The Experts below are selected from a list of 246 Experts worldwide ranked by ideXlab platform
Richard D. Veenstra - One of the best experts on this subject based on the ideXlab platform.
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Differences in Functional Expression of Connexin43 and NaV1.5 by Pan- and Class-Selective Histone Deacetylase Inhibition in Heart
International journal of molecular sciences, 2018Co-Authors: Xian Zhang, Dakshesh Patel, Richard D. VeenstraAbstract:Class-selective histone deacetylase (HDAC) inhibitors were designed to improve safety profiles and therapeutic effectiveness in the treatment of multiple cancers relative to pan-HDAC inhibitors. However, the underlying mechanisms for their therapeutic and cardiotoxic potentials remain poorly understood. Cardiac sodium currents (INa) and gap junction conductance (gj) were measured by whole cell patch clamp techniques on primary cultures of neonatal cardiomyocytes. Cardiac NaV1.5 sodium channel and connexin43 (Cx43) gap junction protein levels were assessed by Western blot analyses. Panobinostat produced concentration-dependent reductions in ventricular gj, peak INa density, and NaV1.5 protein expression levels. Membrane Voltage (Vm)-dependent activation of INa was shifted by +3 to 6 mV with no effect on inactivation. Entinostat (1 μM) did not affect ventricular gj, peak INa density, or INa activation. However, the INa Half-Inactivation Voltage (V½) was shifted by −3.5 mV. Ricolinostat had only minor effects on ventricular gj and INa properties, though INa activation was shifted by −4 mV. Cx43 and NaV1.5 protein expression levels were not altered by class-selective HDAC inhibitors. The lack of effects of class-selective HDAC inhibitors on ventricular gj and INa may help explain the improved cardiac safety profile of entinostat and ricolinostat.
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An amino‐terminal lysine residue of rat connexin40 that is required for spermine block
The Journal of Physiology, 2006Co-Authors: Xianming Lin, Edward Fenn, Richard D. VeenstraAbstract:Spermine blocks connexin40 (Cx40) gap junctions, and two cytoplasmic amino-terminal domain glutamate residues are essential for this inhibitory activity. To further examine the molecular basis for block, we mutated a portion of a basic amino acid (HKH) motif on the Cx40 amino-terminal domain. Replacement of the Cx40 H15 + K16 residues with the Q15 + A16 sequence native to spermine-insensitive connexin43 (Cx43) gap junctions increased the equilibrium dissociation constant (Kd) and reduced the maximum inhibition by spermine. The corresponding electrical distance (δ) approximation was decreased by about 50%. The transjunctional Voltage (Vj)-dependent gating of homotypic Cx40 H15Q + K16A mutant gap junctions was also significantly reduced. The minimum normalized steady-state junctional conductance (Gmin) increased from 0.17 to 0.72, with an increase in the Half-Inactivation Voltage from 48 to 60 mV. However, the unitary junctional conductance (γj; 160 pS) was only slightly altered, and the relative cation/anion conductance and permeability ratios were unchanged from wild-type Cx40 gap junction channels. The relative K+/Cl− permeability (PK/PCl) ratio increased from six to ten when [KCl] was reduced to 25% of normal. These data suggest that the HKH motif at positions 15–17 is important to the conformational structure of the putative Voltage sensor and spermine receptor of Cx40, without causing significant alteration of the electrostatic surface charge potentials that contribute to the ion selectivity of this gap junction channel.
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An amino-terminal lysine residue of rat connexin40 that is required for spermine block.
