Auxiliary Subunits Research Articles

Cadmium activates AMPA and NMDA receptors with M3 helix cysteine substitutions.

AMPA and NMDA receptors are ligand-gated ion channels that depolarize postsynaptic neurons when activated by the neurotransmitter L-glutamate. Changes in the distribution and activity of these receptors underlie learning and memory, but excessive change is associated with an array of neurological disorders, including cognitive impairment, developmental delay, and epilepsy. All of the ionotropic glutamate receptors (iGluRs) exhibit similar tetrameric architecture, transmembrane topology, and basic framework for activation; conformational changes induced by extracellular agonist binding deform and splay open the inner helix bundle crossing that occludes ion flux through the channel. NMDA receptors require agonist binding to all four subunits, whereas AMPA and closely related kainate receptors can open with less than complete occupancy. In addition to conventional activation by agonist binding, we recently identified two locations along the inner helix of the GluK2 kainate receptor subunit where cysteine (Cys) substitution yields channels that are opened by exposure to cadmium ions, independent of agonist site occupancy. Here, we generate AMPA and NMDA receptor subunits with homologous Cys substitutions and demonstrate similar activation of the mutant receptors by Cd. Coexpression of the auxiliary subunit stargazin enhanced Cd potency for activation of Cys-substituted GluA1 and altered occlusion upon treatment with sulfhydryl-reactive MTS reagents. Mutant NMDA receptors displayed voltage-dependent Mg block of currents activated by agonist and/or Cd as well as asymmetry between Cd effects on Cys-substituted GluN1 versus GluN2 subunits. In addition, Cd activation of each Cys-substituted iGluR was inhibited by protons. These results, together with our earlier work on GluK2, reveal a novel mechanism shared among the three different iGluR subtypes for prying open the gate that controls ion entry into the pore.

Analgesia with Gabapentin and Pregabalin May Involve N-Methyl-d-Aspartate Receptors, Neurexins, and Thrombospondins.

The gabapentinoid drugs gabapentin and pregabalin (Neurontin and Lyrica) are mainstay treatments for neuropathic pain and preventing focal seizures. Both drugs have similar effects to each other in animal models and clinically. Studies have shown that a protein first identified as an auxiliary subunit of voltage-gated calcium channels (the α 2 δ-subunit type 1 [α 2 δ-1], or Ca V a2d1) is the high-affinity binding site for gabapentin and pregabalin and is required for the efficacy of these drugs. The α 2 δ-1 protein is required for the ability of gabapentin and pregabalin to reduce neurotransmitter release in neuronal tissue, consistent with a therapeutic mechanism of action via voltage-gated calcium channels. However, recent studies have revealed that α 2 δ-1 interacts with several proteins in addition to voltage-gated calcium channels, and these additional proteins could be involved in gabapentinoid pharmacology. Furthermore, gabapentin and pregabalin have been shown to modify the action of a subset of N-methyl-d-aspartate-sensitive glutamate receptors, neurexin-1α, and thrombospondin proteins by binding to α 2 δ-1. Thus, these effects may contribute substantially to gabapentinoid therapeutic mechanism of action. SIGNIFICANCE STATEMENT: It is widely believed that gabapentin and pregabalin act by modestly reducing the membrane localization and activation of voltage-gated calcium channels at synaptic endings in spinal cord and neocortex via binding to the α 2 δ-1 protein. However, recent findings show that the α 2 δ-1 protein also interacts with N-methyl-d-aspartate-sensitive glutamate receptors, neurexin-1α, thrombospondins (adhesion molecules), and other presynaptic proteins. These newly discovered interactions, in addition to actions at calcium channels, may be important mediators of gabapentin and pregabalin therapeutic effects.

Designer genetically encoded voltage-dependent calcium channel inhibitors inspired by RGK GTPases.

