Opioid Receptor Regulation of Neuronal Voltage-Gated Calcium Channels

Neuronal voltage-gated calcium channels play a pivotal role in the conversion of electrical signals into calcium entry into nerve endings that is required for the release of neurotransmitters. They are under the control of a number of cellular signaling pathways that serve to fine tune synaptic activities, including G-protein coupled receptors (GPCRs) and the opioid system. Besides modulating channel activity via activation of second messengers, GPCRs also physically associate with calcium channels to regulate their function and expression at the plasma membrane. In this mini review, we discuss the mechanisms by which calcium channels are regulated by classical opioid and nociceptin receptors. We highlight the importance of this regulation in the control of neuronal functions and their implication in the development of disease conditions. Finally, we present recent literature concerning the use of novel μ-opioid receptor/nociceptin receptor modulators and discuss their use as potential drug candidates for the treatment of pain.

     

Multiplicity of protein interactions and functions of the voltage-gated calcium channel beta-subunit

For a long time the auxiliary beta-subunit of voltage-gated calcium channels was thought to be engaged exclusively in the regulation of calcium channel function, including gating, intracellular trafficking, assembly and membrane expression. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins (MAGUK) that comprises a series of protein interaction motifs. Two such domains, a Src homology 3 and a guanylate kinase domain are present in the beta-subunit. Recently, it was shown that this subunit interacts with a variety of proteins and regulates diverse cellular processes ranging from gene expression to hormone secretion and endocytosis. In light of these new findings, the beta-subunit deserves to be promoted to the category of multifunctional regulatory protein. Some of these new functions support a tighter regulation of calcium influx through voltage-gated calcium channels and others apparently serve channel unrelated processes. Here we discuss a variety of protein-protein interactions held by the beta-subunit of voltage-gated calcium channels and their functional consequences. Certainly the identification of additional binding partners and effector pathways will help to understand how the different beta-subunit-mediated processes are interwoven.     

Old proteins,developing roles: The regulation of calcium channels by synaptic proteins

Coupling of presynaptic voltage-gated calcium channels to synaptic release machinery is critical for neurotransmission. It was traditionally believed that anchoring calcium channels close to the calcium micro-domain dependent release machinery was the main reason for the physical interactions between channels and synaptic proteins, however in recent years, it is becoming clear that these proteins additionally regulate channel activity, and such processes as channel targeting and alternative splicing, to orchestrate a much broader regulatory role in controlling calcium channel function, calcium influx, and hence neurotransmission. Calcium signalling serves a multitude of cellular functions and therefore requires tight regulation. Specific, often calcium-dependent interactions between synaptic proteins and calcium channels appear to play a significant role in fine-tuning of the synaptic response over development. While it is clear that investigation of a few of the multitude of synaptic proteins will not provide a complete understanding of calcium channel regulation, consideration of the emerging mechanisms by which synaptic protein interactions might regulate calcium channel function is important in order to understand their possible contributions to synaptic transmission. Here, we review the current state of knowledge of the molecular mechanisms by which synaptic proteins regulate presynaptic calcium channel activity.     

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

Coupling of presynaptic voltage-gated calcium channels to the synaptic release machinery is critical for neurotransmission. It was traditionally believed that anchoring calcium channels close to the calcium microdomain dependent release machinery was the main reason for the physical interactions between channels and synaptic proteins, however in recent years, it is becoming clear that these proteins additionally regulate channel activity, and such processes as channel targeting and alternative splicing, to orchestrate a much broader regulatory role in controlling calcium channel function, calcium influx and hence neurotransmission. Calcium signalling serves a multitude of cellular functions and therefore requires tight regulation. Specific, often calcium-dependent interactions between synaptic proteins and calcium channels appear to play a significant role in fine-tuning of the synaptic response over development. While it is clear that investigation of a few of the multitude of synaptic proteins will not provide a complete understanding of calcium channel regulation, consideration of the emerging mechanisms by which synaptic protein interactions might regulate calcium channel function is important in order to understand their possible contributions to synaptic transmission. Here, we review the current state of knowledge of the molecular mechanisms by which synaptic proteins regulate presynaptic calcium channel activity.     

