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P Baldelli

Publications and source records attributed to P Baldelli.

At least 19 recordsLinked to original sources
PubMed2006

Evaluation of the minimum iodine concentration for contrast-enhanced subtraction mammography.

Early manifestation of breast cancer is often very subtle and is displayed in a complex and variable pattern of normal anatomy that may obscure the disease. The use of dual-energy techniques, that can remove the structural noise, and contrast media, that enhance the region surrounding the tumour, could help us to improve the detectability of the lesions. The aim of this work is to investigate the use of an iodine-based contrast medium in mammography with two different double exposure techniques: K-edge subtraction mammography and temporal subtraction mammography. Both techniques have been investigated by using an ideal source, like monochromatic beams produced at a synchrotron radiation facility and a clinical digital mammography system. A dedicated three-component phantom containing cavities filled with different iodine concentrations has been developed and used for measurements. For each technique, information about the minimum iodine concentration, which provides a significant enhancement of the detectability of the pathology by minimizing the risk due to high dose and high concentration of contrast medium, has been obtained. In particular, for cavities of 5 and 8 mm in diameter filled with iodine solutions, the minimum concentration needed to obtain a contrast-to-noise ratio of 5 with a mean glandular dose of 2 mGy has been calculated. The minimum concentrations estimated with monochromatic beams and K-edge subtraction mammography are 0.9 mg ml(-1) and 1.34 mg ml(-1) for the biggest and smallest details, respectively, while for temporal subtraction mammography they are 0.84 mg ml(-1) and 1.31 mg ml(-1). With the conventional clinical system the minimum concentrations for the K-edge subtraction mammography are 4.13 mg ml(-1) (8 mm diameter) and 5.75 mg ml(-1) (5 mm diameter), while for the temporal subtraction mammography they are 1.01 mg ml(-1) (8 mm diameter) and 1.57 mg ml(-1) (5 mm diameter).

Algorithms↗
PubMed2005

Low-threshold exocytosis induced by cAMP-recruited CaV3.2 (alpha1H) channels in rat chromaffin cells.

We have studied the functional role of CaV3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). CaV3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (-50, -40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 microM Ni2+, which selectively blocks T-type currents in RCCs. This suggests that the "low-threshold exocytosis" induced by cAMP is due to increased Ca2+ influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca2+ entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca2+ dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited CaV3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca2+ efficacy similar to that of already existing high-threshold Ca2+ channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca2+ current component recruited by cAMP is selectively associated to the alpha1H (CaV3.2) channel isoform.

Animals↗
PubMed2005

A prototype of a quasi-monochromatic system for mammography applications.

Improvement in image contrast and dose reduction, in mammographic x-ray imaging, can be achieved using narrow energy band x-ray beams in the 16-24 keV range. As part of an Italian Government funded project, a quasi-monochromatic system for mammography applications has been developed. The system is based on a tunable narrow energy band x-ray source operating in the 16-24 keV energy range. The bremsstrahlung beam is monochromatized via Bragg diffraction by a highly oriented pyrolytic graphite mosaic crystal (HOPG). The scanning system provides a large field (18 x 24 cm2) of quasi-monochromatic x-rays with energy resolution ranging from 10% at 18 keV to 17.2% at 24 keV. The system has been characterized in terms of fluence rate and energy resolution. An x-ray tube developed ad hoc allows us to acquire images in a reasonable time to minimize the motion blur. A qualitative analysis has been performed in order to know if the prototype system performances are far from a clinical application, by evaluating the spatial resolution, the field uniformity and the image quality as a function of the quasi-monochromatic beam energy. Dose evaluation has been performed as a function of the energy and compared to a conventional system for mammography. The quasi-monochromatic prototype system can produce comparable image quality at half the dose.

Equipment Design↗
PubMed2004

Dose comparison between conventional and quasi-monochromatic systems for diagnostic radiology.

Several techniques have been introduced in the last year to reduce the dose to the patient by minimizing the risk of tumour induced by radiation. In this work the radiological potential of dose reduction in quasi-monochromatic spectra produced via mosaic crystal Bragg diffraction has been evaluated, and a comparison with conventional spectra has been performed for four standard examinations: head, chest, abdomen and lumbar sacral spine. We have simulated quasi-monochromatic x-rays with the Shadow code, and conventional spectra with the Spectrum Processor. By means of the PCXMC software, we have simulated four examinations according to parameters established by the European Guidelines, and calculated absorbed dose for principal organs and the effective dose. Simulations of quasi-monochromatic laminar beams have been performed without anti-scatter grid, because of their inherent scatter geometry, and compared with simulations with conventional beams with anti-scatter grids. Results have shown that the dose reduction due to the introduction of quasi-monochromatic x-rays depends on different parameters related to the quality of the beam, the organ composition and the anti-scatter grid. With parameters chosen in this study a significant dose reduction can be achieved for two out of four kinds of examination.

