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.

     

Regulation of N-type Voltage-Gated Calcium Channels and Presynaptic Function by Cyclin-Dependent Kinase 5

N-type voltage-gated calcium channels localize to?presynaptic nerve terminals and mediate key events?including synaptogenesis and neurotransmission.?While several kinases have been implicated in the modulation of calcium channels, their impact on presynaptic functions remains unclear. Here we report that the N-type calcium channel is a substrate for cyclin-dependent kinase 5 (Cdk5). The pore-forming α(1) subunit of the N-type calcium channel is phosphorylated in the C-terminal domain, and phosphorylation results in enhanced calcium influx due to increased channel open probability. Phosphorylation of the N-type calcium channel by Cdk5 facilitates neurotransmitter release and alters presynaptic plasticity by increasing the number of docked vesicles at the synaptic cleft. These effects are mediated by an altered interaction between N-type calcium channels and RIM1, which tethers presynaptic calcium channels to the active zone. Collectively, our results highlight a molecular mechanism by which N-type calcium channels are regulated by Cdk5 to affect presynaptic function.     

Regulation of Sodium and Calcium Channels by Signaling Complexes

Membrane depolarization and intracellular calcium transients generated by activation of voltage-gated sodium and calcium channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation-contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. In this article, we review recent experimental results showing that sodium and calcium channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein-protein interactions are important for efficient synaptic transmission and for regulation of ion channels by neurotransmitters and intracellular second messengers. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation-contraction coupling.     

Seeing the Forest through the Trees: Considering Roost-Site Selection at Multiple Spatial Scales

Conservation of bat species is one of the most daunting wildlife conservation challenges in North America, requiring detailed knowledge about their ecology to guide conservation efforts. Outside of the hibernating season, bats in temperate forest environments spend their diurnal time in day-roosts. In addition to simple shelter, summer roost availability is as critical as maternity sites and maintaining social group contact. To date, a major focus of bat conservation has concentrated on conserving individual roost sites, with comparatively less focus on the role that broader habitat conditions contribute towards roost-site selection. We evaluated roost-site selection by a northern population of federally-endangered Indiana bats (Myotis sodalis) at Fort Drum Military Installation in New York, USA at three different spatial scales: landscape, forest stand, and individual tree level. During 2007–2011, we radiotracked 33 Indiana bats (10 males, 23 females) and located 348 roosting events in 116 unique roost trees. At the landscape scale, bat roost-site selection was positively associated with northern mixed forest, increased slope, and greater distance from human development. At the stand scale, we observed subtle differences in roost site selection based on sex and season, but roost selection was generally positively associated with larger stands with a higher basal area, larger tree diameter, and a greater sugar maple (Acer saccharum) component. We observed no distinct trends of roosts being near high-quality foraging areas of water and forest edges. At the tree scale, roosts were typically in American elm (Ulmus americana) or sugar maple of large diameter (>30 cm) of moderate decay with loose bark. Collectively, our results highlight the importance of considering day roost needs simultaneously across multiple spatial scales. Size and decay class of individual roosts are key ecological attributes for the Indiana bat, however, larger-scale stand structural components that are products of past and current land use interacting with environmental aspects such as landform also are important factors influencing roost-tree selection patterns.     

A New Approach to Homeostatic Regulation: Towards a Unified View of Physiological and Ecological Concepts

Stoichiometric homeostasis is the ability of an organism to keep its body chemical composition constant, despite varying inputs. Stoichiometric homeostasis therefore constrains the metabolic needs of consumers which in turn often feed on resources not matching these requirements. In a broader context, homeostasis also relates to the capacity of an organism to maintain other biological parameters (e.g. body temperature) at a constant level over ambient environmental variations. Unfortunately, there are discrepancies in the literature and ecological and physiological definitions of homeostasis are disparate and partly contradictory. Here, we address this matter by reviewing the existing knowledge considering two distinct groups, regulators and conformers and, based on examples of thermo- and osmoregulation, we propose a new approach to stoichiometric homeostasis, unifying ecological and physiological concepts. We suggest a simple and precise graphical way to identify regulators and conformers: for any given biological parameter (e.g. nutrient stoichiometry, temperature), a sigmoidal relation between internal and external conditions can be observed for conformers while an inverse sigmoidal response is characteristic of regulators. This new definition and method, based on well-studied physiological mechanisms, unifies ecological and physiological approaches and is a useful tool for understanding how organisms are affected by and affect their environment.     

