower-conductance state. Coexpression of Cx40tr248 with the CT fragment of either Cx40 (homodomain interactions) or Cx43 (heterodomain interactions) rescued the unitary conductance profile of Cx40. In the N2A cells, the time course of macroscopic junctional current relaxation was best described by a biexponential function in the wild-type Cx40 channels, but it was reduced to a single-exponential function after truncation. However, macroscopic junctional currents recorded in the oocyte expression system were not significantly different between the wild-type and mutant channels. Concatenation of the CT domain of Cx43 to amino acids 1 to 248 of Cx40 yielded a chimeric channel with unitary conductance and voltage-gating profile indistinguishable from that of wild-type Cx40. We conclude that residence of Cx40 channels in the lower-conductance state involves a ball-and-chain type of interaction between the CT domain and the pore-forming region. This interaction can be either homologous (Cx40 truncation with Cx40CT) or heterologous (with the Cx43CT).

Animals↗
PubMed2001

Heterotypic docking of Cx43 and Cx45 connexons blocks fast voltage gating of Cx43.

Immunohistochemical co-localization of distinct connexins (Cxs) in junctional areas suggests the formation of heteromultimeric channels. To determine the docking effects of the heterotypic combination of Cx43 and Cx45 on the voltage-gating properties of their channels, we transfected DNA encoding Cx43 or Cx45 into N2A neuroblastoma or HeLa cells. Using a double whole-cell voltage-clamp technique, we determined macroscopic and single-channel gating properties of the intercellular channels formed. Cx43-Cx45 heterotypic channels had rectifying properties where Cx45 connexons inactivated rapidly upon hyperpolarizing voltage pulses applied to the Cx45-expressing cell. During depolarizing pulses to the Cx45-expressing cell, Cx43 connexons inactivated with substantially reduced kinetics as compared with homotypic Cx43 channels. Similar slow kinetics was observed for homotypic Cx43M257 (truncation mutant). Heterotypic channels had a main conductance whose value was predicted by the sum of corresponding homomeric connexon conductances; it was not voltage dependent and had no detectable residual conductance. The voltage-gating kinetics of heterotypic channels and their single-channel behavior implicate a role for the Cx43 carboxyl-terminal domain in the fast gating mechanism and in the establishment of residual conductance. Our results also suggest that heterotypic docking may lead to conformational changes that inhibit this action of the Cx43 carboxyl-terminal domain.

Animals↗
PubMed2001

Opposing gates model for voltage gating of gap junction channels.

Gap junctions are intercellular channels that link the cytoplasm of neighboring cells. Because a gap junction channel is composed of two connexons docking head-to-head with each other, the channel voltage-gating profile is symmetrical for homotypic channels made of two identical connexons (hemichannels) and asymmetric for the heterotypic channels made of two different connexons (i.e., different connexin composition). In this study we have developed a gating model that allows quantitative characterization of the voltage gating of homotypic and heterotypic channels. This model differs from the present model in use by integrating, rather than separating, the contributions of the voltage gates of the two member connexons. The gating profile can now be fitted over the entire voltage range, eliminating the previous need for data splicing and fusion of two hemichannel descriptions, which is problematic when dealing with heterotypic channels. This model also provides a practical formula to render quantitative several previously qualitative concepts, including a similarity principle for matching a voltage gate to its host connexon, assignment of gating polarity to a connexon, and the effect of docking interactions between two member connexons in an intact gap junction channel.

Algorithms↗
PubMed2001

Heteromeric mixing of connexins: compatibility of partners and functional consequences.

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.

Animals↗
PubMed1999

Junctional communication between isolated pairs of canine atrial cells is mediated by homogeneous and heterogeneous gap junction channels.