High‐voltage‐activated calcium (CaV1/CaV2) channels translate action potentials into Ca2+ influx in excitable cells to control essential biological processes that include; muscle contraction, synaptic transmission, hormone secretion and activity‐dependent regulation of gene expression. Modulation of CaV1/CaV2 channel activity is a powerful mechanism to regulate physiology, and there are a host of intracellular signalling molecules that tune different aspects of CaV channel trafficking and gating for this purpose. Beyond normal physiological regulation, the diverse CaV channel modulatory mechanisms may potentially be co‐opted or interfered with for therapeutic benefits. CaV1/CaV2 channels are potently inhibited by a four‐member sub‐family of Ras‐like GTPases known as RGK (Rad, Rem, Rem2, Gem/Kir) proteins. Understanding the mechanisms by which RGK proteins inhibit CaV1/CaV2 channels has led to the development of novel genetically encoded CaV channel blockers with unique properties; including, chemo‐ and optogenetic control of channel activity, and blocking channels either on the basis of their subcellular localization or by targeting an auxiliary subunit. These genetically encoded CaV channel inhibitors have outstanding utility as enabling research tools and potential therapeutics.

EAT-18 is an essential auxiliary protein interacting with the non-alpha nAChR subunit EAT-2 to form a functional receptor.

Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs.

Role for NADH‐sensitive Kv channels in the myocardial‐vascular signaling axis.

Voltage‐gated potassium (Kv) channels expressed in the coronary vasculature play a pivotal role in coupling oxygen delivery with myocardial metabolic demand. Despite their importance in maintaining adequate perfusion to support proper cardiac function, the processes by which these channels respond to metabolic cues from the myocardium to elicit smooth muscle hyperpolarization and rapid relaxation are unclear. We tested the hypothesis that the heteromeric auxiliary Kvβ subunit (Kvβ1:Kvβ2) complex facilitates increases in Kv1 channel activity and coronary vasodilation during periods of elevated myocardial oxygen consumption. The Kvβ proteins are functional aldo‐keto reductases, and thus catalyze nicotinamide cofactor‐ (i.e., NAD(P)H) dependent reduction of a range of aldehyde substrates. Intriguingly, these subunits also differentially modify the voltage‐sensitivity of Kv1 channels in a manner that depends upon the redox state of bound cofactor. Imaging mass spectrometry revealed that lactate:pyruvate (used as a surrogate for NADH:NAD+) was significantly increased in intramyocardial arteries in hearts from mice acutely treated with dobutamine to drive elevated cardiac workload versus those from vehicle‐treated mice. Elevation of NADH:NAD+ ratio in coronary arterial myocytes following perfusion of sodium lactate reversibly increased whole cell Kv currents and shifted the voltage‐dependence of activation by ~−15 mV. Moreover, application of 1 mM NADH, but not NAD+, increased single Kv1 channel currents in excised membrane patches from coronary arterial myocytes of wild type mice. In contrast, ablation of Kvβ2 abolished the NADH sensitivity of Kv1 activity, whereas ablation of Kvβ1 had little effect. Consistent with this, we found that vasodilation of coronary arteries to reduced bath oxygen (to ~3%) and the relationship between cardiac work and myocardial blood flow was significantly blunted in Kvβ2−/− arteries/mice when compared with wild type arteries/mice. Together, our results suggest that the Kvβ complex is a functional sensing unit that modulates native coronary Kv1 channel activity upon changes in pyridine nucleotide redox ratio, which may impart a profound influence on the control of myocardial blood flow.Support or Funding InformationThis work is supported by grants from the National Institutes of Health (HL142710, GM103492).

Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility

Voltage-gated K+ channels function in macromolecular complexes with accessory subunits to regulate brain function. Here, we describe a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1)-dependent mechanism that regulates the association of the A-type K+ channel subunit Kv4.2 with its auxiliary subunit dipeptidyl peptidase 6 (DPP6), and thereby modulates neuronal excitability and cognitive flexibility. We show that activity-induced Kv4.2 phosphorylation triggers Pin1 binding to, and isomerization of, Kv4.2 at the pThr607-Pro motif, leading to the dissociation of the Kv4.2-DPP6 complex. We generated a novel mouse line harboring a knock-in Thr607 to Ala (Kv4.2TA) mutation that abolished dynamic Pin1 binding to Kv4.2. CA1 pyramidal neurons of the hippocampus from these mice exhibited altered Kv4.2-DPP6 interaction, increased A-type K+ current, and reduced neuronal excitability. Behaviorally, Kv4.2TA mice displayed normal initial learning but improved reversal learning in both Morris water maze and lever press paradigms. These findings reveal a Pin1-mediated mechanism regulating reversal learning and provide potential targets for the treatment of neuropsychiatric disorders characterized by cognitive inflexibility.

