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α2δ proteins serve as auxiliary subunits of voltage‐gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, α2δ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1‐4, the genes encoding for α2δ‐1 to ‐4 isoforms, have been implicated in neurological, neurodevelopmental, and neuropsychiatric disorders. Mutations in CACNA2D2 have been associated with developmental and epileptic encephalopathy (DEE) and cerebellar atrophy. In our present study, we performed a detailed functional characterization of the p.R593P mutation in α2δ‐2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel‐dependent as well as synaptic functions of α2δ‐2. Our data show that the corresponding p.R596P mutation in mouse α2δ‐2 drastically decreases membrane expression and synaptic targeting of α2δ‐2. This defect correlates with altered biophysical properties of postsynaptic CaV1.3 channel but has no effect on presynaptic CaV2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of α2δ‐2_R596P in primary cultures of hippocampal neurons affects the ability of α2δ‐2 to induce a statistically significant increase in the presynaptic abundance of endogenous CaV2.1 channels and presynaptic calcium transients. Moreover, our data demonstrate that in addition to lowering membrane expression, the p.R596P mutation reduces the trans‐synaptic recruitment of GABAA receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the α2δ‐2_R596P reduces the amplitudes of glutamatergic miniature postsynaptic currents in transduced hippocampal neurons. Taken together, our data strongly link the human biallelic p.R593P mutation to the underlying severe neurodevelopmental disorder and highlight the importance of studying α2δ mutations not only in the context of channelopathies but also synaptopathies. [ABSTRACT FROM AUTHOR]

Mammalian Ca2+‐dependent Slo K+ channels can stably associate with auxiliary γ subunits which fundamentally alter their behavior. By a so far unknown mechanism, the four γ subunits reduce the need for voltage‐dependent activation and, thereby, allow Slo to open independently of an action potential. Here, using cryo‐EM, we reveal how the transmembrane helix of γ1/LRRC26 binds and presumably stabilizes the activated voltage‐sensor domain of Slo1. The activation is further enhanced by an intracellular polybasic stretch which locally changes the charge gradient across the membrane. Our data provide a possible explanation for Slo1 regulation by the four γ subunits and also their different activation efficiencies. This suggests a novel activation mechanism of voltage‐gated ion channels by auxiliary subunits. [ABSTRACT FROM AUTHOR]

Voltage-dependent K+ (Kv) channels are diverse, comprising the classical Shab − Kv2, Shaker − Kv1, Shal − Kv4, and Shaw − Kv3 families. The Shaker family alone consists of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, and Kv1.7. Moreover, the Shab family comprises two functional (Kv2.1 and Kv2.2) and several "silent" alpha subunits (Kv2.3, Kv5, Kv6, Kv8, and Kv9), which do not generate K current. However, e.g., Kv8.1, via heteromerization, inhibits outward currents of the same family or even that of Shaw. This property of Kv8.1 is similar to those of designated beta subunits or non-selective auxiliary elements, including ADAM or AMIGO proteins. Kv channels and, in turn, ADAM may modulate the synaptic long-term potentiation (LTP). Prevailingly, Kv1.1 and Kv1.5 are attributed to respective brain and heart pathologies, some of which may occur simultaneously. The aforementioned channel proteins are apparently involved in several brain pathologies, including schizophrenia and seizures. [ABSTRACT FROM AUTHOR]

Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission. They are implicated in nervous system development and function, while their dysfunction is associated with neurological and psychiatric disorders. In this review, we describe recent progress in structural studies of iGluRs and how crystal and cryo-EM structures helped determining the principles of iGluR architecture and assembly, and the mechanisms of activation, desensitization and regulation by auxiliary subunits, positive and negative allosteric modulators, and ion channel blockers. The new structural insights facilitate better understanding of iGluR physiological and pathophysiological roles in functioning of the central nervous system and provide valuable information for structure-based drug design. [ABSTRACT FROM AUTHOR]