INTRODUCTION: The expression of multiple connexins (Cxs) in the canine right atria raises the possibility that heterogeneous gap junction channels might be formed. METHODS AND RESULTS: We compared the unitary conductance (gamma(j)) of gap junction channels between isolated canine atrial cell pairs with those of homogeneous cardiac gap junction channels expressed in other systems. After partial uncoupling with halothane (2 mmol/L), the (gamma)j calculations for atrial isolated cardiocytes ranged from 30 to 220 pS and their distribution in event histograms was spread over the entire range, with a small peak at approximately 100 pS. This distribution deviates from the discrete peaks calculated from (gamma)j of homogeneous channels. All-points histograms of junctional current traces revealed distinct open-state levels. Some of these are related to the main open state of connexin43 (Cx43) (approximately 100 pS), observed between canine ventricular cells, or connexin40 (Cx40) (approximately 215 pS) observed between transfected N2A cells under similar recording conditions. Intermediate values for (gamma)j were not observed in recordings from ventricular cells, which express mostly Cx43, nor in those from N2A cells expressing Cx40, but were observed consistently between atrial cells. Because they were measured as first openings from the nonconductance state, these intermediate values most likely represent main conductance states of heterogeneous channels rather than subconductance states of homogeneous channels. CONCLUSION: This suggests that regulation of cell-to-cell coupling in the heart depends not only on posttranslational modulation of preexisting Cxs, but also on the intracellular assembly mechanisms, and the way individual Cxs interact with others within a connexon and/or with other connexons from adjacent cells.

Action Potentials↗
PubMed1996

Evidence that disruption of connexon particle arrangements in gap junction plaques is associated with inhibition of gap junctional communication by a glycyrrhetinic acid derivative.

Glycyrrhetinic acid exhibits many pharmacological activities, including the ability to block gap junctional communication. However, the mechanism of glycyrrhetinic action is not clear. Others have shown that glycyrrhetinic acid apparently binds to a single proteinatious binding site in the plasma membrane. We present evidence that while exposure to glycyrrhetinic acid derivatives may not affect protein synthesis or location, it does seem to alter connexon particle packing in gap junction plaques.

Animals↗
PubMed1995

Properties of gap junction channels formed of connexin 45 endogenously expressed in human hepatoma (SKHep1) cells.

We have evaluated the voltage dependence and unitary conductance of gap junctional channels that were recorded in a clone isolated from the hepatoma cell line SKHep1. In this clonal population (designated SKHep1A), Northern blots, immunoprecipitation, and immunohistochemical staining demonstrated the expression of connexin (Cx) 45; no other gap junction protein was identified by these techniques, although weak hybridization with Cx40 was detected. Macroscopic junctional conductance (gj) in these cells was low, averaging 1.3 nS, and was steeply voltage dependent. Parameters of voltage sensitivity were as follows: voltage at which voltage-sensitive conductance is reduced by 50%, 13.4 mV; steepness of relation, 0.115 (corresponding to 2.7 gating charges), and voltage-insensitive fraction of residual to total conductance approximately 0.06. Unitary conductance (gamma j) of these junctional channels averaged 32 +/- 8 pS; although gamma j was independent of transjunctional voltage (Vj), at high Vj values (> 50 mV), smaller conductance values were also detected. Open probabilities of the 30-pS channels at various Vj values closely matched the predicted voltage-dependent component of macroscopic gj, the residual conductance at high Vj might be attributable to the smaller conductance events. The voltage dependence of human Cx45 gap junction channels is as steep as that seen for channels formed by Xenopus Cx38 and is much steeper than that previously reported for channels formed of the highly homologous chick Cx45 and for other mammalian connexins expressed either endogenously or exogenously.

Carcinoma, Hepatocellular↗
PubMed1994

Trypanosoma cruzi: effects of infection on receptor-mediated chronotropy and Ca2+ mobilization in rat cardiac myocytes.