Fetal Calcium Regulates Branching Morphogenesis in the Developing Human and Mouse Lung: Involvement of Voltage-Gated Calcium Channels

Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9–17 of human gestation, embryonic days (E)11.5–16.5 in mouse) in a hypercalcaemic environment (∼1.7 in the fetus vs. ∼1.1–1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca²⁺ channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (Ca_V1.2 and Ca_V1.3), P/Q type (Ca_V2.1), N-type (Ca_V2.2), R-type (Ca_V2.3), and T-type (Ca_V3.2 and Ca_V3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Ca_v1.2 and Ca_v1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Ca_v2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Ca_v2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC₂(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match growth and distension within the developing lung.     

Important Role of Asparagines in Coupling the Pore and Votage-Sensor Domain in Voltage-Gated Sodium Channels

Voltage-gated sodium (Na_V) channels contain an α-subunit incorporating the channel’s pore and gating machinery composed of four homologous domains (DI–DIV), with a pore domain formed by the S5 and S6 segments and a voltage-sensor domain formed by the S1–S4 segments. During a membrane depolarization movement, the S4s in the voltage-sensor domains exert downstream effects on the S6 segments to control ionic conductance through the pore domain. We used lidocaine, a local anesthetic and antiarrhythmic drug, to probe the role of conserved Asn residues in the S6s of DIII and DIV in Na_V1.5 and Na_V1.4. Previous studies have shown that lidocaine binding to the pore domain causes a decrease in the maximum gating (Q_max) charge of ∼38%, and three-fourths of this decrease results from the complete stabilization of DIII-S4 (contributing a 30% reduction in Q_max) and one-fourth is due to partial stabilization of DIV-S4 (a reduction of 8–10%). Even though substitutions for the Asn in DIV-S6 in Na_V1.5, N1764A and N1764C, produce little ionic current in transfected mammalian cells, they both express robust gating currents. Anthopleurin-A toxin, which inhibits movement of DIV-S4, still reduced Q_max by nearly 30%, a value similar to that observed in wild-type channels, in both N1764A and N1764C. By applying lidocaine and measuring the gating currents, we demonstrated that Asn residues in the S6s of DIII and DIV are important for coupling their pore domains to their voltage-sensor domains, and that Ala and Cys substitutions for Asn in both S6s result in uncoupling of the pore domains from their voltage-sensor domains. Similar observations were made for Na_V1.4, although substitutions for Asn in DIII-S6 showed somewhat less uncoupling.     

Ancient Origins of RGK Protein Function: Modulation of Voltage-Gated Calcium Channels Preceded the Protostome and Deuterostome Split

RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca²⁺ channel β subunits to modify voltage-gated Ca²⁺ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein–Ca²⁺ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca²⁺ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein – Ca_vβ subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca²⁺ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago.     

Evolution Education: Seeing the Forest for the Trees and Focusing Our Efforts on the Teaching of Evolution

Evolution is the underlying framework upon which all biology is based; however, when it comes to learning evolutionary concepts, many students encounter obstacles. There are many reasons as to why these obstacles occur. These reasons deal with evolution being treated as a discrete topic among many within a biology curriculum, misunderstanding the nature of science, and personal difficulties with understanding due to evolution’s seemingly abstract nature. In this article, we propose a different way of thinking about and teaching evolution in grades K-12, and it surrounds four core areas essential to the understanding of evolution: variation, selection, inheritance, and deep time. Possibilities for how these areas can affect learning are described and implications for assessment are also discussed.     

