Biomedical subjects

E Stefani

Publications and source records attributed to E Stefani.

At least 19 recordsLinked to original sources

PubMed2001

Remodeling of Kv4.3 potassium channel gene expression under the control of sex hormones.

Kv4.3 channels are important molecular components of transient K(+) currents (Ito currents) in brain and heart. They are involved in setting the frequency of neuronal firing and heart pacing. Altered Kv4.3 channel expression has been demonstrated under pathological conditions like heart failure indicating their critical role in heart function. Thyroid hormone studies suggest that their expression in the heart may be hormonally regulated. To explore the possibility that sex hormones control Kv4.3 expression, we investigated whether its expression changes in the pregnant uterus. This organ represents a unique model to study Ito currents, because it possesses this type of K(+) current and undergoes dramatic changes in function and excitability during pregnancy. We cloned Kv4.3 channel from myometrium and found that its protein and transcript expression is greatly diminished during pregnancy. Experiments in ovariectomized rats demonstrate that estrogen is one mechanism responsible for the dramatic reduction in Kv4.3 expression and function prior to parturition. Furthermore, the reduction of plasma membrane Kv4.3 protein is accompanied by a perinuclear localization suggesting that cell trafficking is also controlled by sex hormones. Thus, estrogen remodels the expression of Kv4.3 in myometrium by directly diminishing its transcription and, indirectly, by altering Kv4.3 delivery to the plasma membrane.

Animals↗

PubMed2001

Activation of Trp3 by inositol 1,4,5-trisphosphate receptors through displacement of inhibitory calmodulin from a common binding domain.

Mammalian homologues of Drosophila Trp form plasma membrane channels that mediate Ca(2+) influx in response to activation of phospholipase C and internal Ca(2+) store depletion. Previous studies showed that human Trp3 is activated by inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and identified interacting domains, one on Trp and two on IP(3)R. We now find that Trp3 binds Ca(2+)-calmodulin (Ca(2+)/CaM) at a site that overlaps with the IP(3)R binding domain. Using patch-clamp recordings from inside-out patches, we further show that Trp3 has a high intrinsic activity that is suppressed by Ca(2+)/CaM under resting conditions, and that Trp3 is activated by the following: a Trp-binding peptide from IP(3)R that displaces CaM from Trp3, a myosin light chain kinase Ca(2+)/CaM binding peptide that prevents CaM from binding to Trp3, and calmidazolium, an inactivator of Ca(2+)/CaM. We conclude that inhibition of the inhibitory action of CaM is a key step of Trp3 channel activation by IP(3)Rs.

Amino Acid Sequence↗

PubMed2001

A novel MaxiK splice variant exhibits dominant-negative properties for surface expression.

We identified a novel MaxiK alpha subunit splice variant (SV1) from rat myometrium that is also present in brain. SV1 has a 33-amino acid insert in the S1 transmembrane domain that does not alter S1 overall hydrophobicity, but makes the S0-S1 linker longer. SV1 was transfected in HEK293T cells and studied using immunocytochemistry and electrophysiology. In non-permeabilized cells, N-terminal c-Myc- or C-terminal green fluorescent protein-tagged SV1 displayed no surface labeling or currents. The lack of SV1 functional expression was due to endoplasmic reticulum (ER) retention as determined by colabeling experiments with a specific ER marker. To explore the functional role of SV1, we coexpressed SV1 with the alpha (human SLO) and beta1 (KCNMB1) subunits of the MaxiK channel. Coexpression of SV1 inhibited surface expression of alpha and beta1 subunits approximately 80% by trapping them in the ER. This inhibition seems to be specific for MaxiK channel subunits since SV1 was unable to prevent surface expression of the Kv4.3 channel or to interact with green fluorescent protein. These results indicate a dominant-negative role of SV1 in MaxiK channel expression. Moreover, they reveal down-regulation by splice variants as a new mechanism that may contribute to the diverse levels of MaxiK channel expression in non-excitable and excitable cells.

Alternative Splicing↗

PubMed2001

Decreased expression of voltage- and Ca(2+)-activated K(+) channels in coronary smooth muscle during aging.