Acute Trypanosoma cruzi infection is commonly associated with disorders of impulse conduction and muscle contraction in heart. In order to determine the extent to which receptor function changed in response to infection, infected neonatal rat cardiac myocytes in culture were compared with matched controls with regard to chronotropic response and Ca2+ mobilization following the application of adrenergic agonists. At 7-9 days in culture (5-7 days postinfection), spontaneous beat rates of control myocytes were four times as rapid as those in infected cells. Control cells responded to 10(-5) M isoproterenol (ISO) and 10(-6) M norepinephrine (NE) with increases in beat rate of 34 and 40%, respectively. Effects of ISO on infected cells were similar, and adenylate cyclase activity was similar in control and infected cells when measured in the presence of ISO alone or in combination with Gpp(NH)p. NE produced a more marked chronotropic response in infected cultures and altered Ca2+ mobilization. NE treatment increased Ca2+ levels in control cardiac myocytes from 51.8 +/- 4.4 to 113 +/- 16 nM (in 0 Ca2+ medium) and from 85.2 +/- 6.8 to 131.3 +/- 24.5 nM (1 mM external Ca2+). In infected cardiac myocytes, NE increased Ca2+ from 116.8 +/- 17 to 164.7 +/- 9.6 nM (in 0 Ca2+ medium) and from 132.2 +/- 13.2 to 162.5 +/- 0.3 nM (1 mM Ca2+ medium). Thus, basal and alpha-adrenergic-stimulated Ca2+ levels were higher in infected than uninfected myocytes regardless of the extracellular Ca2+ levels, although the fractional increase in infected myocytes was significantly lower than that in controls (1.4- and 1.2-fold vs 2.2- and 1.5-fold). Therefore, both chronotropic and Ca(2+)-mobilization responses to the alpha-adrenergic agonist NE are altered in T. cruzi-infected cardiac myocytes; the chronotropic response of similarly infected cells to the beta-adrenergic agonist ISO was not affected. These data indicating that T. cruzi infection may be associated with a dissociation in responses to these agonists suggest a possible mechanism to explain, in part, the cardiac dysfunction characteristic of Chagas' disease.

Animals↗
PubMed1994

Gap junction channels: distinct voltage-sensitive and -insensitive conductance states.

All mammalian gap junction channels are sensitive to the voltage difference imposed across the junctional membrane, and parameters of voltage sensitivity have been shown to vary according to the gap junction protein that is expressed. For connexin43, the major gap junction protein in the cardiovascular system, in the uterus, and between glial cells in brain, voltage clamp studies have shown that transjunctional voltages (Vj) exceeding +/- 50 mV reduce junctional conductance (gj). However, substantial gj remains at even very large Vj values; this residual voltage-insensitive conductance has been termed gmin. We have explored the mechanism underlying gmin using several cell types in which connexin43 is endogenously expressed as well as in communication-deficient hepatoma cells transfected with cDNA encoding human connexin43. For pairs of transfectants exhibiting series resistance-corrected maximal gj (gmax) values ranging from < 2 to > 90 nS, the ratio gmin/gmax was found to be relatively constant (about 0.4-0.5), indicating that the channels responsible for the voltage-sensitive and -insensitive components of gj are not independent. Single channel studies further revealed that different channel sizes comprise the voltage-sensitive and -insensitive components, and that the open times of the larger, more voltage-sensitive conductance events declined to values near zero at large voltages, despite the high gmin. We conclude that the voltage-insensitive component of gj is ascribable to a voltage-insensitive substate of connexin43 channels rather than to the presence of multiple types of channels in the junctional membrane. These studies thus demonstrate that for certain gap junction channels, closure in response to specific stimuli may be graded, rather than all-or-none.

Animals↗
PubMed1994

Norepinephrine induces Ca2+ release from intracellular stores in rat pinealocytes.