Action potentials in Xenopus oocytes triggered by blue light.

Voltage-gated sodium (Na+) channels are responsible for the fast upstroke of the action potential of excitable cells. The different α subunits of Na+ channels respond to brief membrane depolarizations above a threshold level by undergoing conformational changes that result in the opening of the pore and a subsequent inward flux of Na+. Physiologically, these initial membrane depolarizations are caused by other ion channels that are activated by a variety of stimuli such as mechanical stretch, temperature changes, and various ligands. In the present study, we developed an optogenetic approach to activate Na+ channels and elicit action potentials in Xenopus laevis oocytes. All recordings were performed by the two-microelectrode technique. We first coupled channelrhodopsin-2 (ChR2), a light-sensitive ion channel of the green alga Chlamydomonas reinhardtii, to the auxiliary β1 subunit of voltage-gated Na+ channels. The resulting fusion construct, β1-ChR2, retained the ability to modulate Na+ channel kinetics and generate photosensitive inward currents. Stimulation of Xenopus oocytes coexpressing the skeletal muscle Na+ channel Nav1.4 and β1-ChR2 with 25-ms lasting blue-light pulses resulted in rapid alterations of the membrane potential strongly resembling typical action potentials of excitable cells. Blocking Nav1.4 with tetrodotoxin prevented the fast upstroke and the reversal of the membrane potential. Coexpression of the voltage-gated K+ channel Kv2.1 facilitated action potential repolarization considerably. Light-induced action potentials were also obtained by coexpressing β1-ChR2 with either the neuronal Na+ channel Nav1.2 or the cardiac-specific isoform Nav1.5. Potential applications of this novel optogenetic tool are discussed.

A homozygous missense variant in CACNB4 encoding the auxiliary calcium channel beta4 subunit causes a severe neurodevelopmental disorder and impairs channel and non-channel functions.

P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and β4 subunits. β4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat β4b cDNA. Heterologously expressed wild-type β4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the β4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, β4b and β4b-L125P augmented the calcium current amplitudes, however, β4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of β4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype β4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of β4b in cultured myotubes and hippocampal neurons. While binding of β4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between β4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of β4b.

L-type Voltage-Gated Calcium Channel Modulators Inhibit Glutamate-Induced Morphology Changes in U118-MG Astrocytoma Cells.

The excitatory neurotransmitter glutamate evokes physiological responses within the astrocytic network that lead to fine morphological changes. However, the mechanism by which astrocytes couple glutamate sensing with cellular calcium rise remains unclear. We tested a possible connection between L-type voltage-gated calcium channels (Cav) and glutamate-induced response in U118-MG astrocytoma cells. While astrocytoma cells differ from primary astrocytes, they demonstrate the same response to glutamate. In this study, the extension of U118-MG processes upon glutamate exposure was shown to depend on extracellular calcium entry via L-type Cav's. Drugs known to bind to the pore-forming subunit of Cav's decreased the astrocytic filopodia extension caused by glutamate, and ligands of the α2δ auxiliary subunit inhibited all process growth (e.g., gabapentinoids). The observed phenotypic responses suggest that α2δ is a main contributor to the role of Cavs in glutamate-dependent filopodiagenesis, thereby opening new avenues of research on the role of α2δ in astrocytic neurochemical signaling.

Sexually Divergent Mortality and Partial Phenotypic Rescue After Gene Therapy in a Mouse Model of Dravet Syndrome.