Algorithms↗
PubMed2004

Exposure to cAMP and beta-adrenergic stimulation recruits Ca(V)3 T-type channels in rat chromaffin cells through Epac cAMP-receptor proteins.

T-type channels are expressed weakly or not at all in adult rat chromaffin cells (RCCs) and there is contrasting evidence as to whether they play a functional role in catecholamine secretion. Here we show that 3-5 days after application of pCPT-cAMP, most RCCs grown in serum-free medium expressed a high density of low-voltage-activated T-type channels without altering the expression and characteristics of high-voltage-activated channels. The density of cAMP-recruited T-type channels increased with time and displayed the typical biophysical and pharmacological properties of low-voltage-activated Ca(2+) channels: (1) steep voltage-dependent activation from -50 mV in 10 mm Ca(2+), (2) slow deactivation but fast and complete inactivation, (3) full inactivation following short conditioning prepulses to -30 mV, (4) effective block of Ca(2+) influx with 50 microM Ni(2+), (5) comparable permeability to Ca(2+) and Ba(2+), and (6) insensitivity to common Ca(2+) channel antagonists. The action of exogenous pCPT-cAMP (200 microM) was prevented by the protein synthesis inhibitor anisomycin and mimicked in most cells by exposure to forskolin and 1-methyl-3-isobutylxanthine (IBMX) or isoprenaline. The protein kinase A (PKA) inhibitor H89 (0.3 microM) and the competitive antagonist of cAMP binding to PKA, Rp-cAMPS, had weak or no effect on the action of pCPT-cAMP. In line with this, the selective Epac agonist 8CPT-2Me-cAMP nicely mimicked the action of pCPT-cAMP and isoprenaline, suggesting the existence of a dominant Epac-dependent recruitment of T-type channels in RCCs that may originate from the activation of beta-adrenoceptors. Stimulation of beta-adrenoceptors occurs autocrinally in RCCs and thus, the neosynthesis of low-voltage-activated channels may represent a new form of 'chromaffin cell plasticity', which contributes, by lowering the threshold of action potential firing, to increasing cell excitability and secretory activity during sustained sympathetic stimulation and/or increased catecholamine circulation.

Action Potentials↗
PubMed2003

Quasi-monochromatic x-rays for diagnostic radiology.

Monochromatic x-ray beams are desirable in various fields of diagnostic radiology; in fact a reduction of the dose and an enhancement of the contrast could be achieved. In this work two different methods to monochromatize x-ray beams produced by conventional tubes have been compared. In the first one the beam is obtained via Bragg diffraction on mosaic crystal and in the second one by attenuating the polychromatic beam with aluminium filters. We have simulated quasi-monochromatic x-ray spectra by setting suitable values of Bragg's angle to obtain beams tuned to 20, 30, 40 and 50 keV with the SHADOW code, an x-ray tracing program designed to study the propagation and the interaction of a photon beam through an optical system. We have validated such a program by comparing some calculated data with measurements carried out on an experimental apparatus. Attenuated polychromatic x-ray spectra have been simulated by setting appropriate values of aluminium filters and potential with the SPECTRUM PROCESSOR, the software version of the Catalogue of Spectral Data for Diagnostic X-Rays, which provides radiographic x-ray spectra that can be attenuated with several material filters. The relation between the energy resolution and the flux as a function of the mean energy has been investigated and results have been compared. Results show that quasi-monochromatic x-ray beams produced via Bragg diffraction exhibit, for a given mean energy and energy resolution, a higher total flux compared to attenuated spectra.

Scattering, Radiation↗
PubMed2003

Dual-energy imaging in full-field digital mammography: a phantom study.

A dual-energy technique which employs the basis decomposition method is being investigated for application to digital mammography. A three-component phantom, made up of plexiglas, polyethylene, and water, was doubly exposed with the full-field digital mammography system manufactured by General Electric. The 'low' and 'high' energy images were recorded with a Mo/Mo anode-filter combination and a Rh/Rh combination, respectively. The total dose was kept within the acceptable levels of conventional mammography. The first hybrid images obtained with the dual-energy algorithm are presented in comparison with a conventional radiograph of the phantom. Image-quality characteristics at contrast cancellation angles between plexiglas and water are discussed. Preliminary results show that a combination of a standard Mo-anode 28 kV radiograph with a Rh-anode 49 kV radiograph provides the best compromise between image-quality and dose in the hybrid image.

