P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mamma... more P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The α1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between α 1A and P- and Q-type channels is unknown.
Peptide toxins have proved to be useful agents, both in discriminating between different componen... more Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently
Human retinoblastoma cells are multipotent retinal precursor cells capable of differentiating int... more Human retinoblastoma cells are multipotent retinal precursor cells capable of differentiating into photoreceptors, neurons, and glia. The current-voltage relation of the undifferentiated cells is dominated by a transient inward current that disappears shortly after differentiation. In 20 mM Ba2+-containing bath solutions, the current has an activation midpoint near −25 mV and appears to be fully inactivated at −20 mV. Sr2+and Ca2+ are preferred charge carriers relative to Ba2+, and the current vanishes in the absence of these divalent cations. Cd2+ blocks the current with an IC50 of 160 μM, and Ni2+ blocks in a biphasic manner with IC50s of 22 and 352 μM. The current is unaffected when sodium is replaced with other monovalent cations, and it is insensitive to nifedipine, ω-conotoxin GVIA, ω-agatoxin IVA, and ω-conotoxin MVIIC. RT-PCR revealed the presence of α1G and α1H mRNA in undifferentiated cells, but following differentiation, a striking reduction of both α1G and α1H mRNA was f...
Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, ye... more Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, yet effective therapies are lacking. We identify the pannexin-1 (Panx1) channel as a therapeutic target for alleviating mechanical allodynia, a cardinal sign of arthritis. In rats, joint pain caused by intra-articular injection of monosodium iodoacetate (MIA) was associated with spinal adenosine 5'-triphosphate (ATP) release and a microglia-specific up-regulation of P2X7 receptors (P2X7Rs). Blockade of P2X7R or ablation of spinal microglia prevented and reversed mechanical allodynia. P2X7Rs drive Panx1 channel activation, and in rats with mechanical allodynia, Panx1 function was increased in spinal microglia. Specifically, microglial Panx1-mediated release of the proinflammatory cytokine interleukin-1β (IL-1β) induced mechanical allodynia in the MIA-injected hindlimb. Intrathecal administration of the Panx1-blocking peptide panx suppressed the aberrant discharge of spinal laminae I-II ...
(1) Local anaesthetics (LA) rely for their clinical actions on state-dependent inhibition of volt... more (1) Local anaesthetics (LA) rely for their clinical actions on state-dependent inhibition of voltage-dependent sodium channels. (2) Single, batrachoxin-modified sodium channels in planar lipid bilayers allow direct observation of drug-channel interactions. Two modes of inhibition of single-channel current are observed: fast block of the open channels and prolongation of a long-lived closed state, some of whose properties resemble those of the inactivated state of unmodified channels. (3) Analogues of different parts of the LA molecule separately mimic each blocking mode: amines-fast block, and water-soluble aromatics-closed state prolongation. (4) Interaction between a v-conotoxin derivative and diethylammonium indicate an intrapore site of fast, open-state block. (5) Site-directed mutagenesis studies suggest that hydrophobic residues in transmembrane segment 6 of repeat domain 4 of sodium channels are critical for both LA binding and stabilization of the inactivated state.
Computational modeling is used to investigate the functional impact of G protein-mediated presyna... more Computational modeling is used to investigate the functional impact of G protein-mediated presynaptic autoinhibition on synaptic filtering properties. It is demonstrated that this form of autoinhibition, which is relieved by depolarization, acts as a high-pass filter. This contrasts with vesicle depletion, which acts as a low-pass filter. Model parameters are adjusted to reproduce kinetic slowing data from different Gbetagamma dimeric isoforms, which produce different degrees of slowing. With these sets of parameter values, we demonstrate that the range of frequencies filtered out by the autoinhibition varies greatly depending on the Gbetagamma isoform activated by the autoreceptors. It is shown that G protein autoinhibition can enhance the spatial contrast between a spatially distributed high-frequency signal and surrounding low-frequency noise, providing an alternate mechanism to lateral inhibition. It is also shown that autoinhibition can increase the fidelity of coincidence dete...
