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Examples of single-channel activity and its analysis, for 1B in combination with 2a in the presence of G12. (a) Top voltage trace: Holding potential 100 mV, test potential in both P1 and P2, to the indicated value, 100 ms, separated by a strong depolarizing prepulse (120 mV, 50 ms). Steps were delivered every 2 s. The voltage protocol is followed by five representative traces and an ensemble of 200 episodes (bottom trace) for each voltage. The zero current line that runs through the traces represents the closed state; openings are downward deflections. The bars to the right of the first trace represent 1 pA and 100 ms, and the bar to the right of the ensemble current trace represents 0.2 pA (this applies to all of the voltages). (b) Open time distribution histograms corresponding to the experiment shown in a for P1 (top histogram) and P2 (bottom histogram). For each voltage 200 episodes were collected. The bin width was 0.5 ms. The mean open time at each voltage is indicated. (c) Open level amplitude histograms for the different voltages in P1 (bars) and P2 (line). The bin width was 0.02 pA. The mean amplitude is indicated for P1 and P2.
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Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the...
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... detection was carried out using the half-amplitude threshold method. Singlechannel amplitude was determined, either by a Gaussian fit to the binned amplitude distributions, or by the mean amplitude in cases when there was a small number of events or multiple conductance modes (see Fig. 2 c). However, the values obtained here (13pS) are smaller than those reported elsewhere (see, for example, Carabelli et al., 1996) and by us ). ...
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... analyzed single-channel records over a wide range of voltages ( Fig. 2 a) and compared a number of parameters, including single-channel ensemble current (Fig. 2 a), mean open time (Fig. 2 b), mean amplitude ( Fig. 2 c), and latency to first opening. The behavior of single channels recorded from cell-attached patches of COS-7 cells transfected with 1B with or without a VDCC auxiliary subunit and either G or ...
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... analyzed single-channel records over a wide range of voltages ( Fig. 2 a) and compared a number of parameters, including single-channel ensemble current (Fig. 2 a), mean open time (Fig. 2 b), mean amplitude ( Fig. 2 c), and latency to first opening. The behavior of single channels recorded from cell-attached patches of COS-7 cells transfected with 1B with or without a VDCC auxiliary subunit and either G or ARK1 (Fig. 3 and 4) was compared using these parameters. These parameters enable one to ...
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... analyzed single-channel records over a wide range of voltages ( Fig. 2 a) and compared a number of parameters, including single-channel ensemble current (Fig. 2 a), mean open time (Fig. 2 b), mean amplitude ( Fig. 2 c), and latency to first opening. The behavior of single channels recorded from cell-attached patches of COS-7 cells transfected with 1B with or without a VDCC auxiliary subunit and either G or ARK1 (Fig. 3 and 4) was compared using these parameters. These parameters enable one to distinguish between the ...
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... analyzed single-channel records over a wide range of voltages ( Fig. 2 a) and compared a number of parameters, including single-channel ensemble current (Fig. 2 a), mean open time (Fig. 2 b), mean amplitude ( Fig. 2 c), and latency to first opening. The behavior of single channels recorded from cell-attached patches of COS-7 cells transfected with 1B with or without a VDCC auxiliary subunit and either G or ARK1 (Fig. 3 and 4) was compared using these parameters. These parameters enable one to distinguish between the effects of either modulatory ...
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... the mean open time and mean amplitude represent compound processes (see Fig. 2 c for the compound amplitude distributions). The open time distribution of expressed ( Wakamori et al., 1999) N-type channels was described by the sum of several exponentials. However, because of the exclusion of the shortest open times in our analysis, the contribution of the shorter exponent could be misinterpreted. We therefore ...
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... The open time distribution of expressed ( Wakamori et al., 1999) N-type channels was described by the sum of several exponentials. However, because of the exclusion of the shortest open times in our analysis, the contribution of the shorter exponent could be misinterpreted. We therefore chose to examine the arithmetic mean open time (Fig. 2 b). Changes in this parameter could therefore be a result of either a change in the distribution between short and long opening populations or in the time constants for channel ...
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... differential coexpression of 1B with either ARK G binding domain or G dimers is advantageous because it allows a comparison between G-depleted and saturated populations, respectively. The ensemble average currents obtained here (Fig. 2 a) represent the changes in ...