Two Voltage-Gated,Calcium Release Channels Coreside in the Vacuolar Membrane of Broad Bean Guard Cells

Voltage-gated, Ca2+ release channels have been characterized at the vacuolar membrane of broad bean guard cells using patch clamps of excised, inside-out membrane patches. The most prevalent Ca2+ release channel had a conductance of 27 pS over voltages negative of the reversal potential (Erev) (cytosol referenced to vacuole), with 5,10, or 20 mM Ca2+ as the charge carrier on the vacuolar side and 50 mM K+ on the cytosolic side. The single-channel current saturated at ~2.6 pA. The relative permeability of the channel was in the range of a Pca2+:Pk+ ratio of 6:1. Divalent cations could act as charge carriers on the vacuolar side with a conductance series of Ba2+ > Mg2+ > Sr2+ > Ca2+ and a selectivity sequence of Ca2+ [approximately equals to] Ba2+ [approximately equals to] Sr2+ > Mg2+. The channel was gated open by cytosol-negative (physiological) transmembrane voltages, increases in vacuolar Ca2+ concentration, and increases in the vacuolar pH. The channel was potently inhibited by the Ca2+ channel blockers Gd3+ (half-maximal inhibition at 10.3 [mu]M) and nifedipine (half-maximal inhibition at 77 [mu]M). The stilbene derivative 4,4[prime]-diisothiocyano-2,2[prime]-stilbene disulfonate was also inhibitory (half-maximal inhibition for a 4-min incubation period at 6.3[mu]M). The 27-pS channel coresides in individual guard cell vacuoles with a less frequently observed 14-pS Ca2+ release channel that had similar, although not identical, voltage dependence and gating characteristics and a lower selectivity for Ca2+ over K+. The requirement for two channels with a similar function at the vacuolar membrane of guard cells is discussed.     

Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl⁻ ion channels that are activated by intracellular Ca²⁺. At present, the structures and mechanisms responsible for Ca²⁺ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca²⁺ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca²⁺ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca²⁺ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca²⁺. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca²⁺ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca²⁺-binding loop (312–323) reduced the apparent Ca²⁺ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca²⁺-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca²⁺ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca²⁺ and suggest a model in which Ca²⁺ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ~100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca²⁺ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca²⁺.     

Depolarization Differentially Affects Allosteric Modulation by Neurotoxins of Scorpion α-Toxin Binding on Voltage-Gated Sodium Channels

Abstract: Voltage-gated sodium channels serve as a target for many neurotoxins that bind to several distinct, allosterically interacting receptor sites. We examined the effect of membrane potentials (incited by increasing external K⁺ concentrations) on the binding modulation by veratridine, brevetoxin, and tetrodotoxin of the scorpion α-toxin AaH II to receptor site 3 on sodium channels of rat brain synaptosomes. Depolarization is shown to differentially modulate neurotoxin effects on AaH II binding: Veratridine increase is potentiated, brevetoxin's inhibitory effect is reduced, and tetrodotoxin enhancement is evident mainly at resting membrane potential (5 m M K⁺). Both tetrodotoxin and veratridine apparently reverse the inhibition of AaH II binding by brevetoxin at resting membrane potential, but only veratridine is able to partially restore AaH II binding at 0 mV (135 m M K⁺). Thus, the allosteric interactions are grouped into two categories, depending on the membrane potential. Under depolarized conditions, the cooperative effects among veratridine and brevetoxin on AaH II binding fit the previously described two-state conformational model. At resting membrane potential, additional interactions are revealed, which may be explained by assuming that toxin binding induces conformational changes on the channel structure, in addition to being state-dependent. Our results provide a new insight into neurotoxin action and the complex dynamic changes underlying allosteric coupling of neurotoxin receptor sites, which may be related to channel gating.     

ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases

Epithelial Na⁺ channels facilitate the transport of Na⁺ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na⁺ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.Many ion channels are silent at rest and are activated in response to a variety of factors, including membrane potential, external ligands, and intracellular signaling processes. The ENaC² has evolved as a channel that is thought to reside primarily in an active state, facilitating the bulk movement of Na⁺ out of renal tubular or airway lumens. The regulated insertion and retrieval of channels at the plasma membrane have important roles in modulating ENaC-dependent Na⁺ transport (1). A number of factors also have a role in regulating ENaC activity via changes in channel P_o or gating. In this regard, it has become increasingly apparent that proteolysis of ENaC subunits has a key role in this process (2). This minireview addresses several questions regarding the role of ENaC subunit proteolysis in regulating channel gating. (i) Where are ENaC subunits cleaved? (ii) Which proteases mediate ENaC cleavage? (iii) Why are channels activated by proteolysis? (iv) Is proteolysis responsible, in part, for the highly variable channel P_o that has been noted for ENaC? (v) Why have ENaCs evolved as channels that require proteolysis for activation?     