Proceedings of the National Academy of Sciences, 1998
We have examined the modulation of cloned and stably expressed rat brain N type calcium channels ... more We have examined the modulation of cloned and stably expressed rat brain N type calcium channels (α 1B + β 1b + α 2 δ subunits) by exogenously applied purified G protein βγ subunits. In the absence of G βγ , barium currents through N type channels are unaffected by application of strong depolarizing prepulses. In contrast, inclusion of purified G βγ in the patch pipette results in N type currents that initially facilitated upon application of positive prepulses followed by rapid reinhibition. Examination of the kinetics of G βγ -dependent reinhibition showed that as the duration between the test pulse and the prepulse was increased, the degree of facilitation was attenuated in a monoexponential fashion. The time constant τ for the recovery from facilitation was sensitive to exogenous G βγ , so that the inverse of τ linearly depended on the G βγ concentration. Overall, the data are consistent with a model whereby a single G βγ molecule dissociates from the channel during the prepulse...
Pflügers Archiv - European Journal of Physiology, 2013
1,4-Dihydropyridines (DHPs) are an important class of L-type calcium channel blockers that are us... more 1,4-Dihydropyridines (DHPs) are an important class of L-type calcium channel blockers that are used to treat conditions such as hypertension and angina. Their primary target in the cardiovascular system is the Cav1.2 L-type calcium channel isoform, however, a number of DHPs also block low-voltage-activated T-type calcium channels. Here, we describe the synthesis of a series of novel DHP derivatives that have a condensed 1,4-DHP ring system (hexahydroquinoline) and report on their abilities to block both L- and T-type calcium channels. Within this series of compounds, modification of a key ester moiety not only regulates the blocking affinity for both L- and T-type channels, but also allows for the development of DHPs with 30-fold selectivity for T-type channels over the L-type. Our data suggest that a condensed dihydropyridine-based scaffold may serve as a pharmacophore for a new class of T-type selective inhibitors.
Pflügers Archiv - European Journal of Physiology, 2013
The L-type voltage-gated calcium channel Cav1.2 is important for excitation-contraction coupling ... more The L-type voltage-gated calcium channel Cav1.2 is important for excitation-contraction coupling in the heart, as well as CREB-mediated transcription in the brain. The ubiquitous calcium-binding protein calmodulin (CaM) is known to modulate calcium-dependent inactivation (CDI) of these channels, thus limiting the amount of calcium entering via Cav1.2 during prolonged or repetitive membrane depolarizations. The proximal N-terminus of Cav1.2 contains a CaMbinding site at residue W52 that is critical for a type of CDI that is mediated by the N-terminal lobe of CaM. Here, we identify a second CaM interaction site in the Cav1.2 Nterminus downstream of the W52 site that is formed by residue C106. We show by site-directed mutagenesis coupled with electrophysiological measurements that this region of the channel functionally partakes in N-lobe CDI, likely by acting as a gating transduction motif. Thus, our data indicate that calcium regulation of Cav1.2 channels is more complex than previously thought, and involves more than one region within the channel's N-terminal domain.
Voltage-and frequency-dependent facilitation of calcium channel activity has been implicated in a... more Voltage-and frequency-dependent facilitation of calcium channel activity has been implicated in a number of key physiological processes. Various mechanisms have been proposed to mediate these regulations, including a switch between channel gating modes, voltage-dependent phosphorylation, and a voltage-dependent deinhibition of G-protein block. Studying such modulation on recombinant Ca channels expressed in oocytes, we previously reported that α 1C L-type calcium channel contrast with non-L type Ca channels by its ability to exhibit facilitation by pre-depolarization (Voltage-dependent facilitation of a neuronal α IC L-type calcium channel, E. Bourinet et al., EMBO Journal, 1994; 13, 5032-5039). To further analyze this effect, we have investigated the molecular determinants which mediate the differences in voltage-dependent facilitation between «facilitable» α 1C and «non facilitable» α 1E calcium channels. We used a series of chimeras which combine the four transmembrane domains of the two channels. Results show that the four domains of α 1C contribute to facilitation, with domain I being most critical. This domain is required but not sufficient alone to generate facilitation. The minimal requirement to observe the effect is the presence of domain I plus one of the three others. We conclude that similarly to activation gating, voltage-dependent facilitation of α 1C is a complex process which involves multiple structural elements were domains I and III play the major role.