Aging is the main risk factor for coronary artery disease. One characteristic of aging coronary arteries is their enhanced contractile responses to endothelial vasoconstricting factors, which increase the risk of coronary vasospasm in older people. Because large-conductance voltage- and Ca(2+)-activated K(+) channels (MaxiK) are key regulators of vascular tone, we explored the possibility that this class of channels is diminished with increasing age. Using site-directed antibodies recognizing the pore-forming alpha subunit and electrophysiological methods, we demonstrate that the number of MaxiK channels is dramatically diminished in aged coronary arteries from old F344 rats. Channel density was reduced from 52+/-9 channels/pF (3 months old) to 18+/-5 channels/pF (25 to 30 months old), which represents a 65% reduction in the older population. Pixel intensity of Western blots was also diminished by approximately 50%. Moreover, the age-related decrease in the channel protein expression was also evident in humans, which showed approximately 80% reduction in 61- to 70-year-old subjects compared with 3- to 18-year-old youngsters and approximately 45% reduction compared with 19- to 56-year-old adults. In agreement with a reduction of MaxiK channel numbers in aging coronary arteries, old coronary arteries from F344 rats contract less effectively ( approximately 70% reduction) than young coronary arteries when exposed to the MaxiK channel blocker iberiotoxin. The contraction studies indicate that under physiological conditions, MaxiK channels are tonically active, serving as a hyperpolarizing force that opposes contraction. Thus, reduced expression of MaxiK channels in aged coronary arteries would lead to a decreased vasodilating capacity and increased risk of coronary spasm and myocardial ischemia in older people.

Adolescent↗

PubMed2001

A conducting state with properties of a slow inactivated state in a shaker K(+) channel mutant.

In Shaker K(+) channel, the amino terminus deletion Delta6-46 removes fast inactivation (N-type) unmasking a slow inactivation process. In Shaker Delta6-46 (Sh-IR) background, two additional mutations (T449V-I470C) remove slow inactivation, producing a noninactivating channel. However, despite the fact that Sh-IR-T449V-I470C mutant channels remain conductive, prolonged depolarizations (1 min, 0 mV) produce a shift of the QV curve by about -30 mV, suggesting that the channels still undergo the conformational changes typical of slow inactivation. For depolarizations longer than 50 ms, the tail currents measured during repolarization to -90 mV display a slow component that increases in amplitude as the duration of the depolarizing pulse increases. We found that the slow development of the QV shift had a counterpart in the amplitude of the slow component of the ionic tail current that is not present in Sh-IR. During long depolarizations, the time course of both the increase in the slow component of the tail current and the change in voltage dependence of the charge movement could be well fitted by exponential functions with identical time constant of 459 ms. Single channel recordings revealed that after prolonged depolarizations, the channels remain conductive for long periods after membrane repolarization. Nonstationary autocovariance analysis performed on macroscopic current in the T449V-I470C mutant confirmed that a novel open state appears with increasing prepulse depolarization time. These observations suggest that in the mutant studied, a new open state becomes progressively populated during long depolarizations (>50 ms). An appealing interpretation of these results is that the new open state of the mutant channel corresponds to a slow inactivated state of Sh-IR that became conductive.

Animals↗

PubMed2000

Expression of the alpha(2)delta subunit interferes with prepulse facilitation in cardiac L-type calcium channels.

We investigated the role of the accessory alpha(2)delta subunit on the voltage-dependent facilitation of cardiac L-type Ca(2+) channels (alpha(1C)). alpha(1C) Channels were coexpressed in Xenopus oocytes with beta(3) and alpha(2)delta calcium channel subunits. In alpha(1C) + beta(3), the amplitude of the ionic current (measured during pulses to 10 mV) was in average approximately 1.9-fold larger after the application of a 200-ms prepulse to +80 mV. This phenomenon, commonly referred to as voltage-dependent facilitation, was not observed when alpha(2)delta was coexpressed with alpha(1C) + beta(3). In alpha(1C) + beta(3), the prepulse produced a left shift ( approximately 40 mV) of the activation curve. Instead, the activation curve for alpha(1C) + beta(3) + alpha(2)delta was minimally affected by the prepulse and had a voltage dependence very similar to the G-V curve of the alpha(1C) + beta(3) channel facilitated by the prepulse. Coexpression of alpha(2)delta with alpha(1C) + beta(3) seems to mimic the prepulse effect by shifting the activation curve toward more negative potentials, leaving little room for facilitation. The facilitation of alpha(1C) + beta(3) was associated with an increase of the charge movement. In the presence of alpha(2)delta, the charge remained unaffected after the prepulse. Coexpression of alpha(2)delta seems to set all the channels in a conformational state from where the open state can be easily reached, even without prepulse.

