Heterodimerization of ORL1 and Opioid Receptors and Its Consequences for N-type Calcium Channel Regulation

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, μ-, δ-, and κ-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The μ-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of μ-opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.     

Receptor-mediated regulation of calcium channels and neurotransmitter release

Ca2+ influx into the nerve terminal is normally the trigger for the release of neurotransmitters. Many neurons possess presynaptic receptors whose activation results in changes in the quantity of neurotransmitter released by an action potential. This paper reviews studies that show that presynaptic receptors can regulate the activity of Ca2+ channels in the nerve terminal, resulting in changes in the influx of Ca2+ and in neurotransmitter release. Neurons possess several different types of voltage-sensitive Ca2+ channels. Ca2+ influx through N-type channels appears to trigger transmitter release in many instances. In other cases Ca2+ influx through L channels can influence transmitter release. Neurotransmitters can inhibit N channels through a G protein-mediated transduction mechanism. The G proteins are frequently pertussis toxin substrates. Inhibition of N channels appears to involve changes in their voltage dependence. Neurotransmitters can also regulate neuronal K+ channels. Activation of these K+ channels can lead to a reduction in Ca2+ influx and neurotransmitter release; these effects are also mediated by G proteins. Thus neurotransmitters may often regulate both presynaptic Ca2+ and K+ channels. These two effects may be synergistic mechanisms for the regulation of Ca2+ influx and neurotransmitter release.     

Ligand-gated ion channels: mechanisms underlying ion selectivity

Anion/cation selectivity is a critical property of ion channels and underpins their physiological function. Recently, there have been numerous mutagenesis studies, which have mapped sites within the ion channel-forming segments of ligand-gated ion channels that are determinants of the ion selectivity. Site-directed mutations to specific amino acids within or flanking the M2 transmembrane segments of the anion-selective glycine, GABA(A) and GABA(C) receptors and the cation-selective nicotinic acetylcholine and serotonin (type 3) receptors have revealed discrete, equivalent regions within the ion channel that form the principal selectivity filter, leading to plausible molecular mechanisms and mathematical models to describe how ions preferentially permeate these channels. In particular, the dominant factor determining anion/cation selectivity seems to be the sign and exposure of charged amino acids lining the selectivity filter region of the open channel. In addition, the minimum pore diameter, which can be influenced by the presence of a local proline residue, also makes a contribution to such ion selectivity in LGICs with smaller diameters increasing anion/cation selectivity and larger ones decreasing it.     

Determinants of the calcium permeation of ligand-gated cation channels.

Fast synaptic transmission in the vertebrate brain is mediated by ligand-gated channel receptors. As some of these receptors have been implicated in learning and memory, it is important to understand their mechanism of action at a molecular level. Excitatory receptors are members of large gene families of related channels that are gated by acetylcholine, serotonin, and the most abundant neurotransmitter, glutamate. Within the last year, a number of important studies have focused on the ability of these channels to flux calcium ions. Calcium entry into neurons through some of these channels triggers biochemical cascades, which can lead to changes in synaptic efficacy, presumed to be a requisite for memory formation, or if it occurs in excess, to cell death. Recent studies that attempt to determine the channel structures responsible for this calcium conductance will be discussed.     

The TRPC3/6/7 subfamily of cation channels

The mammalian transient receptor potential (TRP) proteins consist of a superfamily of Ca2+-permeant non-selective cation channels with structural similarities to Drosophila TRP. The TRP superfamily can be divided into three major families, among them the "canonical TRP" family (TRPC). The seven protein products of the mammalian TRPC family of genes (designated TRPC1-7) share in common the activation through PLC-coupled receptors and have been proposed to encode components of native store-operated channels in different cell types. In addition, the three members of the TRPC3/6/7 subfamily of TRPC channels can be activated by diacylglycerol analogs, providing a possible mechanism of activation of these channels by PLC-coupled receptors. This review summarizes the current knowledge about the mechanism of activation of the TRPC3/6/7 subfamily, as well as the potential role of these proteins as components of native Ca2+-permeant channels.     

Tandem couture

Receptor subunits in the Cys-loop superfamily assemble to form channels as hom opentamers or heteropentamers, expanding functional diversity through modularity. Expression of two or more compatible subunit types can lead to various receptor assemblies or subtypes. However, what may be good for diversity in vivo may be undesirable for the bench scientist, because we often wish to reduce our analyses to a single receptor subtype. By linking two or more subunits, creating tandems or concatamers, we can control stoichiometry and limit expression to exactly one receptor subtype. In this fashion, receptors with mixed subunit subtypes and heterozygous mutations can be separated from a mixture and can be described in detail. However, several recent studies have shown that this may be more easily conceived than accomplished, because several unforeseen problems have arisen. Concatamers can degrade, linkers can sometimes be clipped after or during translation, and one subunit may “loop out” or even become part of a second (now linked) pentamer with different characteristics. Some strategies have been developed to overcome these drawbacks, and the resultant new information that has begun to emerge has revitalized the study of these receptors in heterologous expression systems.     