Opioids are commonly thought of as compounds that alleviate pain. A new study finds that elevated... more Opioids are commonly thought of as compounds that alleviate pain. A new study finds that elevated levels of the opioid dynorphin can unexpectedly activate bradykinin receptors, contributing to the maintenance of neuropathic pain. Endogenous opioid peptides are efficient analgesics that bind to opioid receptors and typically are produced by the body to counteract chronic pain, while sparing the acute pain signals that alert us to impending injury 1. Clinically this is relevant to the use of morphine and other exogenous opioids to achieve analgesia. In this issue, however, Lai and colleagues 2 have uncovered a new action of the opioid dynorphin A, which, by virtue of its
The activity of some voltage-gated calcium channels (VGCCs) can be inhibited by specific G protei... more The activity of some voltage-gated calcium channels (VGCCs) can be inhibited by specific G protein beta subunits. Conversely, in the case of N-type VGCCs, protein kinase C can relieve Gbeta-dependent inhibition by phosphorylating at least one specific site on the calcium channel. A recent publication describes a newly identified method of intracellular regulation of specific VGCCs. Wu et al. have uncovered that VGCC activity can be regulated by phosphatidylinositol-4',5'-bisphosphate (PIP2). Whereas PIP2 is important for maintaining the activity (open state) of Cav2.1 (N-type) and Cav2.2 (P/Q-type) channels, the enzymatic breakdown of PIP2 leads to the inactivation of these channels. Additionally, PIP2 can cause changes in voltage-dependent activation of Cav2.2 (P/Q-type) channels that make it more difficult for these channels to open (from the closed state). Furthermore, protein kinase A activity can circumvent PIP2-mediated inhibition. Thus, the PIP2-mediated regulation of VGCCs is tightly controlled by the functions of kinases (and phosphatases), as well as phospholipases. Wu et al. stress that because PIP2 can be found at synapses, PIP2-dependent control of VGCCs "could have profound consequences on synaptic transmission and plasticity."
Background: Mercury is a well-known neurotoxin implicated in a wide range of neurological or psyc... more Background: Mercury is a well-known neurotoxin implicated in a wide range of neurological or psychiatric disorders including autism spectrum disorders, Alzheimer's disease, Parkinson's disease, epilepsy, depression, mood disorders and tremor. Mercury-induced neuronal degeneration is thought to invoke glutamate-mediated excitotoxicity, however, the underlying mechanisms remain poorly understood. Here, we examine the effects of various mercury concentrations (including pathological levels present in human plasma or cerebrospinal fluid) on cultured, rat cortical neurons. Results: We found that inorganic mercuric chloride (HgCl 2-at 0.025 to 25 μM) not only caused neuronal degeneration but also perturbed neuronal excitability. Whole-cell patch-clamp recordings of pyramidal neurons revealed that HgCl 2 not only enhanced the amplitude and frequency of synaptic, inward currents, but also increased spontaneous synaptic potentials followed by sustained membrane depolarization. HgCl 2 also triggered sustained, 2-5 fold rises in intracellular calcium concentration ([Ca 2+ ] i). The observed increases in neuronal activity and [Ca 2+ ] i were substantially reduced by the application of MK 801, a non-competitive antagonist of N-Methyl-D-Aspartate (NMDA) receptors. Importantly, our study further shows that a pre incubation or co-application of MK 801 prevents HgCl 2-induced reduction of cell viability and a disruption of β-tubulin. Conclusions: Collectively, our data show that HgCl 2-induced toxic effects on central neurons are triggered by an over-activation of NMDA receptors, leading to cytoskeleton instability.