Repetitive synapse activity induces various forms of short-term plasticity. The role of presynapticmechanisms such as residual Ca2+ and vesicle depletion in short-termfacilitation and short-term depression is well established. On the other hand, the contribution of postsynaptic mechanisms such as receptor desensitization and saturation to short-term plasticity is less well known and often ignored. In this review, I will describe short-term plasticity in retinogeniculate synapses of relay neurons of the dorsal lateral geniculate nucleus (dLGN) to exemplify the synaptic properties that facilitate the contribution of AMPA receptor desensitization to short-term plasticity. These include high vesicle release probability, glutamate spillover and, importantly, slow recovery from desensitization of AMPA receptors. The latter is strongly regulated by the interaction of AMPA receptors with auxiliary proteins such as CKAMP44. Finally, I discuss the relevance of short-term plasticity in retinogeniculate synapses for the processing of visual information by LGN relay neurons. [ABSTRACT FROM AUTHOR]

Kainate receptors play fundamental roles in regulating synaptic transmission and plasticity in central nervous system and are regulated by their cognate auxiliary subunits Neuropilin and tolloid-like proteins 1 and 2 (Neto). While electrophysiology-based insights into functions of Neto proteins are known, biophysical and biochemical studies into Neto proteins have been largely missing till-date. Our biochemical, biophysical, and functional characterization of the purified extracellular domain (ECD) of Neto1 shows that Neto1-ECD exists as monomers in solution and has a micromolar affinity for GluK2 receptors in apo state or closed state. Remarkably, the affinity was ~2.8 fold lower for receptors trapped in the desensitized state, highlighting the conformation-dependent interaction of Neto proteins with kainate receptors. SAXS analysis of Neto1-ECD reveals that their dimensions are long enough to span the entire extracellular domain of kainate receptors. The shape and conformation of Neto1-ECD seems to be altered by calcium ions pointing towards its possible role in modulating Neto1 functions. Functional assays using GluK2 receptors and GluK2/GluA2 chimeric receptors reveal a differential role of Neto1 domains in modulating receptor functions. Although the desensitization rate was not affected by the Neto1-ECD, the recovery rates from the desensitized state are altered. [ABSTRACT FROM AUTHOR]

Interleukin-1β (IL-1β) is an important cytokine that modulates peripheral and central pain sensitization at the spinal level. Among its effects, it increases spinal cord excitability by reducing inhibitory Glycinergic and GABAergic neurotransmission. In the brain, IL-1β is released by glial cells in regions associated with pain processing during neuropathic pain. It also has important roles in neuroinflammation and in regulating NMDA receptor activity required for learning and memory. The modulation of glycine-mediated inhibitory activity via IL-1β may play a critical role in the perception of different levels of pain. The central nucleus of the amygdala (CeA) participates in receiving and processing pain information. Interestingly, this nucleus is enriched in the regulatory auxiliary glycine receptor (GlyR) β subunit (βGlyR); however, no studies have evaluated the effect of IL-1β on glycinergic neurotransmission in the brain. Hence, we hypothesized that IL-1β may modulate GlyR-mediated inhibitory activity via interactions with the βGlyR subunit. Our results show that the application of IL-1β (10 ng/ml) to CeA brain slices has a biphasic effect; transiently increases and then reduces sIPSC amplitude of CeA glycinergic currents. Additionally, we performed molecular docking, site-directed mutagenesis, and whole-cell voltage-clamp electrophysiological experiments in HEK cells transfected with GlyRs containing different GlyR subunits. These data indicate that IL-1β modulates GlyR activity by establishing hydrogen bonds with at least one key amino acid residue located in the back of the loop C at the ECD domain of the βGlyR subunit. The present results suggest that IL-1β in the CeA controls glycinergic neurotransmission, possibly via interactions with the βGlyR subunit. This effect could be relevant for understanding how IL-1β released by glia modulates central processing of pain, learning and memory, and is involved in neuroinflammation.