Algorithms↗
PubMed2003

Opposite action of beta1- and beta2-adrenergic receptors on Ca(V)1 L-channel current in rat adrenal chromaffin cells.

Voltage-gated Ca(2+) channels of chromaffin cells are modulated by locally released neurotransmitters through autoreceptor-activated G-proteins. Clear evidence exists in favor of a Ca(2+) channel gating inhibition mediated by purinergic, opioidergic, and alpha-adrenergic autoreceptors. Few and contradictory data suggest also a role of beta-adrenergic autoreceptors (beta-ARs), the action of which, however, remains obscure. Here, using patch-perforated recordings, we show that rat chromaffin cells respond to the beta-AR agonist isoprenaline (ISO) by either upmodulating or downmodulating the amplitude of Ca(2+) currents through two distinct modulatory pathways. ISO (1 microm) could cause either fast inhibition (approximately 25%) or slow potentiation (approximately 25%), or a combination of the two actions. Both effects were completely prevented by propranolol. Slow potentiation was more evident in cells pretreated with pertussis toxin (PTX) or when beta(1)-ARs were selectively stimulated with ISO + ICI118,551. Potentiation was absent when the beta(2)-AR-selective agonist zinterol (1 microm), the protein kinase A (PKA) inhibitor H89, or nifedipine was applied, suggesting that potentiation is associated with a PKA-mediated phosphorylation of L-channels (approximately 40% L-current increase) through beta(1)-ARs. The ISO-induced inhibition was fast and reversible, preserved in cell treated with H89, and mimicked by zinterol. The action of zinterol was mostly on L-channels (38% inhibition). Zinterol action preserved the channel activation kinetics, the voltage-dependence of the I-V characteristic, and was removed by PTX, suggesting that beta(2)AR-mediated channel inhibition was mainly voltage independent and coupled to G(i)/G(o)-proteins. Sequential application of zinterol and ISO mimicked the dual action (inhibition/potentiation) of ISO alone. The two kinetically and pharmacologically distinct beta-ARs signaling uncover alternative pathways, which may serve the autocrine control of Ca(2+)-dependent exocytosis and other related functions of rat chromaffin cells.

Adrenal Glands↗
PubMed2003

Distinct potentiation of L-type currents and secretion by cAMP in rat chromaffin cells.

We have investigated the potentiating action of cAMP on L-currents of rat chromaffin cells and the corresponding increase of Ca(2+)-evoked secretory responses with the aim of separating the action of cAMP on Ca(2+) entry through L-channels and the downstream effects of cAMP/protein kinase A (PKA) on exocytosis. In omega-toxin-treated rat chromaffin cells, exposure to the permeable cAMP analog 8-(4-chlorophenylthio)-adenosine 3',5'-monophosphate (pCPT-cAMP; 1 mM, 30 min) caused a moderate increase of Ca(2+) charge carried through L-channels (19% in 10 mM Ca(2+) at +10 mV) and a drastic potentiation of secretion ( approximately 100%), measured as membrane capacitance increments (deltaC). The apparent Ca(2+) dependency of exocytosis increased with pCPT-cAMP and was accompanied by 83% enhancement of the readily releasable pool of vesicles with no significant change of the probability of release, as evaluated with paired-pulse stimulation protocols. pCPT-cAMP effects could be mimicked by stimulation of beta(1)-adrenoreceptors and reversed by the PKA inhibitor H89, suggesting strict PKA dependence. For short pulses to +10 mV (100 ms), potentiation of exocytosis by pCPT-cAMP was proportional to the quantity of charge entering the cell and occurred independently of whether L, N, or P/Q channels were blocked, suggesting that cAMP acts as a constant amplification factor for secretion regardless of the channel type carrying Ca(2+). Analysis of statistical variations among depolarization-induced capacitance increments indicates that pCPT-cAMP acts downstream of Ca(2+) entry by almost doubling the mean size of unitary exocytic events, most likely as a consequence of an increased granule-to-granule rather than a granule-to-membrane fusion.

Adaptation, Physiological↗
PubMed2002

BDNF up-regulates evoked GABAergic transmission in developing hippocampus by potentiating presynaptic N- and P/Q-type Ca2+ channels signalling.