Glycerotoxin (GLTx) is capable of stimulating neurotransmitter release at the frog neuromuscular ... more Glycerotoxin (GLTx) is capable of stimulating neurotransmitter release at the frog neuromuscular junction by directly interacting with N-type Ca 2+ (Ca v 2.2) channels. Here we have utilized GLTx as a tool to investigate the functionality of Ca v 2.2 channels in various mammalian neuronal preparations. We first adapted a fluorescent-based high-throughput assay to monitor glutamate release from rat cortical synaptosomes. GLTx potently stimulates glutamate secretion and Ca 2+ influx in synaptosomes with an EC 50 of 50 pM. Both these effects were prevented using selective Ca v 2.2 channel blockers suggesting the functional involvement of Ca v 2.2 channels in mediating glutamate release in this system. We further show that both Ca v 2.1 (P/Q-type) and Ca v 2.2 channels contribute equally to depolarization-induced glutamate release. We then investigated the functionality of Ca v 2.2 channels at the neonatal rat neuromuscular junction. GLTx enhances both spontaneous and evoked neurotransmitter release causing a significant increase in the frequency of postsynaptic action potentials. These effects were blocked by specific Ca v 2.2 channel blockers demonstrating that either GLTx or its derivatives could be used to selectively enhance the neurotransmitter release from Ca v 2.2-expressing mammalian neurons.
Internal application of millimolar concentrations of calcium to batrachotoxin (BTX)-activated rat... more Internal application of millimolar concentrations of calcium to batrachotoxin (BTX)-activated rat skeletal muscle sodium channels, bathed symmetrically in 200 mM NaC1, causes a reduction in apparent singlechannel amplitude without visibly increasing noise at a bandwidth of 50 Hz. A greater calcium-induced reduction occurred upon removal of external sodium ions. Internal calcium acted similarly in high ionic strength solutions (3M NaC1), where surface charges are effectively screened, suggesting that calcium acts, in part, by binding within the pore and occluding the conducting pathway. In low ionic strength solutions (20 mu NaC1), internal addition of N-Methyl-Glucamine (NMG) ions decreased the single channel amplitude consistent with screening of negative surface charges. An accurate description of the dose dependence of calcium inhibition, using either a simple blocking model, or rate theory calculations of ion permeation and block, also required surface charge screening. Hence, our data support the view that sodium current inhibition by internal calcium arises from a combination of both open-channel block and surface charge effects.
P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mamma... more P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The α1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between α 1A and P- and Q-type channels is unknown.
Peptide toxins have proved to be useful agents, both in discriminating between different componen... more Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently
Human retinoblastoma cells are multipotent retinal precursor cells capable of differentiating int... more Human retinoblastoma cells are multipotent retinal precursor cells capable of differentiating into photoreceptors, neurons, and glia. The current-voltage relation of the undifferentiated cells is dominated by a transient inward current that disappears shortly after differentiation. In 20 mM Ba2+-containing bath solutions, the current has an activation midpoint near −25 mV and appears to be fully inactivated at −20 mV. Sr2+and Ca2+ are preferred charge carriers relative to Ba2+, and the current vanishes in the absence of these divalent cations. Cd2+ blocks the current with an IC50 of 160 μM, and Ni2+ blocks in a biphasic manner with IC50s of 22 and 352 μM. The current is unaffected when sodium is replaced with other monovalent cations, and it is insensitive to nifedipine, ω-conotoxin GVIA, ω-agatoxin IVA, and ω-conotoxin MVIIC. RT-PCR revealed the presence of α1G and α1H mRNA in undifferentiated cells, but following differentiation, a striking reduction of both α1G and α1H mRNA was f...
Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, ye... more Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, yet effective therapies are lacking. We identify the pannexin-1 (Panx1) channel as a therapeutic target for alleviating mechanical allodynia, a cardinal sign of arthritis. In rats, joint pain caused by intra-articular injection of monosodium iodoacetate (MIA) was associated with spinal adenosine 5'-triphosphate (ATP) release and a microglia-specific up-regulation of P2X7 receptors (P2X7Rs). Blockade of P2X7R or ablation of spinal microglia prevented and reversed mechanical allodynia. P2X7Rs drive Panx1 channel activation, and in rats with mechanical allodynia, Panx1 function was increased in spinal microglia. Specifically, microglial Panx1-mediated release of the proinflammatory cytokine interleukin-1β (IL-1β) induced mechanical allodynia in the MIA-injected hindlimb. Intrathecal administration of the Panx1-blocking peptide panx suppressed the aberrant discharge of spinal laminae I-II ...