The vast majority of fast inhibitory transmission in the brain is mediated by GABA acting on GABA A receptors (GABA A Rs), which provides inhibitory balance to excitatory drive and controls neuronal output. GABA A Rs are also effectively targeted by clinically important drugs for treatment in a number of neurological disorders. It has long been hypothesized that function and pharmacology of GABA A Rs are determined by the channel pore-forming subunits. However, recent studies have provided new dimensions in studying GABA A Rs due to several transmembrane proteins that interact with GABA A Rs and modulate their trafficking and function. In this review, we summarize recent findings on these novel GABA A R transmembrane regulators and highlight a potential avenue to develop new GABA A R psychopharmacology by targeting these receptor-associated membrane proteins. Historically, transmembrane auxiliary subunits of ionotropic receptors have been best characterized at excitatory synapses, but more recent studies extend these observations to inhibitory synapses. We discuss findings on recently discovered transmembrane proteins that interact with GABA A receptors (GABA A Rs), as well as highlight their unique function. Future studies further characterizing the mechanistic and physiological role for these novel transmembrane proteins will not only advance our understanding of GABA A R biology but also potentially lead to the development of more efficacious GABA A R psychopharmacology. [ABSTRACT FROM AUTHOR]

Voltage-gated calcium channels are the principal conduits for depolarization-mediated Ca2+ entry into excitable cells. In this review, the biophysical properties of the relevant members of this family of channels, those that are present in presynaptic terminals, will be discussed in relation to their function in mediating neurotransmitter release. Voltage-gated calcium channels have properties that ensure they are specialized for particular roles, for example, differences in their activation voltage threshold, their various kinetic properties, and their voltage-dependence of inactivation. All these attributes play into the ability of the various voltage-gated calcium channels to participate in different patterns of presynaptic vesicular release. These include synaptic transmission resulting from single action potentials, and longer-term changes mediated by bursts or trains of action potentials, as well as release resulting from graded changes in membrane potential in specialized sensory synapses. [ABSTRACT FROM AUTHOR]

Presynaptic glutamate receptors (GluRs) modulate neurotransmitter release and are physiological targets for regulation during various forms of plasticity. Although much is known about the auxiliary subunits associated with postsynaptic GluRs, far less is understood about presynaptic auxiliary GluR subunits and their functions. At the Drosophila neuromuscular junction, a presynaptic GluR, DKaiR1D, localizes near active zones and operates as an autoreceptor to tune baseline transmission and enhance presynaptic neurotransmitter release in response to diminished postsynaptic GluR functionality, a process referred to as presynaptic homeostatic potentiation (PHP). Here, we identify an auxiliary subunit that collaborates with DKaiR1D to promote these synaptic functions. This subunit, dSol-1, is the homolog of the Caenorhabditis elegans CUB (Complement C1r/C1s, Uegf, Bmp1) domain protein Sol-1. We find that dSol-1 functions in neurons to facilitate baseline neurotransmission and to enable PHP expression, properties shared with DKaiR1D. Intriguingly, presynaptic overexpression of dSol-1 is sufficient to enhance neurotransmitter release through a DKaiR1D-dependent mechanism. Furthermore, dSol-1 is necessary to rapidly increase the abundance of DKaiR1D receptors near active zones during homeostatic signaling. Together with recent work showing the CUB domain protein Neto2 is necessary for the homeostatic modulation of postsynaptic GluRs in mammals, our data demonstrate that dSol-1 is required for the homeostatic regulation of presynaptic GluRs. Thus, we propose that CUB domain proteins are fundamental homeostatic modulators of GluRs on both sides of the synapse. [ABSTRACT FROM AUTHOR]