Chronic application of brain-derived neurotrophic factor (BDNF) induces new selective synthesis of non-L-type Ca2+ channels (N, P/Q, R) at the soma of cultured hippocampal neurons. As N- and P/Q-channels support neurotransmitter release in the hippocampus, this suggests that BDNF-treatment may enhance synaptic transmission by increasing the expression of presynaptic Ca2+ channels as well. To address this issue we studied the long-term effects of BDNF on miniature and stimulus-evoked GABAergic transmission in rat embryo hippocampal neurons. We found that BDNF increased the frequency of miniature currents (mIPSCs) by approximately 40%, with little effects on their amplitude. BDNF nearly doubled the size of evoked postsynaptic currents (eIPSCs) with a marked increase of paired-pulse depression, which is indicative of a major increase in presynaptic activity. The potentiation of eIPSCs was more relevant during the first two weeks in culture, when GABAergic transmission is depolarizing. BDNF action was mediated by TrkB-receptors and had no effects on: (i) the amplitude and dose-response of GABA-evoked IPSCs and (ii) the number of GABA(A) receptor clusters and the total functioning synapses, suggesting that the neurotrophin unlikely acted postsynaptically. In line with this, BDNF affected the contribution of voltage-gated Ca2+ channels mediating evoked GABAergic transmission. BDNF drastically increased the fraction of evoked IPSCs supported by N- and P/Q-channels while it decreased the contribution associated with R- and L-types. This selective action resembles the previously observed up-regulatory effects of BDNF on somatic Ca2+ currents in developing hippocampus, suggesting that potentiation of presynaptic N- and P/Q-channel signalling belongs to a manifold mechanism by which BDNF increases the efficiency of stimulus-evoked GABAergic transmission.

Animals↗
PubMed2001

Direct autocrine inhibition and cAMP-dependent potentiation of single L-type Ca2+ channels in bovine chromaffin cells.

Using the cell-attached recording configuration, we found that in adult bovine chromaffin cells there exists a direct membrane-delimited inhibition of single Bay K-modified L-channels mediated by opioids and ATP locally released in the recording pipette. This autocrine modulation is mediated by pertussis toxin (PTX)-sensitive G-proteins and causes a 50 % decrease of the open channel probability (Po) and an equivalent percentage increase of null sweeps at +10 mV with no changes to the activation kinetics, single channel conductance and mean open time. The decrease in Po is mainly due to an increase in the occurrence and duration of slow closed times (> 40 ms). Addition of purinergic and opioidergic antagonists (suramin and naloxone) or cell pre-treatment with PTX removes the inhibition while addition of ATP and opioids inside the pipette, but not outside, mimics the effect. Strong pre-pulses (+150 mV, 280 ms) followed by short repolarizations are unable to remove the inhibition at test potential (+10 mV). Increasing the level of cAMP by either direct application of 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) or mixtures of forskolin and 1-methyl-3-isobutylxanthine (IBMX) potentiates the activity of L-channels by increasing the mean open time and decreasing the mean closed time and percentage of null sweeps. The cAMP-induced potentiation occurs regardless of whether the G-protein-mediated inhibition is activated by ATP and opioids or inactivated by PTX. Protein kinase inhibitors (H7 and H89) prevent the effects of cAMP without altering the basal autocrine modulation associated with PTX-sensitive G-proteins. Our results provide new evidence for the coexistence of two distinct modulations that may converge on the same neuroendocrine L-channel: a direct G-protein-dependent inhibition and a cAMP-mediated potentiation, which may work in combination to regulate Ca2+ entry during neurosecretion.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2001

G-protein- and cAMP-dependent L-channel gating modulation: a manyfold system to control calcium entry in neurosecretory cells.

Voltage-gated Ca2+ channels are crucial to the control of Ca2+ entry in neurosecretory cells. In the chromaffin cells of adrenal medulla, paracrinally or autocrinally released neurotransmitters induce profound changes in Ca2+ channel gating and Ca2+-dependent events controlling catecholamine secretion and cell activity. The generally held view of these processes is that neurotransmitter-induced modulation of the most widely expressed Ca2+ channels in these cells (N-, P/Q- and L-type) follows two distinct pathways: a direct membrane-delimited Gi/o-protein-induced inhibition of N- and P/Q-type and a remote cAMP-mediated facilitation of L-channels. Both actions depend on voltage, although with remarkably different molecular and kinetic aspects. Recent findings, however, challenge this simple scheme and suggest that L-channels do not require strong pre-pulses to be recruited or facilitated. They are available during normal depolarizations and may be tonically inhibited by Gi/o proteins activated by the released neurotransmitters. Like the N- and P/Q-channels, this autocrine modulation is localized to membrane microareas. Unlike N- and P/Q-channels, however, the inhibition of L-channels is largely independent of voltage and develops in parallel with cAMP-mediated potentiation of channel gating. As L-channels play a crucial role in the control of catecholamine release in chromaffin cells, the two opposite modulations mediated by Gi/o proteins and cAMP may represent an effective way to broaden the dynamic range of Ca2+ signals controlling exocytosis. Here, we review the basic features of this novel L-type channel inhibition comparing it to the well-established forms of L-channel potentiation and voltage-dependent facilitation.