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... facilitation that is a hallmark of voltage-dependent G protein modulation (Dolphin, 1995) was also strongly dependent on VDCC subunit coexpression. With 2a, clear facilitation due to a depolarizing prepulse was evident for whole-cell ( Stephens et al., 1998) (Figs. 1 b and 6, d and e) and single-channel (Figs. 2-5) currents. With the VDCC 1b subunit coexpressed, a more complex behavior was observed, but clear facilitation (although partially overlaid by inactivation at higher potentials) was also evident (Fig. 5 c). The facilitation was time dependent at all voltages (Fig. 5 b), involving the addition of a fast G-unbound population to the gating ...
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This chapter describes a general method for expressing several G-protein α subunits in Escherichia coli (E. coli) at levels 10-100 times higher than achieved previously. G proteins are a family of guanine nucleotide-binding regulatory proteins that link a large number of cell surface receptors to regulation of several intracellular effectors. The c...
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... In practice, both the topology (the number and connectivity of discrete states) and the transition parameters of such models are developed in accordance with experimental data, which mainly come in the form of electrophysiological records [354][355][356]. Pulse from +10 to +50 mV with a duration of 100 ms (holding potential of −100 mV) Pulse from −10 to +10 mV with a duration of 100 ms (holding potential of −100 mV) [364,365] 12 ...
The presence of biological rhythms is a characteristic of all living organisms. Over the past 60 years, scientists around the world have accumulated a huge amount of data on rhythmic processes in living systems at various levels. The acquired knowledge has found applications in human economic activity and medicine. The ultradian (less than a day) rhythms at the organismal, organ, and cellular levels are characterized by high diversity. Unfortunately, biorhythms in different systems are considered, most often, in isolation from each other. Much knowledge about biorhythms was obtained using expert evaluation methods, and later methods of spectral analysis were used to describe biorhythms. Ultradian rhythms have a relatively short duration; therefore, they can be characterized by spectral analysis methods. More and more researchers believe that in order to further expand the understanding of the nature and purpose of biorhythms, the use of more advanced methods of mathematical processing is required, and rhythms in different organs, tissues, and cells should be considered parts of a single system. This review is intended to provide the reader with the variety of ultradian rhythms in living systems (organismal, organ, cellular, molecular levels), the mechanisms of their generation, and their functions to give the reader a picture of the possible relationships between these rhythms. Further, the reader will be able to get acquainted with the variety of mathematical methods for analyzing biorhythms, including bispectral and cross-correlation analyses.
... Direct Gβγ modulation of Cav2.2 is sensitive to the membrane potential and can be reversed by the application of a depolarizing pre-pulse or by trains of action potentials-it is therefore referred to as voltage-dependent inhibition [63][64][65]. The extent of G protein modulation is also dependent on the Cavβ subunit [66][67][68] and critically dependent on the Gβ isoform [61,69], thus allowing receptors to mediate a wide range of inhibitory voltage-dependent processes. Many G protein-coupled receptors also cause voltage-independent modulation of channel activity, most likely via Fig. 1 Key aspects of NOP receptor signalling. A. NOP receptors at synaptic terminals. ...
Activation of nociceptin opioid peptide receptors (NOP, a.k.a. opioid-like receptor-1, ORL-1) by the ligand nociceptin/orphanin FQ, leads to G protein-dependent regulation of Cav2.2 (N-type) voltage-gated calcium channels (VGCCs). This typically causes a reduction in calcium currents, triggering changes in presynaptic calcium levels and thus neurotransmission. Because of the widespread expression patterns of NOP and VGCCs across multiple brain regions, the dorsal horn of the spinal cord, and the dorsal root ganglia, this results in the alteration of numerous neurophysiological features. Here we review the regulation of N-type calcium channels by the NOP-nociceptin system in the context of neurological conditions such as anxiety, addiction, and pain.
... Notably, upon strong membrane depolarisation, Gβγ uncouples from α1. Essential for the VD dissociation of Gβγ is the presence of CaVβ, binding to similar regions in the I-II linker (Meir et al. 2000;Currie 2010). Besides binding to the I-II linker, further binding sites for Gβγ were identified on the N-terminus (Page et al. 1998;Canti et al. 1999) and on the C-terminus of α1B. ...