The Journal of physiology, 2005Co-Authors: Xianming Lin, Edward Fenn, Richard D. VeenstraAbstract:Spermine blocks connexin40 (Cx40) gap junctions, and two cytoplasmic amino-terminal domain glutamate residues are essential for this inhibitory activity. To further examine the molecular basis for block, we mutated a portion of a basic amino acid (HKH) motif on the Cx40 amino-terminal domain. Replacement of the Cx40 H15 + K16 residues with the Q15 + A16 sequence native to spermine-insensitive connexin43 (Cx43) gap junctions increased the equilibrium dissociation constant (K(d)) and reduced the maximum inhibition by spermine. The corresponding electrical distance (delta) approximation was decreased by about 50%. The transjunctional Voltage (V(j))-dependent gating of homotypic Cx40 H15Q + K16A mutant gap junctions was also significantly reduced. The minimum normalized steady-state junctional conductance (G(min)) increased from 0.17 to 0.72, with an increase in the Half-Inactivation Voltage from 48 to 60 mV. However, the unitary junctional conductance (gamma(j); 160 pS) was only slightly altered, and the relative cation/anion conductance and permeability ratios were unchanged from wild-type Cx40 gap junction channels. The relative K(+)/Cl(-) permeability (P(K)/P(Cl)) ratio increased from six to ten when [KCl] was reduced to 25% of normal. These data suggest that the HKH motif at positions 15-17 is important to the conformational structure of the putative Voltage sensor and spermine receptor of Cx40, without causing significant alteration of the electrostatic surface charge potentials that contribute to the ion selectivity of this gap junction channel.
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Regulation of Connexin43 Gap Junctional Conductance by Ventricular Action Potentials
Circulation research, 2003Co-Authors: Xianming Lin, Mark Crye, Richard D. VeenstraAbstract:Transjunctional Voltage regulates cardiac gap junctional conductance, but the kinetics of inactivation were considered too slow to affect cardiac action potential propagation. Connexin43 (Cx43) is abundantly expressed in the atrial and ventricular myocardium and the rapid ventricular conduction tissues (ie, His-Purkinje system) of the mammalian heart and is important to conduction through these cardiac tissues. The kinetics of Cx43 Voltage gating were examined at peak action potential Voltages using simulated ventricular myocardial action potential waveforms or pulse protocols exceeding 100-mV transjunctional potentials. Junctional current responses approximate the action potential morphology but conductance calculations reveal a 50% to 60% decline from peak to near constant plateau values. Junctional conductance recovers during phase 3 repolarization and early diastole to initial values. The bases for these transient changes in junctional conductance are the rapid decay kinetics in tens of milliseconds at peak transjunctional Voltages (V j ) of 130 mV and the gradual increase in junctional conductance as V j returns toward 0 mV. The decay time constants change e-fold per 22.1 mV above the Half-Inactivation Voltage for Cx43 gap junctions of ±58 mV. A realistic dynamic model for changes in junctional resistance between excitable and nonexcitable cells during cardiac action potential propagation was developed based on these findings. This dynamic model of cardiac gap junctions will further our understanding of the role gap junctions play in the genesis and propagation of cardiac arrhythmias. The full text of this article is available online at http://www.circresaha.org.
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Voltage Clamp Limitations of Dual Whole-Cell Gap Junction Current and Voltage Recordings. I. Conductance Measurements
Biophysical journal, 2001Co-Authors: Richard D. VeenstraAbstract:Previous correction methods for series access resistance errors in the dual whole-cell configuration did not take into account the effect of nonzero resting potentials (E(rest)) and junctional reversal potentials (E(rev)). Dual whole-cell currents were modeled according to resistor-circuit analysis and two correction formulas for the measurement of junctional currents (I(j)) were assessed. The equations for I(j), derived from Kirchoff's law before and after baseline subtraction of the nonjunctional current, were assessed for accuracy under a variety of whole-cell patch-clamp recording conditions. Both equations accurately correct for dual whole-cell Voltage-clamp errors provided that the cellular parameters are included in the nonbaseline subtracted I(j) derivations. Junctional conductance (g(j)) estimates are most reliable at high junctional resistance (R(j)) values and minimize the need for corrective methods based on electrode series and cellular input resistances (R(el) and R(in)). In the "open-cell" configuration, low R(j) values relative to R(in) are required for accurate g(j) estimates. These methods provide the basis for accurate quantitative measurements of junctional resistance (or conductance) of gap junction channels or connexin hemichannels in the dual whole-cell or open-cell configurations. Revaluation of V(j)-dependent gating of rat connexin40 g(j) produced nearly identical Boltzmann fits to previously published data. Continuous g(j)-V(j) curves generated by variable slope V(j) ramps provide for more accurate fits and assessment of the time-dependence of the Half-Inactivation Voltage and net gating charge movement.