The largest biomass of channel proteins is located in unicellular organisms and bacteria that have no organs. However, orchestrated bidirectional ionic currents across the cell membrane via the channels are important for the functioning of organs of organisms, and equally concern both fauna or flora. Several ion channels are activated in the course of action potentials. One of the hallmarks of voltage-dependent channels is a 'tail current' − deactivation as observed after prior and sufficient activation predominantly at more depolarized potentials e.g. for Kv while upon hyperpolarization for HCN α subunits. Tail current also reflects the timing of channel closure that is initiated upon termination of stimuli. Finally, deactivation of currents during repolarization could be a selective estimate for given channel as in case of HERG, if dedicated long and more depolarized 'tail pulse' is used. Since from a holding potential of e.g. −70 mV are often a family of outward K+ currents comprising I A and I K are simultaneously activated in native cells. [ABSTRACT FROM AUTHOR]

Kainate type ionotropic glutamate receptors (KARs) are expressed in hippocampal interneurons and regulate interneuron excitability and GABAergic transmission. Neuropilin tolloid-like proteins (NETO1 and NETO2) act as KAR auxiliary subunits; however, their significance for various functions of KARs in GABAergic interneurons is not fully understood. Here we show that NETO1, but not NETO2, is necessary for dendritic delivery of KAR subunits and, consequently, for formation of KAR-containing synapses in cultured GABAergic neurons. Accordingly, electrophysiological analysis of neonatal CA3 stratum radiatum interneurons revealed impaired postsynaptic and metabotropic KAR signaling in Neto1 knockouts, while a subpopulation of ionotropic KARs in the somatodendritic compartment remained functional. Loss of NETO1/KAR signaling had no significant effect on development of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA)-receptor-mediated glutamatergic transmission in CA3 interneurons, contrasting the synaptogenic role proposed for KARs in principal cells. Furthermore, loss of NETO1 had no effect on excitability and characteristic spontaneous network bursts in the immature CA3 circuitry. However, we find that NETO1 is critical for kainate-dependent modulation of network bursts and GABAergic transmission in the hippocampus already during the first week of life. Our results provide the first description of NETO1-dependent subcellular targeting of KAR subunits in GABAergic neurons and indicate that endogenous NETO1 is required for formation of KAR-containing synapses in interneurons. Since aberrant KAR-mediated excitability is implicated in certain forms of epilepsy, NETO1 represents a potential therapeutic target for treatment of both adult and early life seizures. [ABSTRACT FROM AUTHOR]

Modulation of large conductance intracellular ligand-activated potassium (BK) channel family (Slo1–3) by auxiliary subunits allows diverse physiological functions in excitable and non-excitable cells. Cryoelectron microscopy (cryo-EM) structures of voltage-gated potassium (Kv) channel complexes have provided insights into how voltage sensitivity is modulated by auxiliary subunits. However, the modulation mechanisms of BK channels, particularly as ligand-activated ion channels, remain unknown. Slo1 is a Ca2+-activated and voltage-gated BK channel and is expressed in neurons, muscle cells, and epithelial cells. Using cryo-EM and electrophysiology, we show that the LRRC26-γ1 subunit modulates not only voltage but also Ca2+ sensitivity of Homo sapiens Slo1. LRRC26 stabilizes the active conformation of voltage-senor domains of Slo1 by an extracellularly S4-locking mechanism. Furthermore, it also stabilizes the active conformation of Ca2+-sensor domains of Slo1 intracellularly, which is functionally equivalent to intracellular Ca2+ in the activation of Slo1. Such a dual allosteric modulatory mechanism may be general in regulating the intracellular ligand-activated BK channel complexes. [Display omitted] • Structures of the Ca2+-activated and voltage-gated Slo1-LRRC26 complex are revealed • LRRC26 stabilizes the active conformation of voltage sensors by transmembrane helix • LRRC26 stabilizes the active conformation of Ca2+-sensor domains through Arg clusters • LRRC26 modulates Ca2+ and voltage sensitivity of Slo1 by a dual allosteric mechanism Yamanouchi and Kasuya et al. elucidate the mechanism for dual allosteric modulation of Ca2+/voltage sensitivity of Slo1 by LRRC26 from Slo1-LRRC26 complex structures. LRRC26 stabilizes the active conformations of voltage sensors and Ca2+ sensors through its transmembrane helix and intracellular Arg cluster. Structures further provide insights into all-or-none gating modulation by LRRC26. [ABSTRACT FROM AUTHOR]