In rat pinealocytes, an increase in intracellular Ca2+ concentration ([Ca2+]i) due to Ca2+ influx in response to norepinephrine (NE) is a well recognized event involved in regulating several metabolic functions. Since NE also stimulates the metabolism of phosphatidyl inositols in rat pineal gland, it is conceivable that Ca2+ release from intracellular stores also contributes to the NE-induced increase in [Ca2+]i. In this communication, we report that in rat pinealocytes loaded with fura-2, a Ca2+ indicator, NE induced a transient increase in [Ca2+]i that preceded the known Ca2+ influx. This novel [Ca2+]i response to NE was detected in pinealocytes bathed with Ca2+-free saline and prevented by TMB-8, a blocker of Ca2+ release from intracellular stores, supporting the notion that the transient NE-induced Ca2+ response was due to Ca2+ release from intracellular stores. In addition, after an extended exposure to NE a new addition of this neurotransmitter did not elicit the phasic Ca2+ response, and application of increasing amounts of NE induced a Ca2+ response that was progressively smaller, suggesting desensitization. Thus, NE is proposed to increase [Ca2+]i in rat pinealocytes by two mechanisms: (1) phasic release from intracellular stores and (2) tonic influx through a mechanism activated by larger applications of NE than required to evoke the phasic release.

Animals↗
PubMed1994

Characterization of gap junctions between pairs of Leydig cells from mouse testis.

Leydig cells are coupled in vivo by numerous gap junctions. In vivo and in vitro cells were immunolabeled by connexin 43 (Cx43) but not by Cx26 or Cx32 antibodies; immunoblotting confirmed specificity of Cx43 labeling. Pairs of Leydig cells dissociated from mouse testis were studied by dual whole cell voltage clamp, and a high incidence of dye (n = 20) and electrical coupling (n = 60; > 90%) was found. Coupling coefficients were near 1 and junctional conductance (gj) averaged 7.2 +/- 1.2 nS (SE, n = 40). Large transjunctional voltage (Vj) decreased gj; currents decayed exponentially with time constants of seconds that decreased at greater Vj. The residual conductance at large Vj was at least approximately 40% of the initial conductance. Exposure of cell pairs to saline solutions saturated with CO2 (n = 15) or containing 2 mM halothane (n = 15) or 3.5 mM heptanol (n = 15) rapidly and reversibly reduced gj. In eight cell pairs, gating of single junctional channels was observed during halothane-induced reduction in gj. Most gating events at Vj < 40 mV were fit by a Gaussian distribution with a mean of approximately 100 pS. With Vj > 40 mV, smaller transitions of approximately 30 pS were also recorded, and the frequency and duration of the approximately 100-pS transitions decreased. Also, approximately 70-pS transitions between 30- and 100-pS conductances were observed in the absence of 70-pS transitions to or from the baseline, indicating that the 30-pS conductance was a substate induced by large Vj.

Alcohols↗
PubMed1994

Human connexin43 gap junction channels. Regulation of unitary conductances by phosphorylation.

Connexin43 is the major gap protein in the heart and cardiovascular system. Single channel recordings of human connexin43 gap junction channels exogenously expressed in transfected SKHep1 cells demonstrate two discrete classes of channel events, with unitary conductances of predominantly 60 to 70 and 90 to 100 pS when recorded with an internal solution containing CsCl as the major current-carrying ionic species and at moderate transjunctional voltages (< 60 mV). Human connexin43 expressed in SKHep1 cells displays multiple electrophoretic mobilities (apparent M(r), approximately 41 to 45 kD) when resolved in Western blots. Treatment of connexin43 from these cells with alkaline phosphatase collapses the bands into a single 41-kD species; application of alkaline phosphatase to the cell interior through patch pipettes yields channels that are predominantly of the larger unitary conductance. The smaller 60- to 70-pS unitary conductance values correspond to the most common channel size seen in cultured rat cardiac myocytes; these channels were more frequently observed after treatment with the phosphatase inhibitor okadaic acid, which was shown to increase phosphorylation of human connexin43 in these cells under similar conditions. Exposure to the protein kinase inhibitor staurosporine shifted the proportion of events toward the largest unitary conductance and resulted in decreased phosphorylation of human connexin43 in seryl residues in these cells. Thus, the unitary conductance of human connexin43 gap junction channels covaries with the phosphorylation state of the protein. This change in unitary conductance appears to be a unique effect of phosphorylation on gap junction channels, since it has not been observed for other ion channels that have thus far been evaluated.

Alkaloids↗
PubMed1993

Gap junctions formed of connexin43 are found between smooth muscle cells of human corpus cavernosum.