Lynne A. Fieber - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Na+ and Ca2+ currents in bag cells of sexually immature Aplysia californica.
The Journal of Experimental Biology, 1998Co-Authors: Lynne A. FieberAbstract:The neurosecretory bag cells of sexually mature Aplysia californica release egg-laying hormones as part of the reproductive process after a train of action potentials termed afterdischarge. Whole-cell Voltage-clamp experiments were performed in cultured cells from sexually immature A. californica to characterize the inward Voltage-gated currents for Na+ and Ca2+. The goal of these experiments was to investigate the regulation of excitability during sexual maturation. Na+ currents in bag cells of immature A. californica were similar in several ways to those of mature animals. The Na+ currents activated at Voltages less negative than -30 mV and peaked at 10-20 mV in artificial sea water. The time course and pharmacology of bag cell Na+ currents were similar to those of bag cells from mature A. californica, although the Na+ current density was lower in immature A. californica. Na+ currents were inhibited by tetrodotoxin (50 nmol l-1). The Na+ current was relatively insensitive to depolarized holding potentials (Vh), maintaining approximately 50 % of peak current amplitude present at Vh=-70 mV throughout the activation range at Vh=-30 mV. In experiments using a 1 s depolarized Vh prior to a test pulse, the Half-Inactivation Voltage (V1/2) was -27 mV. Recovery of immature Na+ current from steady-state inactivation at Vh=-30 mV had a time constant ( ) of 9.5 ms, significantly slower than in mature animals. Ca2+ currents of immature A. californica activated at approximately -30 mV and peaked at approximately 20 mV with 11 mmol l-1 Ba2+ as the charge carrier. The principle differences from mature Ca2+ currents were the low density of the immature Ca2+ currents and their 'run-down' in whole-cell recordings. The pharmacology and V1/2 of bag cell Ca2+ currents were similar to those of L-type Ca2+ currents in mature cells. The Ca2+ currents were inhibited 61+/-10 % by nifedipine (10 micromol l-1) and were unaffected by -conotoxin GVIA (10 micromol l-1). The Ca2+ currents were relatively insensitive to depolarized Vh, activating maximally at Vh=-90, -70 and -50 mV, and maintaining 50 % of this peak current amplitude throughout the activation range at Vh=-30 mV. The V1/2 was -23 mV in experiments in which cells were subjected to a 1 s depolarized Vh prior to a test pulse. Na+ current amplitudes were maintained or increased during 1 min of 4 Hz test pulses in bag cells at Vh=-70 mV and Vh=-30 mV. In contrast, Ca2+ current run-down occurred during 1 min of 4 Hz test pulses in seven of 10 cells at Vh=-70 mV and in 12 of 12 cells at Vh=-30 mV. The observed scarcity of Na+ and Ca2+ currents in immature bag cells as well as the specific characteristics of immature bag cell Ca2+ currents make repetitive action potential firing and hormone release less likely than in mature bag cells.
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Characterization of Na+ and Ca2+ currents in bag cells of sexually immature Aplysia californica.