(1) Local anaesthetics (LA) rely for their clinical actions on state-dependent inhibition of volt... more (1) Local anaesthetics (LA) rely for their clinical actions on state-dependent inhibition of voltage-dependent sodium channels. (2) Single, batrachoxin-modified sodium channels in planar lipid bilayers allow direct observation of drug-channel interactions. Two modes of inhibition of single-channel current are observed: fast block of the open channels and prolongation of a long-lived closed state, some of whose properties resemble those of the inactivated state of unmodified channels. (3) Analogues of different parts of the LA molecule separately mimic each blocking mode: amines-fast block, and water-soluble aromatics-closed state prolongation. (4) Interaction between a v-conotoxin derivative and diethylammonium indicate an intrapore site of fast, open-state block. (5) Site-directed mutagenesis studies suggest that hydrophobic residues in transmembrane segment 6 of repeat domain 4 of sodium channels are critical for both LA binding and stabilization of the inactivated state.
Computational modeling is used to investigate the functional impact of G protein-mediated presyna... more Computational modeling is used to investigate the functional impact of G protein-mediated presynaptic autoinhibition on synaptic filtering properties. It is demonstrated that this form of autoinhibition, which is relieved by depolarization, acts as a high-pass filter. This contrasts with vesicle depletion, which acts as a low-pass filter. Model parameters are adjusted to reproduce kinetic slowing data from different Gbetagamma dimeric isoforms, which produce different degrees of slowing. With these sets of parameter values, we demonstrate that the range of frequencies filtered out by the autoinhibition varies greatly depending on the Gbetagamma isoform activated by the autoreceptors. It is shown that G protein autoinhibition can enhance the spatial contrast between a spatially distributed high-frequency signal and surrounding low-frequency noise, providing an alternate mechanism to lateral inhibition. It is also shown that autoinhibition can increase the fidelity of coincidence dete...
Proceedings of the National Academy of Sciences, 1998
We have examined the modulation of cloned and stably expressed rat brain N type calcium channels ... more We have examined the modulation of cloned and stably expressed rat brain N type calcium channels (α 1B + β 1b + α 2 δ subunits) by exogenously applied purified G protein βγ subunits. In the absence of G βγ , barium currents through N type channels are unaffected by application of strong depolarizing prepulses. In contrast, inclusion of purified G βγ in the patch pipette results in N type currents that initially facilitated upon application of positive prepulses followed by rapid reinhibition. Examination of the kinetics of G βγ -dependent reinhibition showed that as the duration between the test pulse and the prepulse was increased, the degree of facilitation was attenuated in a monoexponential fashion. The time constant τ for the recovery from facilitation was sensitive to exogenous G βγ , so that the inverse of τ linearly depended on the G βγ concentration. Overall, the data are consistent with a model whereby a single G βγ molecule dissociates from the channel during the prepulse...
Pflügers Archiv - European Journal of Physiology, 2013
1,4-Dihydropyridines (DHPs) are an important class of L-type calcium channel blockers that are us... more 1,4-Dihydropyridines (DHPs) are an important class of L-type calcium channel blockers that are used to treat conditions such as hypertension and angina. Their primary target in the cardiovascular system is the Cav1.2 L-type calcium channel isoform, however, a number of DHPs also block low-voltage-activated T-type calcium channels. Here, we describe the synthesis of a series of novel DHP derivatives that have a condensed 1,4-DHP ring system (hexahydroquinoline) and report on their abilities to block both L- and T-type calcium channels. Within this series of compounds, modification of a key ester moiety not only regulates the blocking affinity for both L- and T-type channels, but also allows for the development of DHPs with 30-fold selectivity for T-type channels over the L-type. Our data suggest that a condensed dihydropyridine-based scaffold may serve as a pharmacophore for a new class of T-type selective inhibitors.