Animals↗

PubMed1999

Hormonal control of protein expression and mRNA levels of the MaxiK channel alpha subunit in myometrium.

Large conductance voltage-dependent and Ca(2+)-modulated K(+) channels play a crucial role in myometrium contractility. Western blots and immunocytochemistry of rat uterine sections or isolated cells show that MaxiK channel protein signals drastically decrease towards the end of pregnancy. Consistent with a transcriptional regulation of channel expression, mRNA levels quantified with the ribonuclease protection assay correlated well with MaxiK protein levels. As a control, Na(+)/K(+)-ATPase protein and RNA levels do not significantly change at different stages of pregnancy. The low numbers of MaxiK channels at the end of pregnancy may facilitate uterine contraction needed for parturition.

Animals↗

PubMed1999

Complete reversal of run-down in rabbit cardiac Ca2+ channels by patch-cramming in Xenopus oocytes; partial reversal by protein kinase A.

The rabbit cardiac Ca2+ channel (alpha1C) expressed in Xenopus oocytes exhibited a complete run-down of ionic currents when cell-attached patches were excised. The alpha1C channel was expressed alone or was coexpressed with the accessory beta2a or beta1b subunit. The catalytic subunit of protein kinase A (PKAc) and MgATP were capable of delaying the run-down of single-channel currents. In 33% of the alpha1C patches, and 26% of the alpha1C+beta2a patches, inclusion of PKAc in the bath solution delayed the run-down for a maximum of 20 min. In experiments where PKAc in the bath was not sufficient to delay the run-down of channel activity, insertion of the patch back into the oocyte (patch-cramming) could restore channel activity. Gating currents were also measured in the alpha1C+beta1b channel and were not subject to any run-down, even after the complete run-down of ionic currents. The results presented here reveal that PKAc is capable of delaying the run-down of currents in a subset of patches. The patch-cramming results suggest that a cytoplasmic factor, in addition to phosphorylation of the channel (by PKAc), may be involved in the maintenance of channel activity.

Adenosine Triphosphate↗

PubMed1999

Substance P release in the dorsal horn assessed by receptor internalization: NMDA receptors counteract a tonic inhibition by GABA(B) receptors.

Inhibitory amino acids have antinociceptive actions in the spinal cord that may involve inhibition of neurotransmitter release from primary afferents. Rat spinal cord slices with dorsal roots were used to study the effect of GABA and glycine on substance P release, assessed by the internalization of neurokinin 1 receptors. After electrical stimulation of the dorsal root at 100 Hz, about half of neurokinin 1 receptor-immunoreactive neurons in laminae I-IIo showed internalization. This internalization was inhibited by GABA (100 microM) and the GABA(B) agonist R-baclofen (10 microM), but not by the GABA(A) agonist muscimol (20 microM) or glycine (100 microM). The GABA(B) antagonist 2-hydroxysaclofen (100 microM) reversed the inhibitory effect of GABA, but not the GABA(A) antagonist bicuculline (100 microM). These findings demonstrate that GABA(B) receptors, but not GABA(A) or glycine receptors, inhibit substance P release induced by dorsal root stimulation. In contrast, R-baclofen did not inhibit the internalization produced by NMDA (100 microM), indicating that the stimulatory effect of NMDA receptors on substance P release is able to surmount the inhibitory effect of GABA(B) receptors. In the presence of the GABA(B) antagonist 2-hydroxysaclofen (100 microM), but not in its absence, stimulation of the dorsal root at 1 or 10 Hz was able to elicit internalization, which was not inhibited by the NMDA receptor antagonist AP-5 (50 microM) or the channel blocker MK-801 (10 microM). Therefore, inhibition of substance P release by GABA(B) receptors is tonic, and in its absence SP release no longer requires NMDA receptor activation.