Despite sparse autonomic innervation, the smooth muscle cells of the corpus cavernosum relax and contract synchronously to achieve penile erection and flaccidity. As with other smooth muscle cell types, the excitation process in the corpora is presumably propagated through gap junctions to allow the diffusion of current-carrying ions and second messenger molecules from cell to cell. Using both molecular and immunocytochemical techniques, we have identified gap junctions between human corporal smooth muscle cells in situ and in culture. Northern analyses demonstrated that corporal smooth muscle cells express the gap junction protein connexin43, but not connexin26 mRNA. Immunoblots showed the presence of connexin43 isoforms, whereas connexin32 was not detected. Immunocytochemical studies in cultured cells identified prominent connexin43 immunoreactive puncta between cells, as well as within the cytoplasm. In addition, gap junction membranes both in situ and in culture were labelled in thin section by anti-connexin43 antibodies using the immunogold technique. We conclude that the presence and distribution of gap junctions in this sparsely innervated tissue may provide an important mechanism of intercellular communication among the smooth muscle cells, and thus play a major role in coordinating tissue contraction and relaxation.

Blotting, Northern↗
PubMed1993

Gap junctions between human corpus cavernosum smooth muscle cells: gating properties and unitary conductance.

We previously showed that corpus cavernosum smooth muscle cells are connected via gap junctions in situ and in culture and that a major protein component of these gap junctions is connexin43. To characterize the physiological properties of the gap junctions between corpus cavernosum smooth muscle cells, we now demonstrate that the cells are dye and electrically coupled and describe some of the gating properties of these gap junctional channels at macroscopic and single-channel levels. Junctional conductance (gj) between corporal smooth muscle cells was moderately voltage sensitive; was reduced rapidly, reversibly, and completely by halothane; and was increased by treatment with a tumor-promoting phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] and decreased by isoproterenol. Histograms of unitary junctional currents revealed multiple conductance peaks with events of approximately 90 pS being the most abundant. TPA and phenylephrine produced large increases in relative frequencies of the smaller events, whereas isoproterenol and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) slightly increased the relative frequencies of the larger events. None of the tested drugs substantially affected the steady-state voltage dependence of gj. These second messenger systems also affected expression of connexin43 by corpus cavernosum smooth muscle cells, as judged by immunoblots. At 6 h of treatment, both TPA- and 8-BrcAMP-treated cultures showed markedly elevated levels of connexin43, whereas at 24 h, the level of connexin43 in TPA-treated cultures had returned to control levels. Together, these data indicate that second messenger molecules involved in penile erection produce changes in gap junction expression and function; it is plausible that these changes could be physiologically relevant in altering and propagating changes in vasomotor tone.

Cell Communication↗
PubMed1993

Biophysical properties of the human cardiac gap junction channel.

1. Gap junction channels interconnect cells of the pacemaking, conduction and contraction elements of the heart and also endothelial and smooth muscle cells of vasculature, thereby providing pathways for electrotonic current spread and for second messenger diffusion. The major gap junction protein in the cardiovascular system is connexin43. 2. When human connexin43 is stably expressed in pairs of a communication-deficient cell line (SKHep1) channels are produced with unitary conductance (gamma j), lipophile sensitivity and voltage-dependent gating similar to those of mammalian systems in which connexin43 is endogenously expressed. 3. At moderate transjunctional voltages (Vj), two gamma j values dominated the recordings, about 60 and 90 pS with CsCl patch solution. The smaller channel size is favored by phosphorylating treatments and the larger channel, by dephosphorylating treatments. 4. Human connexin43 mutants truncated at the carboxy termini display a change in gamma j while a point mutation in the third transmembrane spanning domain appears to change channel selectivity. 5. Voltage dependence of the human connexin43 channel is marked at Vjs, above +/- 50 mV, but large residual conductance remains (due probably to a voltage-insensitive substate) even at the largest Vj values; kinetic but not steady-state behavior is affected by phosphorylation state.

Animals↗