Dravet syndrome (DS) is a neurodevelopmental genetic disorder caused by mutations in the SCN1A gene encoding the α subunit of the NaV1.1 voltage-gated sodium channel that controls neuronal action potential firing. The high density of this mutated channel in GABAergic interneurons results in impaired inhibitory neurotransmission and subsequent excessive activation of excitatory neurons. The syndrome is associated with severe childhood epilepsy, autistic behaviors, and sudden unexpected death in epilepsy. Here, we compared the rescue effects of an adeno-associated viral (AAV) vector coding for the multifunctional β1 sodium channel auxiliary subunit (AAV-NaVβ1) with a control vector lacking a transgene. We hypothesized that overexpression of NaVβ1 would facilitate the function of residual voltage-gated channels and improve the DS phenotype in the Scn1a+/− mouse model of DS. AAV-NaVβ1 was injected into the cerebral spinal fluid of neonatal Scn1a+/− mice. In untreated control Scn1a+/− mice, females showed a higher degree of mortality than males. Compared with Scn1a+/− control mice, AAV-NaVβ1-treated Scn1a+/− mice displayed increased survival, an outcome that was more pronounced in females than males. In contrast, behavioral analysis revealed that male, but not female, Scn1a+/− mice displayed motor hyperactivity, and abnormal performance on tests of fear and anxiety and learning and memory. Male Scn1a+/− mice treated with AAV-NaVβ1 showed reduced spontaneous seizures and normalization of motor activity and performance on the elevated plus maze test. These findings demonstrate sex differences in mortality in untreated Scn1a+/− mice, an effect that may be related to a lower level of intrinsic inhibitory tone in female mice, and a normalization of aberrant behaviors in males after central nervous system administration of AAV-NaVβ1. The therapeutic efficacy of AAV-NaVβ1 in a mouse model of DS suggests a potential new long-lasting biological therapeutic avenue for the treatment of this catastrophic epilepsy.

Sodium channels implement a molecular leaky integrator that detects action potentials and regulates neuronal firing.

Voltage-gated sodium channels play a critical role in cellular excitability, amplifying small membrane depolarizations into action potentials. Interactions with auxiliary subunits and other factors modify the intrinsic kinetic mechanism to result in new molecular and cellular functionality. We show here that sodium channels can implement a molecular leaky integrator, where the input signal is the membrane potential and the output is the occupancy of a long-term inactivated state. Through this mechanism, sodium channels effectively measure the frequency of action potentials and convert it into Na+ current availability. In turn, the Na+ current can control neuronal firing frequency in a negative feedback loop. Consequently, neurons become less sensitive to changes in excitatory input and maintain a lower firing rate. We present these ideas in the context of rat serotonergic raphe neurons, which fire spontaneously at low frequency and provide critical neuromodulation to many autonomous and cognitive brain functions.

α2δ-2 Protein Controls Structure and Function at the Cerebellar Climbing Fiber Synapse.

α2δ proteins (Cacna2d1-4) are auxiliary subunits of voltage-dependent calcium channels that also drive synapse formation and maturation. Because cerebellar Purkinje cells (PCs) predominantly, if not exclusively, express one isoform of this family, α2δ-2 (Cacna2d2), we used PCs as a model system to examine roles of α2δ in excitatory synaptic function in male and female Cacna2d2 knock-out (KO) mice. Whole-cell recordings of PCs from acute cerebellar slices revealed altered climbing fiber (CF)-evoked complex spike generation, as well as increased amplitude and faster decay of CF-evoked EPSCs. CF terminals in the KO were localized more proximally on PC dendrites, as indicated by VGLUT2+ immunoreactive puncta, and computational modeling demonstrated that the increased EPSC amplitude can be partly attributed to the more proximal location of CF terminals. In addition, CFs in KO mice exhibited increased multivesicular transmission, corresponding to greater sustained responses during repetitive stimulation, despite a reduction in the measured probability of release. Electron microscopy demonstrated that mutant CF terminals had twice as many vesicle release sites, providing a morphologic explanation for the enhanced glutamate release. Though KO CFs evoked larger amplitude EPSCs, the charge transfer was the same as wild-type as a result of increased glutamate reuptake, producing faster decay kinetics. Together, the larger, faster EPSCs in the KO explain the altered complex spike responses, which degrade information transfer from PCs and likely contribute to ataxia in Cacna2d2 KO mice. Our results also illustrate the multidimensional synaptic roles of α2δ proteins.SIGNIFICANCE STATEMENT α2δ proteins (Cacna2d1-4) regulate synaptic transmission and synaptogenesis, but coexpression of multiple α2δ isoforms has obscured a clear understanding of how various α2δ proteins control synaptic function. We focused on roles of the α2δ-2 protein (Cacna2d2), the deletion of which causes cerebellar ataxia and epilepsy in mice and humans. Because cerebellar Purkinje cells (PCs) only express this single isoform, we studied excitatory climbing fiber synaptic function onto PCs in Cacna2d2 KO mice. Using optical and electrophysiological analysis, we provide a detailed description of the changes in PCs lacking α2δ-2, and provide a comprehensive mechanistic explanation for how functional synaptic phenotypes contribute to the altered cerebellar output.