Animals↗

PubMed1998

Role of the S4 segment in a voltage-dependent calcium-sensitive potassium (hSlo) channel.

We investigated the role of individual charged residues of the S4 region of a MaxiK channel (hSlo) in channel gating. We measured macroscopic currents induced by wild type (WT) and point mutants of hSlo in inside-out membrane patches of Xenopus laevis oocytes. Of all the residues tested, only neutralizations of Arg-210 and Arg-213 were associated with a reduction in the number of gating charges as determined using the limiting slope method. Channel activation in WT and mutant channels was interpreted using an allosteric model. Mutations R207Q, R207E, and R210N facilitated channel opening in the absence of Ca2+; however, this facilitation was not observed in the channels Ca2+-bound state. Mutation R213Q behaved similarly to the WT channel in the absence of Ca2+, but Ca2+ was unable to stabilize the open state to the same extent as it does in the WT. Mutations R207Q, R207E, R210N, and R213Q reduced the coupling between Ca2+ binding and channel opening when compared with the WT. Mutations L204R, L204H, Q216R, E219Q, and E219K in the S4 domain showed a similar phenotype to the WT channel. We conclude that the S4 region in the hSlo channel is part of the voltage sensor and that only two charged amino acid residues in this region (Arg-210 and Arg-213) contribute to the gating valence of the channel.

Amino Acid Sequence↗

PubMed1998

Pore accessibility during C-type inactivation in Shaker K+ channels.

Shaker K+ channels inactivate through two distinct molecular mechanisms: N-type, which involves the N-terminal domain and C-type that appears to involve structural modifications at the external mouth of the channel. We have tested pore accessibility of the Shaker K+ channel during C-type inactivation using Ba2+ as a probe. We determined that external Ba2+ binds to C-type inactivated channels forming an extremely stable complex; i.e. there is Ba2+ trapping by C-type inactivated channels. The structural changes Shaker channels undergo during C-type inactivation create high energy barriers that hinder Ba2+ exit to either the extracellular solution or to the intracellular solution.

Barium↗

PubMed1998

Functional coupling between human E-type Ca2+ channels and mu opioid receptors expressed in Xenopus oocytes.

Neuronal alpha1E Ca2+ channels were expressed in Xenopus laevis oocytes alone and in combination with the mu opioid receptor. Macroscopic currents were recorded under voltage clamp conditions. The stimulation of the morphine receptor by the synthetic [D-Ala2,N-Me-Phe4,Gly-ol5] enkephalin (DAMGO) produced a 20% reduction in the alpha1E ionic current. This effect was associated with a large change in the decay phase of the Ba2+ current. The effect of 1 microM DAMGO was fully antagonized by the universal mu opioid receptor antagonist naloxone and by the selective antagonist beta-funaltrexamine. The ionic current inhibition induced by DAMGO was partially recovered by preceding strong depolarizations. The injection of the catalytic subunit of pertussis toxin (A-protomer) abolished the effect of DAMGO, suggesting the involvement of a GTP binding protein in the alpha1E modulation. The coexpression of the regulatory beta2a Ca2a channel subunit, together with the alpha1E subunit and the mu opioid receptor, prevented the reduction of the ionic current following the receptor stimulation with DAMGO, whereas the coexpression with the beta3 subunit reduced by approximately 50% the modulatory effect of DAMGO. The effect produced by the stimulation of the opioid receptor could be mimicked by coexpressing the alpha1E channel with the G-protein betagamma subunits.

Analgesics, Opioid↗

PubMed1998

Unique regulatory properties of the type 2a Ca2+ channel beta subunit caused by palmitoylation.

Beta subunits of voltage-gated Ca2+ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for beta2a, which differs in that it does not support prepulse facilitation of alpha1C Ca2+ channels, inhibits voltage-induced inactivation of neuronal alpha1E Ca2+ channels, and is more effective in blocking inhibition of alpha1E channels by G protein-coupled receptors. We show that the distinguishing properties of beta2a, rather than interaction with a distinct site of alpha1, are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of beta2a were lost in a mutant that could not be palmitoylated [beta2a(Cys3,4Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three beta2 splice variants differing only in their N-termini.