Animals↗
PubMed2000

BDNF, NT-3 and NGF induce distinct new Ca2+ channel synthesis in developing hippocampal neurons.

Neurotrophins exert short- and long-term effects on synaptic transmission. The mechanism underlying these forms of synaptic plasticity is unknown although it is likely that intracellular Ca2+ and presynaptic Ca2+ channels play a critical role. Here we show that BDNF, NGF and NT-3 (10-100 ng/mL) exhibit a selective long-term up-regulation of voltage-gated Ca2+ current densities in developing hippocampal neurons of 6-20 days in culture. NGF and NT-3 appear more effective in up-regulating L-currents, while BDNF predominantly acts on non-L-currents (N, P/Q and R). The effects of the three neurotrophins were time- and dose-dependent. The EC50 was comparable for BDNF, NGF and NT-3 (10-16 ng/mL) while the time of half-maximal activation was significantly longer for NGF compared to BDNF (58 vs. 25 h). Despite the increased Ca2+ current density, the neurotrophins did not alter the voltage-dependence of channel activation, the kinetics parameters or the elementary properties of Ca2+ channels (single-channel conductance, probability of opening and mean open time). Neurotrophin effects were completely abolished by coincubation with the nonspecific Trk-receptor inhibitor K252a, the protein synthesis blocker anisomycin and the MAP-kinase inhibitor PD98059, while cotreatment with the PLC-gamma blocker, U73122, was without effect. Immunocytochemistry and Western blotting revealed that neurotrophins induced an increased MAP-kinase phosphorylation and its translocation to the nucleus. The present findings suggest that on a long time scale different neurotrophins can selectively up-regulate different Ca2+ channels. The action is mediated by Trk-receptors/MAP-kinase pathways and induces an increased density of newly available Ca2+ channels with unaltered gating activity.

Animals↗
PubMed2000

Modulation of acetylcholinesterase and voltage-gated Na(+) channels in choline acetyltransferase- transfected neuroblastoma clones.

Neurotransmitters appear early in the developing embryo and may play a role in the regulation of neuronal differentiation. To study potential effects of acetylcholine production in neuronal differentiation, we used the FB5 subclone of N18TG2 murine neuroblastoma cells stably transfected with cDNA for choline acetyltransferase. We tested whether the forced acetylcholine production can modify the expression or the cellular localization of different neuronal markers. We studied the activity, localization, and secretion of acetylcholinesterase in view of its possible role in the modulation of the morphogenetic action of acetylcholine and of its proposed role of a regulator of neurite outgrowth. FB5 cells are characterized by a high level of acetylcholinesterase, predominantly released into the culture medium. Acetylcholinesterase secretion into the medium was lower in choline acetyltransferase-transfected clones than in nontransfected and antisense-transfected controls. Moreover, sequential extraction of acetylcholinesterase revealed that detergent-extracted, i.e., membrane-associated, activity was higher in the transfected clones expressing choline acetyltransferase activity than in both control groups. These observations suggest that a shift occurs in the utilization of acetylcholinesterase in choline acetyltransferase-transfected clones from a secretion pathway to a pathway leading to membrane localization. In addition, the choline acetyltransferase-positive clones showed higher densities of voltage-gated Na(+) channels and enhanced high-affinity choline uptake, suggesting the accomplishment of a more advanced differentiated neuronal phenotype. Finally, binding experiments demonstrated the presence of muscarinic acetylcholine receptors in all examined clones. This observation is consistent with the proposed existence of an autocrine loop, which may be important for the enhancement in the expression of neurospecific traits.

Acetylcholinesterase↗
PubMed1999

Selective up-regulation of P- and R-type Ca2+ channels in rat embryo motoneurons by BDNF.