AMPA receptor structure and auxiliary subunits

Fast excitatory synaptic transmission in the mammalian brain is largely mediated by AMPA-type ionotropic glutamate receptors (AMPARs), which are activated by the neurotransmitter glutamate. In synapses, the function of AMPARs is tuned by their auxiliary subunits, a diverse set of membrane proteins associated with the core pore-forming subunits of the AMPARs. Each auxiliary subunit provides distinct functional modulation of AMPARs, ranging from regulation of trafficking to shaping ion channel gating kinetics. Understanding the molecular mechanism of the function of these complexes is key to decoding synaptic modulation and their global roles in cognitive activities, such as learning and memory. Here, we review the structural and molecular complexity of AMPAR-auxiliary subunit complexes, as well as their functional diversity in different brain regions. We suggest that the recent structural information provides new insights into the molecular mechanisms underlying synaptic functions of AMPAR-auxiliary subunit complexes.

Molecular basis for N-terminal acetylation by human NatE and its modulation by HYPK

The human N-terminal acetyltransferase E (NatE) contains NAA10 and NAA50 catalytic, and NAA15 auxiliary subunits and associates with HYPK, a protein with intrinsic NAA10 inhibitory activity. NatE co-translationally acetylates the N-terminus of half the proteome to mediate diverse biological processes, including protein half-life, localization, and interaction. The molecular basis for how NatE and HYPK cooperate is unknown. Here, we report the cryo-EM structures of human NatE and NatE/HYPK complexes and associated biochemistry. We reveal that NAA50 and HYPK exhibit negative cooperative binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substrate-binding site. NAA50 activity is increased through NAA15 tethering, but is inhibited by HYPK through structural alteration of the NatE substrate-binding site. These studies reveal the molecular basis for coordinated N-terminal acetylation by NatE and HYPK.

Cav2.1 C-terminal fragments produced in Xenopus laevis oocytes do not modify the channel expression and functional properties.

The sequence and genomic organization of the CACNA1A gene that encodes the Cav2.1 subunit of both P and Q-type Ca2+ channels are well conserved in mammals. In human, rat and mouse CACNA1A, the use of an alternative acceptor site at the exon 46-47 boundary results in the expression of a long Cav2.1 splice variant. In transfected cells, the long isoform of human Cav2.1 produces a C-terminal fragment, but it is not known whether this fragment affects Cav2.1 expression or functional properties. Here, we cloned the long isoform of rat Cav2.1 (Cav2.1(e47)) and identified a novel variant with a shorter C-terminus (Cav2.1(e47s)) that differs from those previously described in the rat and mouse. When expressed in Xenopus laevis oocytes, Cav2.1(e47) and Cav2.1(e47s) displayed similar functional properties as the short isoform (Cav2.1). We show that Cav2.1 isoforms produced short (CT1) and long (CT1(e47)) C-terminal fragments that interacted in vivo with the auxiliary Cavβ4a subunit. Overexpression of the C-terminal fragments did not affect Cav2.1 expression and functional properties. Furthermore, the functional properties of a Cav2.1 mutant without the C-terminal Cavβ4 binding domain (Cav2.1ΔCT2) were similar to those of Cav2.1 and were not influenced by the co-expression of the missing fragments (CT2 or CT2(e47)). Our results exclude a functional role of the C-terminal fragments in Cav2.1 biophysical properties in an expression system widely used to study this channel.

Association of the CACNA2D2 gene with schizophrenia in Chinese Han population.