G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase

A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.     

G-protein-coupled receptors, channels, and Na+-H+ exchanger in nuclear membranes of heart, hepatic, vascular endothelial, and smooth muscle cells

The action of several peptides and drugs is thought to be primarily dependent on their interactions with specific cell surface G-protein-coupled receptors and ionic transporters such as channels and exchangers. Recent development of 3-D confocal microscopy allowed several laboratories, including ours, to identify and study the localization of receptors, channels, and exchangers at the transcellular level of several cell types. Using this technique, we demonstrated in the nuclei of several types of cells the presence of Ca(2+) channels as well as Na(+)-H(+) exchanger and receptors such as endothelin-1 and angiotensin II receptors. Stimulation of these nuclear membrane G-protein-coupled receptors induced an increase of nuclear Ca(2+). Our results suggest that, similar to the plasma membrane, nuclear membranes possess channels, exchangers and receptors such as those for endothelin-1 and angiotensin II, and that the nucleus seems to be a cell within a cell. This article will emphasize these findings.     

Development of different electrophysiological mechanisms for muscarinic inhibition of atria and ventricles

The negative inotropic effect of acetylcholine (ACh) in atrial muscle can be accounted for by a decrease of a voltage- and time-dependent slow inward current (Isi) carried by Ca2+/Na+ and an increase of outward time-dependent current carried by K+ (IK1) through inwardly rectifying channels. The negative inotropic effect of ACh in ventricular muscle is associated with a reduction of Isi; there is no important effect of ACh on IK1 in ventricular muscle. Because atrial and ventricular muscles display IK1 that is sensitive to Ba2+ and have similar numbers of muscarinic receptor sites, it is concluded that ventricular muscle lacks a metabolic link between the muscarinic receptor and inwardly rectifying K+ channels. Although there is much evidence for cyclic nucleotides as the mediator between muscarinic receptors and Isi channels, cyclic nucleotides do not seem to connect these receptors with inwardly rectifying K+ channels. According to this hypothesis, identification of a metabolic link between muscarinic receptors and IK1 channels should be demonstrable in atrial but not ventricular muscle.     

Structure and assembly mechanism for heteromeric kainate receptors

Native glutamate receptor ion channels are tetrameric assemblies containing two or more different subunits. NMDA receptors are obligate heteromers formed by coassembly of two or three divergent gene families. While some AMPA and kainate receptors can form functional homomeric ion channels, the KA1 and KA2 subunits are obligate heteromers which?function only in combination with GluR5-7. The mechanisms controlling glutamate receptor assembly involve an initial step in which the amino terminal domains (ATD) assemble as dimers. Here, we establish by sedimentation velocity that the ATDs of GluR6 and KA2 coassemble as a heterodimer of K(d) 11?nM, 32,000-fold lower than the K(d) for homodimer formation by KA2; we solve crystal structures for the GluR6/KA2 ATD heterodimer and heterotetramer assemblies. Using these structures as a guide, we perform a mutant cycle analysis to probe the energetics of assembly and show that high-affinity ATD interactions are required for biosynthesis of functional heteromeric receptors.     

Toxin entry: how bacterial proteins get into mammalian cells

Certain bacteria secrete protein toxins that catalytically modify and disrupt essential processes in mammalian cells, often leading to cell death. As the substrates modified by these toxins are located in the mammalian cell cytosol, a catalytically active toxin polypeptide must reach this compartment in order to act. The toxins bind to receptors on the surface of susceptible cells and enter them by endocytic uptake. Endocytosed toxins initially accumulate in endosomes, where some of these proteins take advantage of the acidic environment within these organelles to form, or contribute to the formation of, protein-conducting channels through which the catalytic polypeptide is able to translocate into the cytosol. Other toxins are unable to respond to low pH in this way and must undergo intracellular vesicular transport to reach a compartment where pre-existing protein-conducting channels occur and can be exploited for membrane translocation — the endoplasmic reticulum. In this way, cell entry by this second group of toxins demonstrates that the secretory pathway of mammalian cells is completely reversible.     