The Journal of experimental biology, 1998Co-Authors: Lynne A. FieberAbstract:The neurosecretory bag cells of sexually mature Aplysia californica release egg-laying hormones as part of the reproductive process after a train of action potentials termed afterdischarge. Whole-cell Voltage-clamp experiments were performed in cultured cells from sexually immature A. californica to characterize the inward Voltage-gated currents for Na+ and Ca2+. The goal of these experiments was to investigate the regulation of excitability during sexual maturation. Na+ currents in bag cells of immature A. californica were similar in several ways to those of mature animals. The Na+ currents activated at Voltages less negative than -30 mV and peaked at 10-20 mV in artificial sea water. The time course and pharmacology of bag cell Na+ currents were similar to those of bag cells from mature A. californica, although the Na+ current density was lower in immature A. californica. Na+ currents were inhibited by tetrodotoxin (50 nmol l-1). The Na+ current was relatively insensitive to depolarized holding potentials (Vh), maintaining approximately 50% of peak current amplitude present at Vh = -70 mV throughout the activation range at Vh = -30 mV. In experiments using a 1 s depolarized Vh prior to a test pulse, the Half-Inactivation Voltage (V1/2) was -27 mV. Recovery of immature Na+ current from steady-state inactivation at Vh = -30 mV had a time constant (tau) of 9.5 ms, significantly slower than in mature animals. Ca2+ currents of immature A. californica activated at approximately -30 mV and peaked at approximately 20 mV with 11 mmol l-1 Ba2+ as the charge carrier. The principle differences from mature Ca2+ currents were the low density of the immature Ca2+ currents and their 'run-down' in whole-cell recordings. The pharmacology and V1/2 of bag cell Ca2+ currents were similar to those of L-type Ca2+ currents in mature cells. The Ca2+ currents were inhibited 61 +/- 10% by nifedipine (10 mumol l-1) and were unaffected by omega-conotoxin GVIA (10 mumol l-1). The Ca2+ currents were relatively insensitive to depolarized Vh, activating maximally at Vh = -90, -70 and -50 mV, and maintaining 50% of this peak current amplitude throughout the activation range at Vh = -30 mV. The V1/2 was -23 mV in experiments in which cells were subjected to a 1 s depolarized Vh prior to a test pulse. Na+ current amplitudes were maintained or increased during 1 min of 4 Hz test pulses in bag cells at Vh = -70 mV and Vh = -30 mV. In contrast, Ca2+ current run-down occurred during 1 min of 4 Hz test pulses in seven of 10 cells at Vh = -70 mV and in 12 of 12 cells at Vh = -30 mV. The observed scarcity of Na+ and Ca2+ currents in immature bag cells as well as the specific characteristics of immature bag cell Ca2+ currents make repetitive action potential firing and hormone release less likely than in mature bag cells.
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Characterization and modulation of Na+ and Ca2+ currents underlying the action potential in bag cells of two species of Aplysia.
The Journal of Experimental Biology, 1995Co-Authors: Lynne A. FieberAbstract:The neurosecretory bag cells of Aplysia produce long trains of action potentials (afterdischarge) to release hormones important to egg laying. These ionic currents are modulated by second messengers. Modulation of excitability in bag cells is incompletely understood partly because the currents that are modulated have not been fully characterized. Whole-cell Voltage-clamp experiments were executed in cultured cells from sexually mature A. californica and A. brasiliana to characterize the inward Voltage-gated currents for Na+ and Ca2+. These species had similar Na+ and Ca2+ current characteristics. The Na+ currents activated at Voltages less negative than -30 mV and peaked at between +10 and +20 mV in artificial sea water. The time course and pharmacology of bag cell Na+ currents were similar to those of fast Na+ current in other excitable cells. Na+ currents were abolished in Na(+)-free extracellular solution and were not inhibited by Cd2+. The KD for inhibition by tetrodotoxin was 2.6 nmol l-1. The Na+ current was relatively insensitive to depolarized holding potentials (Vh), maintaining approximately 65% of peak current amplitude throughout the activation range at Vh = -30 mV. In experiments using a 1 s depolarized Vh prior to a test pulse, the Half-Inactivation Voltage (V1/2) was -21 mV. The time constant of recovery from steady-state activation was 2.9 ms at Vh = -70 mV and 6.8 ms at Vh = -30 mV. The Ca2+ currents activated near -10 mV and peaked at approximately +20 mV with 11 mmol l-1 Ba2+ as the charge carrier. The pharmacology and V1/2 of bag cell Ca2+ current were similar to those of L-type Ca2+ currents. In extracellular solution without Na+, but containing Ba2+, Cs+ and tetraethylammonium, the Ca2+ current was inhibited by 25-100% by nifedipine (10 mumol l-1), mean 42%, and was unaffected in the majority of cells by omega-conotoxin (10 mumol l-1). The Ca2+ current was insensitive to Ni2+ (100 mumol l-1), but was abolished by 100 mumol l-1 Cd2+. Like the Na+ current, the Ca2+ current was relatively insensitive to depolarized Vh, maintaining more than 80% of peak current amplitude throughout the activation range at Vh = -40 mV. With a 1 s depolarized Vh prior to a test pulse, the V1/2 was -30 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Xianming Lin - One of the best experts on this subject based on the ideXlab platform.