BackgroundSchizophrenia (SCZ) is a severely complex psychiatric disorder in which ~80% can be explained by genetic factors. Single nucleotide polymorphisms (SNPs) in calcium channel genes are potential genetic risk factors for a spectrum of psychiatric disorders including SCZ. This study evaluated the association between SNPs in the voltage-gated calcium channel auxiliary subunit alpha2delta 2 gene (CACNA2D2) and SCZ in the Han Chinese population of Northeast China.MethodsA total of 761 SCZ patients and 775 healthy controls were involved in this case-control study. Three SNPs (rs3806706, rs45536634 and rs12496815) of CACNA2D2 were genotyped by the MALDI-TOF-MS technology. Genotype distribution and allele frequency differences between cases and controls were tested by Chi-square (χ2) in males and females respectively using SPSS 24.0 software. Linkage disequilibrium and haplotype analyses were conducted using Haploview4.2. The false discovery rate correction was utilized to control for Type I error by R3.2.3.ResultsThere was a significant difference in allele frequencies (χ2 = 9.545, Padj = 0.006) and genotype distributions (χ2 = 9.275, Padj = 0.006) of rs45536634 between female SCZ patients and female healthy controls after adjusting for multiple comparisons. Minor allele A (OR = 1.871, 95% CI [1.251–2.798]) and genotype GA + AA (OR = 1.931, 95% CI [1.259–2.963]) were associated with an increased risk of SCZ. Subjects with haplotype AG consisting of rs45536634 and rs12496815 alleles had a higher risk of SCZ (OR = 1.91, 95% CI [1.26–2.90]) compared those with other haplotypes.ConclusionsThis study provides evidence that CACNA2D2 polymorphisms may influence the susceptibility to SCZ in Han Chinese women.

A Potent Voltage-gated Calcium Channel Inhibitor Engineered from a Nanobody Targeted to Auxiliary CaVβ Subunits

Inhibiting high-voltage-activated calcium channels (HVACCs; CaV1/CaV2) is therapeutic for myriad cardiovascular and neurological diseases. For particular applications, genetically-encoded HVACC blockers may enable channel inhibition with greater tissue-specificity and versatility than is achievable with small molecules. Here, we engineered a genetically-encoded HVACC inhibitor by first isolating an immunized llama nanobody (nb.F3) that binds auxiliary HVACC CaVβ subunits. Nb.F3 by itself is functionally inert, providing a convenient vehicle to target active moieties to CaVβ-associated channels. Nb.F3 fused to the catalytic HECT domain of Nedd4L (CaV-aβlator), an E3 ubiquitin ligase, ablated currents from diverse reconstituted HVACCs, and from endogenous CaV1/CaV2 channels in cardiomyocytes, dorsal root ganglion neurons, and pancreatic β cells. In cardiomyocytes, CaV-aβlator redistributed CaV1.2 channels from dyads to Rab-7-positive late endosomes. This work introduces CaV-aβlator as a potent genetically-encoded HVACC inhibitor, and describes a general approach that can be broadly adapted to generate versatile modulators for macro-molecular membrane protein complexes.

Exploring the Role of the First Extracellular Loop of CaV2.3 in Mediating the Interaction with Auxiliary Subunits

Presynaptic calcium entry occurs through voltage-gated calcium (CaV) channels which are activated by membrane depolarization. The presynaptic CaV2 channel gene family includes the pharmaco-resistant CaV2.3 or R-type Ca2+ channel. Like other high-voltage-activated CaV2 channels, CaV2.3 is formed by the association of the pore-forming CaVα1 subunit with auxiliary CaVβ and CaVα2δ1. CaVβ promotes cell surface expression, and co-expression with CaVα2δ1 increases by 2-3 fold the peak current density of CaV2.3. In contrast to CaV1.2, CaVα2δ1 does not modulate the activation potential of CaV2.3. Herein we explored the molecular basis for these differences. Homology-based model of CaV2.3 identified the first extracellular loop of CaVα1 (IS1S2) as a potential interface with the VWA domain of CaVα2δ1 (Briot et al., JBC 2018). IS1S2 of CaV2.3 contains a cluster of three consecutive negatively charged residues 113-PEDDK-117 that bears strong homology with the IS1S2 loop of CaV1.2 (178-PEDDS-182). Previous studies from our group have identified CaV1.2 Asp-181 (equivalent to CaV2.3 Asp-116) as anchoring the interaction between CaV1.2 and CaVα2δ1 (Bourdin et al., JBC 2017). We are herein showing that substitutions of Asp-116 disrupt the co-immunoprecipitation of CaVβ3/CaV2.3 with CaVα2δ1 whereas single substitutions to alanine residues in neighboring positions (Pro-113, Glu-114, Asp-115, and Lys-117) did not impair the interaction between CaVα2δ1 and CaV2.3. We are currently carrying out patch-clamp experiments to investigate the functional modulation of single-substituted CaV2.3 proteins by CaVα2δ1. Preliminary data suggest that channels substituted at positions Asp-115 or Lys-117 displayed altered activation properties when coexpressed with CaVα2δ1. We conclude that the functional modulation of CaV2.3 by CaVα2δ1 involves a broader range of residues than previously described for the L-type CaV1.2 channel and suggests family specific differences in channel modulation.