In the mammalian brain, presynaptic CaV2.2 channels play a pivotal role for synaptic transmission by mediating fast neurotransmitter exocytosis via influx of Ca2+ into the active zone at the presynaptic terminal. The distribution and activity of CaV2.2 channels at different synapses and maturity stages in the brain remains to be elucidated. In this study, I first show high levels of CaV2.2 channels in mouse cortex and hippocampus throughout development, persisting into adulthood. In contrast, CaV2.2 channels in the cerebellum and brain stem decreased as the brain matured. I thereafter assessed CaV2.2 channels during homeostatic synaptic plasticity, a compensatory form of homeostatic control preventing excessive or insufficient neuronal activity during which extensive active zone remodelling has been described. In this work I show that chronic silencing of neuronal activity in mature hippocampal cultures resulted in elevated presynaptic Ca2+ transients, mediated by a 30 % increase in CaV2.2 channel levels at the presynapse. Next, this work focussed on α2δ-1 subunits, important regulators of synaptic transmission and CaV2.2 channel abundance at the presynaptic membrane. Here, I show that α2δ-1- overexpression reduces the contribution of CaV2.2 channels to total Ca2+ flux without altering the amplitude of the Ca2+ transients. Finally, levels of endogenous α2δ-1 decreased during homeostatic synaptic plasticity, whereas the overexpression of α2δ-1 prevented homeostatic synaptic plasticity in hippocampal neurons. Together, this study reveals a key role for CaV2.2 channels and novel roles for α2δ-1 during plastic synaptic adaptation.
... High-voltage activated VGCCs are heteromers composed of up to four subunits (Ca V α1, Ca V β, Ca V α 2 -δ, Ca V γ) (Buraei and Yang 2010;Campiglio and Flucher 2015). The subunit of particular importance for the regulation of channel function is the Ca V β-subunit, with four different isoforms (Ca V β 1-4 ) involved in the regulation of voltage dependence, channel activity, current kinetics, and also the membrane targeting of functional channels (Meir et al. 2000;Dolphin 2012). Structurally, Ca V β-subunits consist of five domains: a variable N-terminus, a conserved Src homology 3 domain (SH3), a conserved but alternatively spliced HOOK domain, a guanylate kinase domain (GK), and a variable C-terminus (Buraei and Yang 2010). ...
Voltage-gated calcium channel (VGCC) subunits have been genetically associated with autism spectrum disorders (ASD). The properties of the pore-forming VGCC subunit are modulated by auxiliary β-subunits, which exist in four isoforms (Ca V β 1-4 ). Our previous findings suggested that activation of L-type VGCCs is a common feature of Ca V β 2 subunit mutations found in ASD patients. In the current study, we functionally characterized a novel Ca V β 1b variant (p.R296C) identified in an ASD patient. We used whole-cell and single-channel patch clamp to study the effect of Ca V β 1b_R296C on the function of L- and N-type VGCCs. Furthermore, we used co-immunoprecipitation followed by Western blot to evaluate the interaction of the Ca V β 1b -subunits with the RGK-protein Gem. Our data obtained at both, whole-cell and single-channel levels, show that compared to a wild-type Ca V β 1b , the Ca V β 1b_R296C variant inhibits L- and N-type VGCCs. Interaction with and modulation by the RGK-protein Gem seems to be intact. Our findings indicate functional effects of the Ca V β 1b_R296C variant differing from that attributed to Ca V β 2 variants found in ASD patients. Further studies have to detail the effects on different VGCC subtypes and on VGCC expression.
... Inhibition by Gβγ is voltage-dependent and large or repeated depolarizations of the presynaptic terminal could overcome the inhibition. 41 Gβγ also activates voltage-gated potassium channels (Kv's) via a mechanism involving phospholipase A (PLA). The overall effect of decrease calcium ion influx and increased potassium ion efflux is hyperpolarization and inhibition of neurotransmitter release, therefore decreasing GABA-mediated inhibition of output neurons concentrations in the neurons, which has a variety of cellular effects. ...
Opioids are a commonly prescribed and efficacious medication for the treatment of chronic pain but major side effects such as addiction, respiratory depression, analgesic tolerance, and paradoxical pain hypersensitivity make them inadequate and unsafe for patients requiring long‐term pain management. This review summarizes recent advances in our understanding of the outcomes of chronic opioid administration to lay the foundation for the development of novel pharmacological strategies that attenuate opioid tolerance and hypersensitivity; the two main physiological mechanisms underlying the inadequacies of current therapeutic strategies. We also explore mechanistic similarities between the development of neuropathic pain states, opioid tolerance, and hypersensitivity which may explain opioids’ lack of efficacy in certain patients. The findings challenge the current direction of analgesic research in developing non‐opioid alternatives and we suggest that improving opioids, rather than replacing them, will be a fruitful avenue for future research. The PAG‐RVM descending system under normal conditions. In the naïve state, GABAergic interneurons are tonically active, thus both PAG output neurons and OFF‐cells have low spontaneous firing rates. Activity in OFF‐cells causes antinociception, and activity in ON‐cells represents descending facilitation of pain. The normal activity of ON‐cells is also low, such that the overall balance between ON‐cell and OFF‐cell output to the dorsal horn (DH) is equal and there is no net hypersensitive or antinociceptive state in the individual.