The KCNQ1 channel is a voltage-dependent potassium channel and is ubiquitously expressed throughout the human body including the heart, lung, kidney, pancreas, intestine and inner ear. Gating properties of the KCNQ1 channel are modulated by KCNE auxiliary subunits. For example, the KCNQ1-KCNE1 channel produces a slowly-activating potassium current, while KCNE3 makes KCNQ1 a voltage-independent, constitutively open channel. Thus, physiological functions of KCNQ1 channels are greatly dependent on the type of KCNE protein that is co-expressed in that organ. It has long been debated how the similar single transmembrane KCNE proteins produce quite different gating behaviors. Recent applications of voltage-clamp fluorometry (VCF) for the KCNQ1 channel have shed light on this question. The VCF is a quite sensitive method to detect structural changes of membrane proteins and is especially suitable for tracking the voltage sensor domains of voltage-gated ion channels. In this short review, I will introduce how the VCF technique can be applied to detect structural changes and what have been revealed by the recent VCF applications to the gating modulation of KCNQ1 channels by KCNE proteins.

Voltage-gated Ca2+ channels are composed of a pore-forming α1 subunit and auxiliary β and α2δ subunits, which modulate Ca2+ current properties and channel trafficking. So far, the partial redundancy and specificity of α1 for α2δ subunits in the CNS have remained largely elusive. Mature spiral ganglion (SG) neurons express α2δ subunit isoforms 1, 2, and 3 and multiple Ca2+ channel subtypes. Differentiation and in vivo functions of their endbulb of Held synapses, which rely on presynaptic P/Q channels (Lin et al., 2011), require the α2δ3 subunit (Pirone et al., 2014). This led us to hypothesize that P/Q channels may preferentially co-assemble with α2δ3. Using a dissociated primary culture, we analyzed the effects of α2δ3 deletion on somatic Ca2+ currents (ICa) of SG neurons isolated at postnatal day 20 (P20), when the cochlea is regarded to be mature. P/Q currents were the dominating steady-state Ca2+ currents (54% of total) followed by T-type, L-type, N-type, and R-type currents. Deletion of α2δ3 reduced P/Q- and R-type currents by 60 and 38%, respectively, whereas L-type, N-type, and T-type currents were not altered. A subset of ICa types was also analyzed in SG neurons isolated at P5, i.e., before the onset of hearing (P12). Both L-type and N-type current amplitudes of wildtype SG neurons were larger at P5 compared with P20. Deletion of α2δ3 reduced L-type and N-type currents by 23 and 44%, respectively. In contrast, small P/Q currents, which were just being up-regulated at P5, were unaffected by the lack of α2δ3. In summary, α2δ3 regulates amplitudes of L- and N-type currents of immature cultured SG neurons, whereas it regulates P/Q- and R-type currents at P20. Our data indicate a developmental switch from dominating somatic N- to P/Q-type currents in cultured SG neurons. A switch from N- to P/Q-type channels, which has been observed at several central synapses, may also occur at developing endbulbs of Held. In this case, reduction of both neonatal N- (P5) and more mature P/Q-type currents (around/after hearing onset) may contribute to the impaired morphology and function of endbulb synapses in α2δ3-deficient mice. [ABSTRACT FROM AUTHOR]