Voltage-gated calcium channels

The article concentrates on representatives of voltage-gated calcium ion channels that are present in practically all cells. Regarded is the molecular arrangement of a voltage-gated calcium channel that consists of pore forming trans-membrane alpha1 subunit and auxiliary alpha2delta-, beta-, and gamma-subunits. Under discussion are the structure and functions of each subunit. The principles of subunits interaction are considered. The research represents modern classification of voltage-gated calcium channels, draws parallels with the earlier classifications and discusses calcium currents going through various calcium channels. Considered are the problems of regulating the activity of voltage-gated channels by proteinkinases. The issues of blockers and activators of voltage-gated calcium channels are brought up. The article gives a detailed analysis of the mechanisms of voltage-gated calcium channels selectivity. The molecular organization of the selectivity filter is considered. Presented are the basic theories of permeability of voltage-gated calcium channels.     

质膜钙离子ATP酶

钙离子(Ca2+)是重要的第二信使,通过与效应蛋白的结合和解离,以及在不同细胞器之间的穿梭运动而精确调控细胞活动,参与多种重要生命过程。细胞内具有精确调节Ca2+时空分布的调控系统。在静息状态下,细胞内的游离Ca2+浓度约为100 nmol/L;而当细胞受到信号刺激后,胞内的Ca2+浓度可上升至1000 nmol/L甚至更高。细胞中存在多种跨膜运送Ca2+的膜蛋白,以精确调节Ca2+浓度的时空动态变化,其中,细胞质膜上的多种Ca2+通道(包括电压门控通道、受体门控通道、储存控制通道等),以及内质网/肌质网和线粒体等胞内"钙库"膜上的雷诺丁受体、三磷酸肌醇受体等膜蛋白复合物,均可提升胞内Ca2+浓度,而细胞质膜上的钠钙交换体、质膜Ca2+-ATP酶、"钙库"膜上的内质网Ca2+-ATP酶、线粒体Ca2+单向转运体等,可将Ca2+浓度降低至静息态水平。质膜钙ATP酶是向细胞外运送Ca2+的关键膜蛋白,本文将对其结构、功能及其酶活性的调控机制做一简要综述。     

SOC and now also SIC: Store-operated and store-inhibited channels

There is a specialized form of calcium influx that involves a close communication between endoplasmic reticulum and the channels at the plasma membrane. In one side store depletion activates channels known as store-operated channels (SOC), which are responsible of the well-studied store-operated calcium entry (SOCE). SOC comprises two different types of channels. Orai, which is exclusively activated by store depletion being the channel responsible of the calcium release-activated calcium current, and transient receptor potential canonical channel, which in contrast, is activated by store depletion only under specific conditions and carries nonselective cationic currents. On the other hand, it has been recently shown that store depletion also inhibits calcium channels. The first member identified, of what we named as store-inhibited channels (SIC), is the L-type voltage-gated calcium channel. Stores control both SOC and SIC by means of the multifunctional protein STIM1. The identification of SOC and SIC opens a new scenario for the role of store depletion in the modulation of different calcium entry pathways, which may satisfy different cellular processes.     

Palmitoylation and Membrane Association of the Stress Axis Regulated Insert (STREX) Controls BK Channel Regulation by Protein Kinase C

Large-conductance, calcium- and voltage-gated potassium (BK) channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis regulated exon (STREX) at splice site 2 inverts BK channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal channel activity by PKC in the insertless (ZERO) splice variant, STREX channels were completely resistant to PKC. STREX channel mutants in which Ser(700), located between the two regulatory domains of K(+) conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ～50% decrease in maximal channel activity, whereas the S700A mutant retained its normal activity. BK channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK channels where PKC fails to phosphorylate and to inhibit channel activity. Importantly, PKA which inhibits channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion channel palmitoylation and phosphorylation in regulation of cellular excitability.     

The Src homology 3 domain of the beta-subunit of voltage-gated calcium channels promotes endocytosis via dynamin interaction

High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the alpha-interaction-domain in the pore-forming alpha1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the alpha-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the alpha1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing alpha1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the alpha-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltage-gated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the alpha1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.     