... In our work, we concentrated initially on the role of the calcium channel b subunits . We then uncovered an essential role for the N-terminus of Ca V 2 calcium channels in their G-protein modulation (Page et al., 1998;Canti et al., 1999), and we demonstrated the importance of calcium channel b subunits in the G-protein modulation of Ca V 2.2 channels (Meir et al., 2000;Leroy et al., 2005), and also in PI3 kinase-mediated calcium channel modulation (Viard et al., 2004). ...
In this short review, I describe from personal experience how every step in the career of any scientist, no matter how disjointed and pragmatic each might seem at the time, will almost inevitably meld together, to help us all tackle novel projects. My postdoctoral research in Paul Greengard's lab, where I investigated neurotransmitter-mediated phosphorylation of Synapsin I, was instrumental in my career progression, and Paul's support was instrumental in my ability to make a leap into independent research.
... Four isoforms of the Ca V β subunit are described (β 1 -β 4 ), each having different splice variants that might interact with any α 1 channel isoform in a tissue-dependent manner [11,47]. In addition to their essential effect on the forward trafficking of VGCCs [48,49], β subunits can also modulate the channel's kinetics, reported as a shift of the activation potential and raise of the opening probability of HVA channels resulting in larger current densities [14,50,51]. More surprisingly, several studies uncovered a calcium channel-independent function of β 4 which has been shown to translocate to the nucleus and might be directly involved in activity-dependent gene regulation. ...
Voltage-gated calcium channels (VGCCs) represent key regulators of the calcium influx through the plasma membrane of excitable cells, like neurons. Activated by the depolarization of the membrane, the opening of VGCCs induces very transient and local changes in the intracellular calcium concentration, known as calcium nanodomains, that in turn trigger calcium-dependent signaling cascades and the release of chemical neurotransmitters. Based on their central importance as concierges of excitation-secretion coupling and therefore neuronal communication, VGCCs have been studied in multiple aspects of neuronal function and malfunction. However, studies on molecular interaction partners and recent progress in omics technologies have extended the actual concept of these molecules. With this review, we want to illustrate some new perspectives of VGCCs reaching beyond their function as calcium-permeable pores in the plasma membrane. Therefore, we will discuss the relevance of VGCCs as voltage sensors in functional complexes with ryanodine receptors, channel-independent actions of auxiliary VGCC subunits, and provide an insight into how VGCCs even directly participate in gene regulation. Furthermore, we will illustrate how structural changes in the intracellular C-terminus of VGCCs generated by alternative splicing events might not only affect the biophysical channel characteristics but rather determine their molecular environment and downstream signaling pathways.
... Experiments using global m-opioid receptor knockout mice indicate that m-opioid receptors are the primary mediators of both the analgesic and respiratory depressant effects of morphine (Dahan et al., 2001;Matthes et al., 1996). At the intracellular level, opioid agonist binding to the m-opioid receptor leads to the activation of G-protein-coupled inwardly rectifying potassium (GIRK) channels (Reuveny et al., 1994;Whorton and MacKinnon, 2011), and inhibition of specific presynaptic voltagegated calcium channels (Herlitze et al., 1996;Meir et al., 2000). GIRK channel activation has been described as the primary mechanism for respiratory depression by opioids (Levitt et al., 2015;Montandon et al., 2011Montandon et al., , 2016a. ...
Various drugs alter respiratory function however patients receiving chronic opioid therapy for pain control are at risk for exceptionally complex and potentially lethal disorders of breathing during sleep. In addition to central apneas in as many as 75% of these patients, other disturbances of respiration during sleep are also frequently observed including ataxic breathing (Biot's respiration), hypoventilation and obstructive apneas. All commercially available narcotics are μ-opioid receptor agonists and reduce neuronal activity of both pain and respiratory neurons including those found in discrete regions of the brainstem involved with respiratory control. These drugs suppress all parameters of respiration: breathing frequency, tidal volume, chest and abdominal wall compliance, upper airway patency, cough reflex, and chemoresponsiveness to hypercapnia and hypoxia. The clinical presentation of patients with opioid induced central apneas during sleep is further complicated by numerous co-morbidities and the use of other medications. There is currently no consensus regarding optimal therapy. Patients receiving opioids respond paradoxically to continuous positive airway pressure. Some may respond satisfactorily to adaptive servo-ventilation or bilevel positive airway pressure with a back-up rate. Oxygen therapy, alone or with airway pressure, is indicated to correct hypoxemia.