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An amino‐terminal lysine residue of rat connexin40 that is required for spermine block
The Journal of Physiology, 2006Co-Authors: Xianming Lin, Edward Fenn, Richard D. VeenstraAbstract:Spermine blocks connexin40 (Cx40) gap junctions, and two cytoplasmic amino-terminal domain glutamate residues are essential for this inhibitory activity. To further examine the molecular basis for block, we mutated a portion of a basic amino acid (HKH) motif on the Cx40 amino-terminal domain. Replacement of the Cx40 H15 + K16 residues with the Q15 + A16 sequence native to spermine-insensitive connexin43 (Cx43) gap junctions increased the equilibrium dissociation constant (Kd) and reduced the maximum inhibition by spermine. The corresponding electrical distance (δ) approximation was decreased by about 50%. The transjunctional Voltage (Vj)-dependent gating of homotypic Cx40 H15Q + K16A mutant gap junctions was also significantly reduced. The minimum normalized steady-state junctional conductance (Gmin) increased from 0.17 to 0.72, with an increase in the Half-Inactivation Voltage from 48 to 60 mV. However, the unitary junctional conductance (γj; 160 pS) was only slightly altered, and the relative cation/anion conductance and permeability ratios were unchanged from wild-type Cx40 gap junction channels. The relative K+/Cl− permeability (PK/PCl) ratio increased from six to ten when [KCl] was reduced to 25% of normal. These data suggest that the HKH motif at positions 15–17 is important to the conformational structure of the putative Voltage sensor and spermine receptor of Cx40, without causing significant alteration of the electrostatic surface charge potentials that contribute to the ion selectivity of this gap junction channel.
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An amino-terminal lysine residue of rat connexin40 that is required for spermine block.
The Journal of physiology, 2005Co-Authors: Xianming Lin, Edward Fenn, Richard D. VeenstraAbstract:Spermine blocks connexin40 (Cx40) gap junctions, and two cytoplasmic amino-terminal domain glutamate residues are essential for this inhibitory activity. To further examine the molecular basis for block, we mutated a portion of a basic amino acid (HKH) motif on the Cx40 amino-terminal domain. Replacement of the Cx40 H15 + K16 residues with the Q15 + A16 sequence native to spermine-insensitive connexin43 (Cx43) gap junctions increased the equilibrium dissociation constant (K(d)) and reduced the maximum inhibition by spermine. The corresponding electrical distance (delta) approximation was decreased by about 50%. The transjunctional Voltage (V(j))-dependent gating of homotypic Cx40 H15Q + K16A mutant gap junctions was also significantly reduced. The minimum normalized steady-state junctional conductance (G(min)) increased from 0.17 to 0.72, with an increase in the Half-Inactivation Voltage from 48 to 60 mV. However, the unitary junctional conductance (gamma(j); 160 pS) was only slightly altered, and the relative cation/anion conductance and permeability ratios were unchanged from wild-type Cx40 gap junction channels. The relative K(+)/Cl(-) permeability (P(K)/P(Cl)) ratio increased from six to ten when [KCl] was reduced to 25% of normal. These data suggest that the HKH motif at positions 15-17 is important to the conformational structure of the putative Voltage sensor and spermine receptor of Cx40, without causing significant alteration of the electrostatic surface charge potentials that contribute to the ion selectivity of this gap junction channel.