Pflügers Archiv - European Journal of Physiology, 2013
The L-type voltage-gated calcium channel Cav1.2 is important for excitation-contraction coupling ... more The L-type voltage-gated calcium channel Cav1.2 is important for excitation-contraction coupling in the heart, as well as CREB-mediated transcription in the brain. The ubiquitous calcium-binding protein calmodulin (CaM) is known to modulate calcium-dependent inactivation (CDI) of these channels, thus limiting the amount of calcium entering via Cav1.2 during prolonged or repetitive membrane depolarizations. The proximal N-terminus of Cav1.2 contains a CaMbinding site at residue W52 that is critical for a type of CDI that is mediated by the N-terminal lobe of CaM. Here, we identify a second CaM interaction site in the Cav1.2 Nterminus downstream of the W52 site that is formed by residue C106. We show by site-directed mutagenesis coupled with electrophysiological measurements that this region of the channel functionally partakes in N-lobe CDI, likely by acting as a gating transduction motif. Thus, our data indicate that calcium regulation of Cav1.2 channels is more complex than previously thought, and involves more than one region within the channel's N-terminal domain.
Voltage-and frequency-dependent facilitation of calcium channel activity has been implicated in a... more Voltage-and frequency-dependent facilitation of calcium channel activity has been implicated in a number of key physiological processes. Various mechanisms have been proposed to mediate these regulations, including a switch between channel gating modes, voltage-dependent phosphorylation, and a voltage-dependent deinhibition of G-protein block. Studying such modulation on recombinant Ca channels expressed in oocytes, we previously reported that α 1C L-type calcium channel contrast with non-L type Ca channels by its ability to exhibit facilitation by pre-depolarization (Voltage-dependent facilitation of a neuronal α IC L-type calcium channel, E. Bourinet et al., EMBO Journal, 1994; 13, 5032-5039). To further analyze this effect, we have investigated the molecular determinants which mediate the differences in voltage-dependent facilitation between «facilitable» α 1C and «non facilitable» α 1E calcium channels. We used a series of chimeras which combine the four transmembrane domains of the two channels. Results show that the four domains of α 1C contribute to facilitation, with domain I being most critical. This domain is required but not sufficient alone to generate facilitation. The minimal requirement to observe the effect is the presence of domain I plus one of the three others. We conclude that similarly to activation gating, voltage-dependent facilitation of α 1C is a complex process which involves multiple structural elements were domains I and III play the major role.
Opioids are commonly thought of as compounds that alleviate pain. A new study finds that elevated... more Opioids are commonly thought of as compounds that alleviate pain. A new study finds that elevated levels of the opioid dynorphin can unexpectedly activate bradykinin receptors, contributing to the maintenance of neuropathic pain. Endogenous opioid peptides are efficient analgesics that bind to opioid receptors and typically are produced by the body to counteract chronic pain, while sparing the acute pain signals that alert us to impending injury 1. Clinically this is relevant to the use of morphine and other exogenous opioids to achieve analgesia. In this issue, however, Lai and colleagues 2 have uncovered a new action of the opioid dynorphin A, which, by virtue of its
The activity of some voltage-gated calcium channels (VGCCs) can be inhibited by specific G protei... more The activity of some voltage-gated calcium channels (VGCCs) can be inhibited by specific G protein beta subunits. Conversely, in the case of N-type VGCCs, protein kinase C can relieve Gbeta-dependent inhibition by phosphorylating at least one specific site on the calcium channel. A recent publication describes a newly identified method of intracellular regulation of specific VGCCs. Wu et al. have uncovered that VGCC activity can be regulated by phosphatidylinositol-4',5'-bisphosphate (PIP2). Whereas PIP2 is important for maintaining the activity (open state) of Cav2.1 (N-type) and Cav2.2 (P/Q-type) channels, the enzymatic breakdown of PIP2 leads to the inactivation of these channels. Additionally, PIP2 can cause changes in voltage-dependent activation of Cav2.2 (P/Q-type) channels that make it more difficult for these channels to open (from the closed state). Furthermore, protein kinase A activity can circumvent PIP2-mediated inhibition. Thus, the PIP2-mediated regulation of VGCCs is tightly controlled by the functions of kinases (and phosphatases), as well as phospholipases. Wu et al. stress that because PIP2 can be found at synapses, PIP2-dependent control of VGCCs "could have profound consequences on synaptic transmission and plasticity."