A TM2 residue in the beta1 subunit determines spontaneous opening of homomeric and heteromeric gamma-aminobutyric acid-gated ion channels

Gamma-aminobutyric acid type A (GABAA) receptors are major inhibitory neurotransmitter-gated ion channels in the central nervous system. GABAA receptors consist of multiple subunits and exhibit distinct pharmacological and channel properties. Of all GABAA receptor subunits, the beta subunit is thought to be a key component for the functionality of the receptors. Certain types of GABAA receptors have been found to be constitutively active. However, the molecular basis for spontaneous opening of channels of these receptors is not totally understood. In this study, we showed that channels that contain the beta1 but not beta3 subunits opened spontaneously when these subunits were expressed homomerically or co-expressed with other types of GABAA receptor subunits in Xenopus oocytes. Using subunit chimeras and site-directed mutagenesis, we localized a key amino acid residue, a serine at position 265, that is critical in conferring an open state of the beta1 subunit-containing GABAA receptors in the absence of agonist. Moreover, some point mutations of Ser-265 also produced constitutively active channels. The magnitude of spontaneous activity of these receptors was correlated with the molecular volume of the residue at 265 for both homomeric and heteromeric GABAA receptors, suggesting that the spontaneous activity of the beta1 subunit-containing GABAA receptors may be mediated through a similar molecular mechanism that is dependent on the molecular volume of the residue at 265.     

D2 dopamine receptors interact directly with N-type calcium channels and regulate channel surface expression levels

N-type channels are located on dendrites and at pre-synaptic nerve terminals where they play a fundamental role in neurotransmitter release. They are potently regulated by the activation of a number of different types of pertussis toxin (PTX)-sensitive G alpha(i/o) coupled receptors, which results in voltage-dependent inhibition of channel activity via G betagamma subunits. Using heterologous expression in HEK 293T cells, we show via whole cell patch clamp recordings that D2 receptors mediate both G betagamma (i.e., voltage-dependent) and voltage-independent inhibition of channel activity. Furthermore, using co-immunoprecipitation and pull down assays involving the intracellular regions of each protein, we show that D2 receptors and N-type channels form physical signaling complexes. Finally, we use confocal microscopy to demonstrate that D2 receptors regulate N-type channel trafficking to affect the number of calcium channels available at the plasma membrane. Taken together, these data provide evidence for multiple voltage-dependent and voltage-independent mechanisms by which D2 receptor subtypes influence N-type channel activity.     

Functional Characterization of the Cardiac Ryanodine Receptor Pore-Forming Region

Ryanodine receptors are homotetrameric intracellular calcium release channels. The efficiency of these channels is underpinned by exceptional rates of cation translocation through the open channel and this is achieved at the expense of the high degree of selectivity characteristic of many other types of channel. Crystallization of prokaryotic potassium channels has provided insights into the structures and mechanisms responsible for ion selection and movement in these channels, however no equivalent structural detail is currently available for ryanodine receptors. Nevertheless both molecular modeling and cryo-electron microscopy have identified the probable pore-forming region (PFR) of the ryanodine receptor (RyR) and suggest that this region contains structural elements equivalent to those of the PFRs of potassium-selective channels. The aim of the current study was to establish if the isolated putative cardiac RyR (RyR2) PFR could form a functional ion channel. We have expressed and purified the RyR2 PFR and shown that function is retained following reconstitution into planar phospholipid bilayers. Our data provide the first direct experimental evidence to support the proposal that the conduction pathway of RyR2 is formed by structural elements equivalent to those of the potassium channel PFR.     

Use of optical biosensors to detect modulation of Slack potassium channels by G protein-coupled receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ion to flow through the plasma membrane. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex. Traditional assays examining the interaction between channels and regulatory proteins generally provide little information on the time-course of interactions in living cells. We have now used a novel label-free technology to detect changes in the distribution of mass close to the plasma membrane following modulation of potassium channels by G protein-coupled receptors (GPCRs). This technology uses optical sensors embedded in microplates to detect changes in the refractive index at the surface of cells. Although the activation of GPCRs has been studied with this system, protein-protein interactions due to modulation of ion channels have not yet been characterized. Here we present data that the characteristic pattern of mass distribution following GPCR activation is significantly modified by the presence of a sodium-activated potassium channel, Slack-B, a channel that is known to be potently modulated by activation of these receptors.     

D2 dopamine receptors interact directly with N-type calcium channels and regulate channel surface expression levels

N-type channels are located on dendrites and at pre-synaptic nerve terminals where they play a fundamental role in neurotransmitter release. They are potently regulated by the activation of a number of different types of pertussis toxin (PTX)-sensitive Gαi/o coupled receptors, which results in voltage-dependent inhibition of channel activity via Gβγ subunits. Using heterologous expression in HEK 293T cells, we show via whole cell patch clamp recordings that D2 receptors mediate both Gβγ (i.e. voltage-dependent) and voltage-independent inhibition of channel activity. Furthermore, using co-immunoprecipitation and pull down assays involving the intracellular regions of each protein, we show that D2 receptors and N-type channels form physical signaling complexes. Finally, we use confocal microscopy to demonstrate that D2 receptors regulate N-type channel trafficking to affect the number of calcium channels available at the plasma membrane. Taken together, these data provide evidence for multiple voltage-dependent and voltage-independent mechanisms by which D2 receptor subtypes influence N-type channel activity.     