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Regulation of Connexin43 Gap Junctional Conductance by Ventricular Action Potentials
Circulation research, 2003Co-Authors: Xianming Lin, Mark Crye, Richard D. VeenstraAbstract:Transjunctional Voltage regulates cardiac gap junctional conductance, but the kinetics of inactivation were considered too slow to affect cardiac action potential propagation. Connexin43 (Cx43) is abundantly expressed in the atrial and ventricular myocardium and the rapid ventricular conduction tissues (ie, His-Purkinje system) of the mammalian heart and is important to conduction through these cardiac tissues. The kinetics of Cx43 Voltage gating were examined at peak action potential Voltages using simulated ventricular myocardial action potential waveforms or pulse protocols exceeding 100-mV transjunctional potentials. Junctional current responses approximate the action potential morphology but conductance calculations reveal a 50% to 60% decline from peak to near constant plateau values. Junctional conductance recovers during phase 3 repolarization and early diastole to initial values. The bases for these transient changes in junctional conductance are the rapid decay kinetics in tens of milliseconds at peak transjunctional Voltages (V j ) of 130 mV and the gradual increase in junctional conductance as V j returns toward 0 mV. The decay time constants change e-fold per 22.1 mV above the Half-Inactivation Voltage for Cx43 gap junctions of ±58 mV. A realistic dynamic model for changes in junctional resistance between excitable and nonexcitable cells during cardiac action potential propagation was developed based on these findings. This dynamic model of cardiac gap junctions will further our understanding of the role gap junctions play in the genesis and propagation of cardiac arrhythmias. The full text of this article is available online at http://www.circresaha.org.
Paweł Szulczyk - One of the best experts on this subject based on the ideXlab platform.
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Opioid μ receptor activation inhibits sodium currents in prefrontal cortical neurons via a protein kinase A- and C-dependent mechanism
Brain research, 2006Co-Authors: Grzegorz Witkowski, Paweł SzulczykAbstract:Opioid transmission in the medial prefrontal cortex is involved in mood regulation and is altered by drug dependency. However, the mechanism by which ionic channels in cortical neurons are controlled by mu opioid receptors has not been elucidated. In this study, the effect of mu opioid receptor activation on Voltage-dependent Na(+) currents was assessed in medial prefrontal cortical neurons. In 66 out of 98 nonpyramidal neurons, the application of 1 microM of DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-OL]-enkephalin), a specific mu receptor agonist, caused a decrease in the Na(+) current amplitude to approximately 79% of that observed in controls (half blocking concentration = 0.094 microM). Moreover, DAMGO decreased the maximum current activation rate, prolonged its time-dependent inactivation, and shifted the half inactivation Voltage from -63.4 mV to -71.5 mV. DAMGO prolonged the time constant of recovery from inactivation from 5.4 ms to 7.4 ms. The DAMGO-evoked inhibition of Na(+) current was attenuated when GDP-beta-S (0.4 mM, Guanosine 5-[beta-thio]diphosphate trilithium salt) was included in the intracellular solution. Inhibitors of kinase A and C greatly attenuated the DAMGO-induced inhibition, while adenylyl cyclase and kinase C activators mirrored the DAMGO inhibitory effect. Na(+) currents in pyramidal neurons were insensitive to DAMGO. We conclude that the activation of mu opioid receptors inhibits the Voltage-dependent Na(+) currents expressed in nonpyramidal neurons of the medial prefrontal cortex, and that kinases A and C are involved in this inhibitory pathway.
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Effects of ATP and GTP on Voltage-gated K+ currents in glandular and muscular sympathetic neurons.
Brain research, 2005Co-Authors: Bartłomiej Szulczyk, Grzegorz Witkowski, Rafał Rola, Paweł SzulczykAbstract:Abstract This study assesses the effects of ATP and GTP on the kinetic properties of Voltage-gated K + currents in anatomically identified postganglionic sympathetic neurons innervating the submandibular gland and the masseter muscle in rats. Three types of K + currents were isolated: the I Af steady-state inactivating at more hyperpolarized potentials, I As steady-state inactivating at less hyperpolarized potentials than I Af and the I K current independent of membrane potential. The kinetic properties of these currents were tested in neurons with ATP (4 mM) and GTP (0.5 mM) or without ATP and GTP in the intracellular solution.In glandular and muscular neurons in the absence of ATP and GTP in the intracellular solution, the current density of I Af was significantly larger (142 pA/pF and 166 pA/pF, respectively) comparing to cells with ATP and GTP (96 pA/pF and 100 pA/pF, respectively). The I As was larger only in glandular neurons (52 pA/pF vs. 37 pA/pF).Conversely, I K current density was smaller in glandular and muscular neurons without ATP and GTP (17 pA/pF and 31 pA/pF, respectively) comparing to cells with ATP and GTP (57 pA/pF and 58 pA/pF, respectively). In glandular (15.5 nA/ms vs. 6.9 nA/ms) and muscular (10.9 nA/ms vs. 7.5 nA/ms) neurons, the I Af activated faster in the absence of ATP and GTP. Half inactivation Voltage of I Af in glandular (−110.0 mV vs. −119.7 mV) and muscular (−108.4 vs. −117.3 mV) neurons was shifted towards depolarization in the absence of ATP and GTP. We suggest that the kinetic properties of K + currents in glandular and muscular sympathetic neurons change markedly in the absence of ATP and GTP in the cytoplasm. Effectiveness of steady-state inactivated currents ( I Af and I AS ) increased, while effectiveness of steady-state noninactivated currents decreased in the absence of ATP and GTP. The effects were more pronounced in glandular than in muscular neurons.