Amino Acid Sequence↗

PubMed1998

Long lasting facilitation of the rabbit cardiac Ca2+ channel: correlation with the coupling efficiency between charge movement and pore opening.

Facilitation of Ca2+ entry into cells enhances Ca(2+)-activated events such as transmitter release and stimulation of second messenger systems. We have found a long lasting prepulse facilitation (up to 3-fold) of the cardiac Ca2+ channel alpha1Cbeta1b that lasts for tens of seconds without altering current kinetics. The voltage- and time-dependence of the installation of facilitation was characterized as well as the time- and use-dependence of the decay of facilitation. The degree of facilitation was correlated with the coupling efficiency between the charge movement and pore opening channels that were poorly coupled prior to facilitation exhibited the largest facilitation.

Animals↗

PubMed1998

Facilitation by the beta2a subunit of pore openings in cardiac Ca2+ channels.

1. Single channel recordings were performed on the cardiac calcium channel (alpha1C) in order to study the effect of coexpression of the accessory beta2a subunit. On-cell patch clamp recordings were performed after expression of these channels in Xenopus oocytes. 2. The alpha1C subunit, when expressed alone, had similar single channel properties to native cardiac channels. Slow transitions between low and high open probability (Po) gating modes were found as well as fast gating transitions between the open and closed states. 3. Coexpression of the beta2a subunit caused changes in the fast gating during high Po mode. In this mode, open time distributions reveal at least three open states and the beta2a subunit favours the occupancy of the longest, 10-15 ms open state. No effect of the beta2a subunit was found when the channel was gating in the low Po mode. 4. Slow gating transitions were also affected by the beta2a subunit. The high Po mode was maintained for the duration of the depolarizing pulse in the presence of the beta2a subunit; while the alpha1C channel when expressed alone, frequently switched into and out of the high Po mode during the course of a sweep. 5. The beta2a subunit also affected mode switching that occurred between sweeps. Runs analysis revealed that the alpha1C subunit has a tendency toward non-random mode switching. The beta2a subunit increased this tendency. A chi2 analysis of contingency tables indicated that the beta2a subunit caused the alpha1C channel to gain 'intrinsic memory', meaning that the mode of a given sweep can be non-independent of the mode of the previous sweep. 6. We conclude that the beta2a subunit causes changes to the alpha1C channel in both its fast and slow gating behaviour. The beta2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the beta2a subunit decreases the slow switching between low and high Po modes.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1998

Ionic currents underlying HTRP3 mediated agonist-dependent Ca2+ influx in stably transfected HEK293 cells.

hTrp3 is a human homologue of the Drosophila gene responsible for a transient receptor potential (trp) mutation. When stably expressed in HEK293 cells, hTrp3 formed ion channels that were active under resting conditions but could be further stimulated by carbachol or ATP via endogenous muscarinic or purinergic receptors, respectively. Agonist evoked currents reversed polarity near 0 mV in physiological ionic conditions and were associated with a significant increase in the current variance. These results suggest the involvement of a non-selective cation channel with relatively large unitary amplitude. Consistent with this, resolved unitary events had a conductance of approximately 60 pS in the negative voltage range and an extrapolated reversal potential near 0 mV.

Adenosine Triphosphate↗

PubMed1998

Cut-open oocyte voltage-clamp technique.

In this article, we described the use and limitations of the COVG technique. The advantages of the present method as follows: 1. High-frequency response and low noise recording (24-microseconds time constant and 1.2-nA rms at 5 kHz). This allows the accurate description of small gating currents and fast activation or deactivation of ionic currents to be adequately resolved. Currents up to 20-30 microA can be adequately clamped. 2. Stable recording conditions lasting for several hours. Even though this technique has internal access, rundown is minimized compared to excised patches. 3. Access to the cell interior. The intracellular medium can be exchanged with various solutions. This was of invaluable help in recording K+ gating currents, an experimental condition that required the complete blockade of all ionic currents. This advantage will make the present method suitable for the study of channel modulation by second messengers and drugs. In addition, channel selectivity properties can be defined by ion substitution experiments. 4. Channel localization. A study of the membrane compartmentalization of channels can be easily obtained with this method, since it allows the voltage clamping of different regions of the oocyte surface.

Animals↗

PubMed1998

Gating currents.

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Animals↗