... Thus, D 2 R activation leads to an attenuation of postsynaptic excitation. This "inhibition of excitation" is likely caused by the D 2 R activating the G βγ protein, which inhibits the N-type Ca 2+ channels (Meir et al., 2000;Davila et al., 2003;Bettler et al., 2004). The N-type Ca 2+ channels are involved in the vesicular release cascade (Miller, 1987;Weber et al., 2010), which may gate glutamate release from the OSN and M/TCs (Isaacson and Strowbridge, 1998). ...
In the central nervous system, dopamine is well-known as the neuromodulator that is involved with regulating reward, addiction, motivation, and fine motor control. Yet, decades of findings are revealing another crucial function of dopamine: modulating sensory systems. Dopamine is endogenous to subsets of neurons in the retina and olfactory bulb (OB), where it sharpens sensory processing of visual and olfactory information. For example, dopamine modulation allows the neural circuity in the retina to transition from processing dim light to daylight and the neural circuity in the OB to regulate odor discrimination and detection. Dopamine accomplishes these tasks through numerous, complex mechanisms in both neural structures. In this review, we provide an overview of the established and emerging research on these mechanisms and describe similarities and differences in dopamine expression and modulation of synaptic transmission in the retinas and OBs of various vertebrate organisms. This includes discussion of dopamine neurons’ morphologies, potential identities, and biophysical properties along with their contributions to circadian rhythms and stimulus-driven synthesis, activation, and release of dopamine. As dysregulation of some of these mechanisms may occur in patients with Parkinson’s disease, these symptoms are also discussed. The exploration and comparison of these two separate dopamine populations shows just how remarkably similar the retina and OB are, even though they are functionally distinct. It also shows that the modulatory properties of dopamine neurons are just as important to vision and olfaction as they are to motor coordination and neuropsychiatric/neurodegenerative conditions, thus, we hope this review encourages further research to elucidate these mechanisms.
... Gβ-mediated inhibition of Cav2 channels is voltage-dependent in that it can be relieved by strong or repeated depolarizations which cause the unbinding of Gβϒ from the channel 158 . It was subsequently found that CaV β subunits are required for voltage-dependent inhibition of Cav2 channels by Gβ 159,160 . Although Gβ was found to interact with a QxxER motif within the AID, this site was previously identified to be partially occupied by CaVβ 161 . ...
... Some G protein-coupled receptors (GPCRs) inhibit CaV2 channel activity via their Gßγ subunits. This requires the presence of a conserved RAR motif in the N-terminal sequence of the channel, and also involves Cav ß binding to the I-II linker 159,162 . GPCRs coupled to Gq/11 inhibit Cav2 channels by reducing levels of PIP2 (and activating PKC, which phosphorylates the AID 214 a| Cav2 channel-protein interactions that promote or inhibit presynaptic Cav channel function (depicted along the color-coded gradient to indicate strength of stimulatory (red) and inhibitory (blue) modulation) are major determinants of the amount of neurotransmitter release at the synapse 88,100,105,106,108,113,115,117,118,[121][122][123]153,154,[170][171][172]175,179,182,183,186 . ...
Chemical synapses are heterogeneous junctions formed between neurons that are specialized for the conversion of electrical impulses into the exocytotic release of neurotransmitters. Voltage-gated Ca2+ channels play a pivotal role in this process as they are the major conduits for the Ca2+ ions that trigger the fusion of neurotransmitter-containing vesicles with the presynaptic membrane. Alterations in the intrinsic function of these channels and their positioning within the active zone can profoundly alter the timing and strength of synaptic output. Advances in optical and electron microscopic imaging, structural biology and molecular techniques have facilitated recent breakthroughs in our understanding of the properties of voltage-gated Ca2+ channels that support their presynaptic functions. Here we examine the nature of these channels, how they are trafficked to and anchored within presynaptic boutons, and the mechanisms that allow them to function optimally in shaping the flow of information through neural circuits. Voltage-gated calcium channels have an essential role in the regulation of neurotransmitter release. Dolphin and Lee describe here how advances in the techniques available to study presynaptic voltage-gated calcium channels have provided insight into their composition, trafficking, regulation and contributions to presynaptic function.