Non-canonical Role of CaVα2δ1 in Cardiac Hypertrophy

Excessive neurohormonal activity contributes to the progression of pathological hypertrophy and subsequent transition to heart failure. Although increased diastolic Ca2+ represents a hallmark feature of heart failure, the role of L-type CaV1.2 channels in this process remains controversial. CaV1.2 is an oligomeric protein complex formed by the distinctive pore-forming subunit CaVα1 with auxiliary subunits (CaVβ, calmodulin, and CaVα2δ1). CaVα2δ1 is a large extracellular GPI-anchored protein that facilitates channel activation at physiological voltages and stimulates (≈ 5-fold) peak current density. The present study examined the time-dependent changes in the expression of CaV auxiliary subunits in an in vitro model of cardiac hypertrophy. Cultured 1-day old neonatal rat ventricular cardiomyocytes (NRVMs) were treated for 24-48 h with 1 μM norepinephrine or 50 nM endothelin-1. In the presence of norepinephrine or endothelin-1, cardiac troponin-T protein levels and cell surface area (confocal imaging) were increased 1.8 ± 0.3-fold (n=3) and 1.5 ± 0.3-fold (n=4), respectively. Analysis of the CaV1.2 subunits revealed that CaVα2δ1 protein levels were 1.4 ± 0.15-fold higher (n=3) in hypertrophic cardiomyocytes compared to untreated NRVMs without a concomitant change in the expression of CaVα1 and CaVβ subunits. CaVα2δ1 was identified at the plasma membrane and unexpectedly detected at the perinuclear region co-localized with calreticulin. The increased expression of CaVα2δ1 protein levels persisted in hypertrophic NRVMs treated with 1 μM nifedipine. Lastly, confocal imaging identified modest CaV1.2 and CaVα2δ1 immunoreactivity in ventricular fibroblasts. Experiments are currently examining the identity of CaVα2δ1 protein partners in NRVMs. Collectively, these data demonstrate that the heavily glycosylated CaVα2δ1 protein is upregulated in hypertrophic NRVMs and localization at the perinuclear region may contribute in part to the progression of pathological cardiac hypertrophy via a potential non-canonical paradigm.

Mechanism of Modulation of AMPA Receptors by TARPγ8

Fast excitatory synaptic transmission in the mammalian central nervous system is mediated by glutamate activated AMPA receptors (AMPARs). The core GluA subunits of AMPARs in neurons co-assemble with auxiliary subunits, including transmembrane AMPAR regulatory proteins (TARPs), several of which modifiy AMPA receptor-channel gating kinetics. In the case of AMPARs containing four copies of TARP γ8, achieved by expression of the tandem GluA2Q∗TARPγ8 subunit developed in the lab, AMPARs exhibit resensitization, or partial recovery from desensitization in continued presence of glutamate. We have recorded single channel currents in cell-attached patch recordings and show that under resensitizing conditions, these AMPARs show transitions primarily from closed to higher conductance levels, similar to activation of homomeric GluA receptors in the presence of cyclothiazide. To study the conformation associated with these states we used single molecule FRET and show that this high conductance state produced by GluA2Q∗TARPγ8 channels exhibits tighter coupling between subunits in the extracellular parts of the receptor.