The Mechanism of Na+/K+ Selectivity in Mammalian Voltage-Gated Sodium Channels Based on Molecular Dynamics Simulation

Voltage-gated sodium (Na_v) channels and their Na⁺/K⁺ selectivity are of great importance in the mammalian neuronal signaling. According to mutational analysis, the Na⁺/K⁺ selectivity in mammalian Na_v channels is mainly determined by the Lys and Asp/Glu residues located at the constriction site within the selectivity filter. Despite successful molecular dynamics simulations conducted on the prokaryotic Na_v channels, the lack of Lys at the constriction site of prokaryotic Na_v channels limits how much can be learned about the Na⁺/K⁺ selectivity in mammalian Na_v channels. In this work, we modeled the mammalian Na_v channel by mutating the key residues at the constriction site in a prokaryotic Na_v channel (Na_vRh) to its mammalian counterpart. By simulating the mutant structure, we found that the Na⁺ preference in mammalian Na_v channels is collaboratively achieved by the deselection from Lys and the selection from Asp/Glu within the constriction site.     

Oxaliplatin Modulates the Characteristics of Voltage-Gated Calcium Channels and Action Potentials in Small Dorsal Root Ganglion Neurons of Rats

Oxaliplatin is important for treating colorectal cancer. Although oxaliplatin is highly effective, it has severe side effects, of which neurotoxicity in dorsal root ganglion (DRG) neurons is one of the most common. The key mechanisms of this neurotoxicity are still controversial. However, disturbances of calcium homeostasis in DRG neurons have been suggested to mediate oxaliplatin neurotoxicity. By using whole-cell patch-clamp and current-clamp techniques, as well as immunocytochemical staining, we examined the influence of short- and long-term exposure to oxaliplatin on voltage-gated calcium channels (VGCC) and different VGCC subtypes in small DRG neurons of rats in vitro. Exposure to oxaliplatin reduced VGCC currents (I_Ca(V)) in a concentration-dependent manner (1–500 μM; 13.8–63.3%). Subtype-specific measurements of VGCCs showed differential effects on I_Ca(V). While acute treatment with oxaliplatin led to a reduction in I_Ca(V) for P/Q-, T-, and L-type VGCCs, I_Ca(V) of N-type VGCCs was not affected. Exposure of DRG neurons to oxaliplatin (10 or 100 μM) for 24 h in vitro significantly increased the I_Ca(V) current density, with a significant influence on L- and T-type VGCCs. Immunostaining revealed an increase of L- and T-type VGCC protein levels in DRG neurons 24 h after oxaliplatin exposure. This effect was mediated by calcium-calmodulin-protein kinase II (CaMKII). Significant alterations in action potentials (AP) and their characteristics were also observed. While the amplitude increased after oxaliplatin treatment, the rise time and time-to-peak decreased, and these effects were reversed by treatment with pimozide and nimodipine, which suggests that VGCCs are critically involved in oxaliplatin-mediated neurotoxicity.     

Seeing the Forest for the Trees: Integrated Conservation and Development Projects and Local Perceptions of Conservation in Madagascar

This paper explores local perceptions of internationally financed conservation and development projects in Madagascar and the success of these projects at influencing perceptions. Interviews, surveys, and focus group sessions were conducted in the peripheral zones of three Malagasy national parks: Ranomafana, Andohahela, and Masoala. Relevant questions explored community demographics, socioeconomic status, and local perceptions of the parks. The principal finding is that while a majority of people living in the peripheral zones do find conservation a valuable goal, they see it as a luxury they cannot afford. Despite their efforts and innovation, conservation and development projects have had a minimal impact on socioeconomic or associational life in the Ranomafana and Andohahela peripheral zones, and a significant but modest impact in the Masoala peripheral zone, by providing economic alternatives to destructive resource use. As a result, they are limited in their success at promoting conservation outcomes.