Cultured spinal cord motoneurons from day 15 rat embryos (E15) represent a useful model to study Ca2+ channel diversities and their regulation by neurotrophins. Besides the previously identified L-, N- and P-type channels, E15 rat motoneurons also express high densities of R-type channels. We have previously shown that the P-type channel is nearly absent in 60% of these cells, while the R-type contributes to approximately 35% of the total current. Here, we show that chronic preincubation of cultured rat motoneurons with high concentrations (20-100 ng/mL) of brain-derived neurotrophic factor (BDNF) caused a selective up-regulation of the P- and R-type current density available after blocking N- and L-type channels, with no changes to cell membrane capacitance. N- and L-type channels were either not affected or slightly down-modulated by the neurotrophin. The onset of BDNF up-regulation of P/R-type currents had a half-time of 12 h and reached maximal values of approximately 80%. High concentrations of nerve growth factor (NGF; 50-100 ng/mL) had no effect on P/R currents, while BDNF action was prevented by the kinase inhibitor K252a and by the protein synthesis inhibitor anisomycin. These results suggest that chronic applications of BDNF selectively up-regulates the Ca2+ channel types which are most likely to be involved in the control of neurotransmitter release in mammalian neuromuscular junctions. The signal transduction mechanism is probably mediated by TrkB receptors and involves the synthesis of newly functionally active P- and R-type channels. Our data furnish a rationale for a number of recent observations in other laboratories, in which prolonged applications of neurotrophins were shown to potentiate the presynaptic response in developing synapses.

Animals↗
PubMed1998

Antagonists-resistant calcium currents in rat embryo motoneurons.

Ca2+ channels diversity of cultured rat embryo motoneurons was investigated with whole-cell current recordings. In 5-20 mM Ba2+, the whole-cell currents were separated in low- (LVA) and high-voltage-activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed approximately 20% L-, approximately 45% N- and approximately 35% P- and R-type currents. P-type currents were revealed only in 40% of motoneurons, in which 20-200 nM omega-Aga-IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nM in 5 mM Ba2+), suggesting weak expression and heterogeneous distribution of P-type channels compensated by high densities of HVA Ca2+ channels resistant to all the antagonists (R-type). A significant residual current could also be resolved after prolonged applications of 5 microM omega-CTx-MVIIC, which allowed separation of N- and P-type currents by the distinct onset of toxin block. The antagonists-resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the alphaE channel. The current activates around -20 mV in 20 mM Ba2+; inactivates slowly and independently of Ca2+; is blocked by low [Cd2+] and high [Ni2+]; and is larger with Ba2+ than Ca2+. The uncovered R-type calcium current can account for part of the presynaptic Ca2+ current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP-and omega-CTx-GVIA, and displays anomalous sensitivity to omega-Aga-IVA and omega-CTx-MVIIC.

Animals↗
PubMed1996

Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels.

In voltage-dependent ion channels, the gating of the channels is determined by the movement of the voltage sensor. This movement reflects the rearrangement of the protein in response to a voltage stimulus, and it can be thought of as a net displacement of elementary charges (e0) through the membrane (z: effective number of elementary charges). In this paper, we measured z in Shaker IR (inactivation removed) K+ channels, neuronal alpha 1E and alpha 1A, and cardiac alpha 1C Ca2+ channels using two methods: (a) limiting slope analysis of the conductance-voltage relationship and (b) variance analysis, to evaluate the number of active channels in a patch, combined with the measurement of charge movement in the same patch. We found that in Shaker IR K+ channels the two methods agreed with a z congruent to 13. This suggests that all the channels that gate can open and that all the measured charge is coupled to pore opening in a strictly sequential kinetic model. For all Ca2+ channels the limiting slope method gave consistent results regardless of the presence or type of beta subunit tested (z = 8.6). However, as seen with alpha 1E, the variance analysis gave different results depending on the beta subunit used. alpha 1E and alpha 1E beta 1a gave higher z values (z = 14.77 and z = 15.13 respectively) than alpha 1E beta 2a (z = 9.50, which is similar to the limiting slope results). Both the beta 1a and beta 2a subunits, coexpressed with alpha 1E Ca2+ channels facilitated channel opening by shifting the activation curve to more negative potentials, but only the beta 2a subunit increased the maximum open probability. The higher z using variance analysis in alpha 1E and alpha 1E beta 1a can be explained by a set of charges not coupled to pore opening. This set of charges moves in transitions leading to nulls thus not contributing to the ionic current fluctuations but eliciting gating currents. Coexpression of the beta 2a subunit would minimize the fraction of nulls leading to the correct estimation of the number of channels and z.

Animals↗