Background: Mercury is a well-known neurotoxin implicated in a wide range of neurological or psyc... more Background: Mercury is a well-known neurotoxin implicated in a wide range of neurological or psychiatric disorders including autism spectrum disorders, Alzheimer's disease, Parkinson's disease, epilepsy, depression, mood disorders and tremor. Mercury-induced neuronal degeneration is thought to invoke glutamate-mediated excitotoxicity, however, the underlying mechanisms remain poorly understood. Here, we examine the effects of various mercury concentrations (including pathological levels present in human plasma or cerebrospinal fluid) on cultured, rat cortical neurons. Results: We found that inorganic mercuric chloride (HgCl 2-at 0.025 to 25 μM) not only caused neuronal degeneration but also perturbed neuronal excitability. Whole-cell patch-clamp recordings of pyramidal neurons revealed that HgCl 2 not only enhanced the amplitude and frequency of synaptic, inward currents, but also increased spontaneous synaptic potentials followed by sustained membrane depolarization. HgCl 2 also triggered sustained, 2-5 fold rises in intracellular calcium concentration ([Ca 2+ ] i). The observed increases in neuronal activity and [Ca 2+ ] i were substantially reduced by the application of MK 801, a non-competitive antagonist of N-Methyl-D-Aspartate (NMDA) receptors. Importantly, our study further shows that a pre incubation or co-application of MK 801 prevents HgCl 2-induced reduction of cell viability and a disruption of β-tubulin. Conclusions: Collectively, our data show that HgCl 2-induced toxic effects on central neurons are triggered by an over-activation of NMDA receptors, leading to cytoskeleton instability.
Glycerotoxin (GLTx) is capable of stimulating neurotransmitter release at the frog neuromuscular ... more Glycerotoxin (GLTx) is capable of stimulating neurotransmitter release at the frog neuromuscular junction by directly interacting with N-type Ca 2+ (Ca v 2.2) channels. Here we have utilized GLTx as a tool to investigate the functionality of Ca v 2.2 channels in various mammalian neuronal preparations. We first adapted a fluorescent-based high-throughput assay to monitor glutamate release from rat cortical synaptosomes. GLTx potently stimulates glutamate secretion and Ca 2+ influx in synaptosomes with an EC 50 of 50 pM. Both these effects were prevented using selective Ca v 2.2 channel blockers suggesting the functional involvement of Ca v 2.2 channels in mediating glutamate release in this system. We further show that both Ca v 2.1 (P/Q-type) and Ca v 2.2 channels contribute equally to depolarization-induced glutamate release. We then investigated the functionality of Ca v 2.2 channels at the neonatal rat neuromuscular junction. GLTx enhances both spontaneous and evoked neurotransmitter release causing a significant increase in the frequency of postsynaptic action potentials. These effects were blocked by specific Ca v 2.2 channel blockers demonstrating that either GLTx or its derivatives could be used to selectively enhance the neurotransmitter release from Ca v 2.2-expressing mammalian neurons.
Internal application of millimolar concentrations of calcium to batrachotoxin (BTX)-activated rat... more Internal application of millimolar concentrations of calcium to batrachotoxin (BTX)-activated rat skeletal muscle sodium channels, bathed symmetrically in 200 mM NaC1, causes a reduction in apparent singlechannel amplitude without visibly increasing noise at a bandwidth of 50 Hz. A greater calcium-induced reduction occurred upon removal of external sodium ions. Internal calcium acted similarly in high ionic strength solutions (3M NaC1), where surface charges are effectively screened, suggesting that calcium acts, in part, by binding within the pore and occluding the conducting pathway. In low ionic strength solutions (20 mu NaC1), internal addition of N-Methyl-Glucamine (NMG) ions decreased the single channel amplitude consistent with screening of negative surface charges. An accurate description of the dose dependence of calcium inhibition, using either a simple blocking model, or rate theory calculations of ion permeation and block, also required surface charge screening. Hence, our data support the view that sodium current inhibition by internal calcium arises from a combination of both open-channel block and surface charge effects.
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Papers by Gerald Zamponi