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Stellate neurones innervating the rat heart express N, L and P/Q calcium channels
Journal of The Autonomic Nervous System, 1998Co-Authors: Wojciech Kukwa, T Macioch, Paweł SzulczykAbstract:Abstract The aim of the study was to investigate the kinetic properties and identify the subtypes of Ca2+ currents in the cardiac postganglionic sympathetic neurones of rats. Neurones were labelled with a fluorescent tracer—Fast-Blue, injected into the pericardial cavity. Voltage-dependent Ca2+ currents were recorded from dispersed stellate ganglion cells that showed Fast Blue labelling. Only high threshold Voltage-dependent Ca2+ currents were found in the somata of cardiac sympathetic neurones. Their maximum amplitude, mean cell capacitance and current density were respectively: 0.67 nA, 19.3 pF and 36.4 pA/pF (n=21). The maximum Ca2+ conductance was 51.3 nS (n=14). Half activation Voltage equalled +11.0 mV and the slope factor for conductance 11.1 (n=14). As tested with a 10 s pre-pulse, the Ca2+ current began to inactivate at −80 mV. Half inactivation Voltage and slope factor for steady-state inactivation were −36.6 mV and 14.1 (n=9), respectively. Saturating concentration of L channel blocker (nifedipine), N channel blocker (ω-conotoxin-GVIA), P/Q channel blocker (ω-Agatoxin-IVA) and N/P/Q channel blocker (ω-conotoxin-MVIIC) reduced the total Ca2+ current by 26.8% (n=7), 57.1% (n=12), 25.9% (n=6) and 69.4% (n=6), respectively. These results show that the somata of cardiac postganglionic cardiac sympathetic neurones contain significant populations of N, L and P/Q high threshold Ca2+ channels.
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Stellate neurones innervating the rat heart express N, L and P/Q calcium channels.
Journal of the autonomic nervous system, 1998Co-Authors: Wojciech Kukwa, T Macioch, Paweł SzulczykAbstract:The aim of the study was to investigate the kinetic properties and identify the subtypes of Ca2+ currents in the cardiac postganglionic sympathetic neurones of rats. Neurones were labelled with a fluorescent tracer--Fast-Blue, injected into the pericardial cavity. Voltage-dependent Ca2+ currents were recorded from dispersed stellate ganglion cells that showed Fast Blue labelling. Only high threshold Voltage-dependent Ca2+ currents were found in the somata of cardiac sympathetic neurones. Their maximum amplitude, mean cell capacitance and current density were respectively: 0.67 nA, 19.3 pF and 36.4 pA/pF (n = 21). The maximum Ca2+ conductance was 51.3 nS (n = 14). Half activation Voltage equalled +11.0 mV and the slope factor for conductance 11.1 (n = 14). As tested with a 10 s pre-pulse, the Ca2+ current began to inactivate at -80 mV. Half inactivation Voltage and slope factor for steady-state inactivation were -36.6 mV and 14.1 (n = 9), respectively. Saturating concentration of L channel blocker (nifedipine), N channel blocker (omega-conotoxin-GVIA), P/Q channel blocker (omega-Agatoxin-IVA) and N/P/Q channel blocker (omega-conotoxin-MVIIC) reduced the total Ca2+ current by 26.8% (n = 7), 57.1% (n = 12), 25.9% (n = 6) and 69.4% (n = 6), respectively. These results show that the somata of cardiac postganglionic cardiac sympathetic neurones contain significant populations of N, L and P/Q high threshold Ca2+ channels.