Comparison of quantitative calcium flux through NMDA, ATP, and ACh receptor channels.

NMDA receptors, ATP receptors, and nicotinic ACh receptors respond to agonist by undergoing conformational changes that open weakly selective cationic channels that are permeable to calcium. We determined the fraction of the current carried by calcium by simultaneously measuring membrane current using whole-cell patch-clamp techniques and intracellular Ca2+ using the fluorescent indicator Fura-2. The Fura-2 response to free Ca2+ was calibrated individually for each cell. Two different calibration methods are compared: one uses voltage-activated Ca2+ channels, and the other uses the same ligand-gated channels that are being tested but in a pure Ca2+ solution. The two methods give quantitatively different results. The method using pure Ca2+ currents through ligand-gated channels calibrates the Fura-2 signal through the same influx pathway that generates the test response, thus controlling for the distribution of channels and ensuring a similar interaction between the incoming Ca2+ and Fura-2. In a physiologic solution containing 2.5 mM Ca2+ at a holding potential of -50 mV, the percentage of inward current carried by Ca2+ through NMDA receptors in hippocampal neurons is 12.4%. By comparison, in sympathetic neurons the percentage of current carried by Ca2+ through neuronal nAChRs is 4.7%, and through ATP-activated purinergic receptors it is 6.5%. These percentages can be used to estimate the amount of Ca2+ entry through these receptors during synaptic activation, but care must be exercised in considering the many subtypes of each receptor.     

Abnormal intracellular ca(2+)homeostasis and disease

A whole range of cell functions are regulated by the free cytosolic Ca(2+)concentration. Activator Ca(2+)from the extracellular space enters the cell through various types of Ca(2+)channels and sometimes the Na(+)/Ca(2+)-exchanger, and is actively extruded from the cell by Ca(2+)pumps and Na(+)/Ca(2+)-exchangers. Activator Ca(2+)can also be released from internal Ca(2+)stores through inositol trisphosphate or ryanodine receptors and is taken up into these organelles by means of Ca(2+)pumps. The resulting Ca(2+)signal is highly organized in space, frequency and amplitude because the localization and the integrated free cytosolic Ca(2+)concentration over time contain specific information. Mutations or functional abnormalities in the various Ca(2+)transporters, which in vitro seem to induce trivial functional alterations, therefore, often lead to a plethora of diseases. Skeletal-muscle pathology can be caused by mutations in ryanodine receptors (malignant hyperthermia, porcine stress syndrome, central-core disease), dihydropyridine receptors (familial hypokalemic periodic paralysis, malignant hyperthermia, muscular dysgenesis) or Ca(2+)pumps (Brody disease). Ca(2+)-pump mutations in cutaneous epidermal keratinocytes and cochlear hair cells lead to, skin diseases (Darier and Hailey-Hailey) and hearing/vestibular problems respectively. Mutated Ca(2+)channels in the photoreceptor plasma membrane cause vision problems. Hemiplegic migraine, spinocerebellar ataxia type-6, one form of episodic ataxia and some forms of epilepsy can be due to mutations in plasma-membrane Ca(2+)channels, while antibodies against these channels play a pathogenic role in all patients with the Lambert-Eaton myasthenic syndrome and may be of significance in sporadic amyotrophic lateral sclerosis. Brain inositol trisphosphate receptors have been hypothesized to contribute to the pathology in opisthotonos mice, manic-depressive illness and perhaps Alzheimer's disease. Various abnormalities in Ca(2+)-handling proteins have been described in heart during aging, hypertrophy, heart failure and during treatment with immunosuppressive drugs and in diabetes mellitus. In some instances, disease-causing mutations or abnormalities provide us with new insights into the cell biology of the various Ca(2+)transporters.     

Ion channels and receptors: molecular targets for behavioral evolution

Ion channels and receptors play critical roles in shaping neuronal activity, and thus are appropriate targets for evolutionary change to generate new behaviors. In this review, the evolution and differentiation of the many voltage-gated ion channels and transmitter-activated receptors is summarized; these channels and receptors evolved very early, and with some exceptions all species with nervous systems use similar sets of channels and receptors. Several examples are given of mechanisms for species-specific behavioral evolution that arise from mutations involving the structure, alternative splicing, level of expression, targeting and modulation of these important neural proteins.