Mechanisms of Gain Control by Voltage-Gated Channels in Intrinsically-Firing Neurons

Gain modulation is a key feature of neural information processing, but underlying mechanisms remain unclear. In single neurons, gain can be measured as the slope of the current-frequency (input-output) relationship over any given range of inputs. While much work has focused on the control of basal firing rates and spike rate adaptation, gain control has been relatively unstudied. Of the limited studies on gain control, some have examined the roles of synaptic noise and passive somatic currents, but the roles of voltage-gated channels present ubiquitously in neurons have been less explored. Here, we systematically examined the relationship between gain and voltage-gated ion channels in a conductance-based, tonically-active, model neuron. Changes in expression (conductance density) of voltage-gated channels increased (Ca²⁺ channel), reduced (K⁺ channels), or produced little effect (h-type channel) on gain. We found that the gain-controlling ability of channels increased exponentially with the steepness of their activation within the dynamic voltage window (voltage range associated with firing). For depolarization-activated channels, this produced a greater channel current per action potential at higher firing rates. This allowed these channels to modulate gain by contributing to firing preferentially at states of higher excitation. A finer analysis of the current-voltage relationship during tonic firing identified narrow voltage windows at which the gain-modulating channels exerted their effects. As a proof of concept, we show that h-type channels can be tuned to modulate gain by changing the steepness of their activation within the dynamic voltage window. These results show how the impact of an ion channel on gain can be predicted from the relationship between channel kinetics and the membrane potential during firing. This is potentially relevant to understanding input-output scaling in a wide class of neurons found throughout the brain and other nervous systems.     

Slow vacuolar channels from barley mesophyll cells are regulated by 14-3-3 proteins

The conductance of the vacuolar membrane at elevated cytosolic Ca(2+) levels is dominated by the slow activating cation selective (SV) channel. At physiological, submicromolar Ca(2+) concentrations the SV currents are very small. Only recently has the role of 14-3-3 proteins in the regulation of voltage-gated and Ca(2+)-activated plasma membrane ion channels been investigated in Drosophila, Xenopus and plants. Here we report the first evidence that plant 14-3-3 proteins are involved in the down-regulation of ion channels in the vacuolar membrane as well. Using the patch-clamp technique we have demonstrated that 14-3-3 protein drastically reduces the current carried by SV channels. The current decline amounted to 80% and half-maximal reduction was reached within 5 s after 14-3-3-addition to the bath. The voltage sensitivity of the channel was not affected by 14-3-3. A coordinating role for 14-3-3 proteins in the regulation of plasma membrane and tonoplast ion transporters is discussed.     

Expression of the voltage-gated sodium channel NaV1.5 in the macrophage late endosome regulates endosomal acidification

Voltage-gated sodium channels expressed on the plasma membrane activate rapidly in response to changes in membrane potential in cells with excitable membranes such as muscle and neurons. Macrophages also require rapid signaling mechanisms as the first line of defense against invasion by microorganisms. In this study, our goal was to examine the role of intracellular voltage-gated sodium channels in macrophage function. We demonstrate that the cardiac voltage-gated sodium channel, NaV1.5, is expressed on the late endosome, but not the plasma membrane, in a human monocytic cell line, THP-1, and primary human monocyte-derived macrophages. Although the neuronal channel, NaV1.6, is also expressed intracellularly, it has a distinct subcellular localization. In primed cells, NaV1.5 regulates phagocytosis and endosomal pH during LPS-mediated endosomal acidification. Activation of the endosomal channel causes sodium efflux and decreased intraendosomal pH. These results demonstrate a functionally relevant intracellular voltage-gated sodium channel and reveal a novel mechanism to regulate macrophage endosomal acidification.     

L-type calcium channel modulates mechanosensitivity of the cardiomyocyte cell line H9c2

The application of mechanical stimuli to cells often induce increases in intracellular calcium, affecting the regulation of a variety of cell functions. Although the mechanism of mechanotransduction-induced calcium increases has not been fully resolved, the involvement of mechanosensitive ion channels in the plasma membrane and the endoplasmic reticulum has been reported. Here, we demonstrate that voltage-gated L-type calcium channels play a critical role in the mechanosensitive calcium response in H9c2 rat cardiomyocytes. The intracellular calcium level in H9c2 cells increased in a reproducible dose-dependent manner in response to uniaxial stretching. The stretch-activated calcium response (SICR) completely disappeared in calcium-free medium, whereas thapsigargin and cyclopiazonic acid, inhibitors of sarcoendoplasmic reticulum calcium ATPase, partially reduced the SICR. These findings suggest that both calcium influx across the cell membrane and calcium release from the sarcoendoplasmic reticulum are involved in the SICR. Nifedipine, diltiazem, and verapamil, inhibitors of L-type calcium channels, reduced the SICR in a dose-dependent manner. Furthermore, small interfering RNA against the L-type calcium channel α1c subunit diminished the SICR dramatically. Nifedipine also diminished the mechanosensitivity of Langendorff-perfused rat heart. These results suggest that the SICR in H9c2 cardiomyocytes involves the activation of L-type calcium channels and subsequent calcium release from the sarcoendoplasmic reticulum.     