Roman Shirokov - One of the best experts on this subject based on the ideXlab platform.
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Regulation of CaV1.2 Channels by Sphingolipids and Cholesterol. Specific Role of the Gamma Subunit
Biophysical Journal, 2012Co-Authors: Anna Angelova, Naga Chalamalasetty, Roman ShirokovAbstract:Previoulsy (Biophys. Meetings 2010 and 2011), we showed that inactivation of L-type Ca2+ channels containing the γ1 subunit changes during cell cycle. The Half-Inactivation Voltage is very negative (in the −70 - −50 mV range) in cells at the G1 phase, but also after serum starvation, or after the ER shock.Here we report that the novel regulatory pathway is membrane-delimited because it depends on cholesterol and sphingolipids. Alterations of the membrane lipid fluidity (e.g., addition of Triton X-100) affected inactivation regardless of the γ1 subunit. However, various manipulations with membrane cholesterol, ceramide, and/or shpingosine were efficient only when the γ1 subunit was present. Although tested positively in our experimental conditions, phosphoinositides, C-8-ceramide-1-phosphate, sphingosine-1-phosphate, and glycosphingolipid GM1 did not act specifically on inactivation with the γ1 subunit.When cholesterol was depleted from the membrane, or upon application of sphingosine, ceramide-C2, or ceramide-C8, inactivation shifted to the −70 - −50 mV range. Isomers epi-cholesterol and dihydro-ceramide-C2 did not act. Because ceramide and sphingosine strongly activate phosphatases PP1 and PP2A, phosphatase inhibitors calyculin A, or tautomycetin, were applied in order to prevent a rapid current run-down. Thus, a role of phosphorylation-dependent pathways can be excluded as well.We compared the membrane cholesterol content in cells at different cell-cycle stages, but did not find any significant difference. However, the membrane contents of sphingosine and ceramide are well known to change during cell cycle.Therefore, we propose that the enhancement of inactivation of L-type channels by the γ1 subunit is dependent on sphingosine and ceramide. Possibly, the sphingolipids interact directly with the subunit. The large scale of changes of inactivation properties due to these interactions allows efficient spatiotemporal tuning of functional availability of L-type Ca2+ channels.Supported by R01MH079406.
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Thermodynamic Linkage with Voltage Sensing Explains the Large Variability of Inactivation in L-Type Ca Channels with γ1 Subunit
Biophysical Journal, 2011Co-Authors: Anna Angelova, Roman ShirokovAbstract:The Voltage-dependence of steady-state inactivation (a.k.a. the inactivation curve) is an important determinant of functional availability of calcium channels. Some experimental and pathological conditions change the Half-Inactivation Voltage (V1/2) but not the slope of the inactivation curve. We observed that the V1/2 varies from cell to cell by as much as 40 mV when CaV1.2 channels are co-expressed with γ1 subunit in tsA-201 cells. This parallel shift cannot be explained by a simple mixing of channels with different V1/2 values (e.g., with and without the γ1).We found that the γ1 subunit had a relatively small effect on inactivation assessed at the level of gating currents. It caused the Voltage-dependence of the intramembrane charge movement in inactivated channels to shift by about −10 mV, and this effect did not vary from cell to cell. Therefore, the large shift of the inactivation curve seen at the level of ionic currents appears to be unrelated to the small changes observed at the level of the Voltage sensor for activation/inactivation gating. We explain this paradox in the framework of a minimal four-state model of inactivation, in which the V1/2 parameter of the inactivation curve is determined by both the Voltage-dependence of gating currents and the maximal extent (efficacy) of inactivation. The parallel shift of the inactivation curve may result solely from a change in the efficacy.Therefore, we propose that functional availability of CaV1.2 channels with the γ1 subunit is controlled by a cell-specific molecular modification that affects similarly all channels in a particular cell. This modification primarily alters the efficacy of inactivation and, thus, leads to large changes of the availability of channels to activate.Supported by NIH R01MH079406.