Structural Model of the CaV1.2 Pore

Understanding the structure and functional mechanisms of voltage-gated calcium channels remains a major task in membrane biophysics. In the absence of three dimensional structures, homology modelling techniques are the method of choice, to address questions concerning the structure of these channels. We have developed models of the open Cav1.2 pore, based on the crystal structure of the mammalian voltage-gated potassium channel Kv1.2 and a model of the bacterial sodium channel NaChBac. Our models are developed to be consistent with experimental data and modelling criteria. The models highlight major differences between voltage-gated potassium and calcium channels, in the P segments, as well as the inner pore helices. Molecular dynamics simulations support the hypothesis of a clockwise domain arrangement and experimental observations of asymmetric calcium channel behaviour. In the accompanying paper these models were used to study structural effects of a channelopathy mutation.     

Structural model of the Ca V 1.2 pore

Understanding the structure and functional mechanisms of voltage-gated calcium channels remains a major task in membrane biophysics. In the absence of three dimensional structures, homology modeling techniques are the method of choice, to address questions concerning the structure of these channels. We have developed models of the open Ca(V)1.2 pore, based on the crystal structure of the mammalian voltage-gated potassium channel K(V)1.2 and a model of the bacterial sodium channel NaChBac. Our models are developed to be consistent with experimental data and modeling criteria. The models highlight major differences between voltage-gated potassium and calcium channels in the P segments, as well as the inner pore helices. Molecular dynamics simulations support the hypothesis of a clockwise domain arrangement and experimental observations of asymmetric calcium channel behavior. In the accompanying paper these models were used to study structural effects of a channelopathy mutation.     

Hysteresis in voltage-gated channels

Ion channels constitute a superfamily of membrane proteins found in all living creatures. Their activity allows fast translocation of ions across the plasma membrane down the ion's transmembrane electrochemical gradient, resulting in a difference in electrical potential across the plasma membrane, known as the membrane potential. A group within this superfamily, namely voltage-gated channels, displays activity that is sensitive to the membrane potential. The activity of voltage-gated channels is controlled by the membrane potential, while the membrane potential is changed by these channels' activity. This interplay produces variations in the membrane potential that have evolved into electrical signals in many organisms. These signals are essential for numerous biological processes, including neuronal activity, insulin release, muscle contraction, fertilization and many others. In recent years, the activity of the voltage-gated channels has been observed not to follow a simple relationship with the membrane potential. Instead, it has been shown that the activity of voltage-gated channel displays hysteresis. In fact, a growing number of evidence have demonstrated that the voltage dependence of channel activity is dynamically modulated by activity itself. In spite of the great impact that this property can have on electrical signaling, hysteresis in voltage-gated channels is often overlooked. Addressing this issue, this review provides examples of voltage-gated ion channels displaying hysteretic behavior. Further, this review will discuss how Dynamic Voltage Dependence in voltage-gated channels can have a physiological role in electrical signaling. Furthermore, this review will elaborate on the current thoughts on the mechanism underlying hysteresis in voltage-gated channels.     

Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I

Calcium plays a critical part in the regulation of cell growth, and growth factors stimulate calcium entry into cells through calcium-permeable channels. However, the molecular nature and regulation of calcium-permeable channels are still unclear at present. Here we report the molecular characterization of a calcium-permeable cation channel that is regulated by insulin-like growth factor-I (IGF-I). This channel, which we name growth-factor-regulated channel (GRC), belongs to the TRP-channel family and localizes mainly to intracellular pools under basal conditions. Upon stimulation of cells by IGF-I, GRC translocates to the plasma membrane. Thus, IGF-I augments calcium entry through GRC by regulating trafficking of the channel.