• Ion channels consist of conducting and non-conducting (auxiliary) subunits. • Auxiliary subunits regulate ion conductance and have non-conducting roles. • Ion channels control diverse cellular processes and are aberrantly expressed in cancer. • Auxiliary subunits play major roles in cancer cells, including regulating adhesion, migration, invasion and gene expression. Several superfamilies of plasma membrane channels which regulate transmembrane ion flux have also been shown to regulate a multitude of cellular processes, including proliferation and migration. Ion channels are typically multimeric complexes consisting of conducting subunits and auxiliary, non-conducting subunits. Auxiliary subunits modulate the function of conducting subunits and have putative non-conducting roles, further expanding the repertoire of cellular processes governed by ion channel complexes to processes such as transcellular adhesion and gene transcription. Given this expansive influence of ion channels on cellular behaviour it is perhaps no surprise that aberrant ion channel expression is a common occurrence in cancer. This review will focus on the conducting and non-conducting roles of the auxiliary subunits of various Ca2+, K+, Na+ and Cl− channels and the burgeoning evidence linking such auxiliary subunits to cancer. Several subunits are upregulated (e.g. Ca v β, Ca v γ) and downregulated (e.g. K v β) in cancer, while other subunits have been functionally implicated as oncogenes (e.g. Na v β 1 , Ca v α 2 δ 1) and tumour suppressor genes (e.g. CLCA2, KCNE2, BKγ 1) based on in vivo studies. The strengthening link between ion channel auxiliary subunits and cancer has exposed these subunits as potential biomarkers and therapeutic targets. However further mechanistic understanding is required into how these subunits contribute to tumour progression before their therapeutic potential can be fully realised. [ABSTRACT FROM AUTHOR]

The auxiliary α2δ calcium channel subunits play key roles in voltage-gated calcium channel function. Independent of this, α2δ-1 has also been suggested to be important for synaptogenesis. Using an epitope-tagged knockin mouse strategy, we examined the effect of α2δ-1 on CaV2.2 localization in the pain pathway in vivo, where CaV2.2 is important for nociceptive transmission and α2δ-1 plays a critical role in neuropathic pain. We find CaV2.2 is preferentially expressed on the plasma membrane of calcitonin gene-related peptide-positive small nociceptors. This is paralleled by strong presynaptic expression of CaV2.2 in the superficial spinal cord dorsal horn. EM-immunogold localization shows CaV2.2 predominantly in active zones of glomerular primary afferent terminals. Genetic ablation of α2δ-1 abolishes CaV2.2 cell-surface expression in dorsal root ganglion neurons and dramatically reduces dorsal horn expression. There was no effect of α2δ-1 knockout on other dorsal horn pre- and postsynaptic markers, indicating the primary afferent pathways are not otherwise affected by α2δ-1 ablation. [ABSTRACT FROM AUTHOR]

Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

In the brain, AMPA receptors (AMPARs)-mediated excitatory synaptic transmission is critically regulated by the receptor auxiliary subunits. Recent proteomic studies have identified that Ferric Chelate Reductase 1 Like protein (FRRS1L), whose mutations in human lead to epilepsy, choreoathetosis, and cognitive deficits, is present in native AMPAR complexes in the brain. Here we have characterized FRRS1L in both heterologous cells and in mouse neurons. We found that FRRS1L interacts with both GluA1 and GluA2 subunits of AMPARs, but does not form dimers/oligomers, in HEK cells. In mouse hippocampal neurons, recombinant FRRS1L at the neuronal surface partially co-localizes with GluA1 and primarily localizes at non-synaptic membranes. In addition, native FRRS1L in hippocampus is localized at dynein, but not kinesin5B, vesicles. Functionally, over-expression of FRRS1L in hippocampal neurons does not change glutamatergic synaptic transmission. In contrast, single-cell knockout (KO) of FRRS1L strongly reduces the expression levels of the GluA1 subunit at the neuronal surface, and significantly decreases AMPAR-mediated synaptic transmission in mouse hippocampal pyramidal neurons. Taken together, these data characterize FRRS1L in heterologous cells and neurons, and reveal an important role of FRRS1L in the regulation of excitatory synaptic strength.

Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal a subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering. [ABSTRACT FROM AUTHOR]

The γ-aminobutyric acid receptors (GABARs) mediate fast inhibitory transmission in central nervous system of insects and are important targets of insecticides. An auxiliary subunit, Shisa7, was identified in mammals as a single-passing transmembrane protein. However, the homology gene(s) of Shisa in invertebrates has not been reported to date. In the present study, a homolog Shisa gene was identified from the two-spotted spider mite, Tetranychus urticae Koch. Its open reading frame had 927 base pairs and encoded 308 amino acid residues, which has a typical Shisa domain at 13th-181st amino acid residues. According to the phylogenetic tree, the invertebrate Shisa was categorized apart with those of vertebrate, and Tu Shisa showed closest relationship with the Shisa9 of velvet mite, Dinothrombium tinctorium (L.). In the electrophysiological assay with two-electrode voltage clamp, the GABA-activated Tu RDL channel was functionally formed in the Africa clawed frog Xenopus laevis (Daudin) oocytes (EC 50 = 53.34 μM). No GABA-activated current could be observed in Tu Shisa-expressed oocytes, whereas Tu Shisa could reduce the sensitivity of TuRDL / TuShisa (mass ratio of 1: 4) channel to GABA. The homology structural models of Tu RDL and Tu Shisa were built by the SWISS-MODEL server, their interaction was predicted using Z -DOCK and three predicted hydrogen bonds and interface residues were analysed by PyMOL. Meanwhile, the key interface residues of Tu Shisa affected the stability of complex were calculated by Discovery Studio 2019. In conclusion, the Tu Shisa, as the first reported invertebrate Shisa, was explored and functionally examined as the GABARs auxiliary subunit. Our findings provide a basis for research of invertebrate Shisa. [Display omitted] • Tu Shisa was first isolated from two-spotted spider mite Tetranychus urticae Koch. • Tu Shisa could reduce the sensitivity of Tu RDL channel expressed in Xenopus laevis oocytes to GABA. • Tu Shisa interacted with Tu RDL by three hydrogen bonds. • GLY29 and GLY18 of Tu Shisa were two key interface residues affected the stability of Tu RDL- Tu Shisa complex. [ABSTRACT FROM AUTHOR]

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated cation channels that mediate excitatory signal transmission in the central nervous system (CNS) of vertebrates. The members of the iGluR subfamily of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPARs) mediate most of the fast excitatory signal transmission, and their abundance in the postsynaptic membrane is a major determinant of the strength of excitatory synapses. Therefore, regulation of AMPAR trafficking to the postsynaptic membrane is an important constituent of mechanisms involved in learning and memory formation, such as long-term potentiation (LTP) and long-term depression (LTD). Auxiliary subunits play a critical role in the facilitation and regulation of AMPAR trafficking and function. The currently identified auxiliary subunits of AMPARs are transmembrane AMPA receptor regulatory proteins (TARPs), suppressor of lurcher (SOL), cornichon homologues (CNIHs), synapse differentiation-induced gene I (SynDIG I), cysteine-knot AMPAR modulating proteins 44 (CKAMP44), and germ cell-specific gene 1-like (GSG1L) protein. In this review we summarize our current knowledge of the modulatory influence exerted by these important but still underappreciated proteins.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are assembled of four core subunits and several additional interacting proteins. Cystine-knot AMPA receptor-modulating proteins (CKAMPs) constitute a family of four proteins that influence the trafficking, subcellular localization and function of AMPA receptors. The four CKAMP family members CKAMP39/shisa8, CKAMP44/shisa9, CKAMP52/shisa6 and CKAMP59/shisa7 differ in their expression profile and their modulatory influence on AMPA receptor function. In this review, I report about recent findings on the differential roles of CKAMP family members.

Key points The AMPA-type ionotropic glutamate receptors (AMPARs) mediate the majority of excitatory synaptic transmission and their function impacts learning, cognition and behaviour., The gating of AMPARs occurs in milliseconds, precisely controlled by a variety of auxiliary subunits that are expressed differentially in the brain, but the difference in mechanisms underlying AMPAR gating modulation by auxiliary subunits remains elusive and is investigated., The elements of the AMPAR that are functionally recruited by auxiliary subunits, stargazin and cornichon 3, are located not only in the extracellular domains but also in the lipid-accessible surface of the AMPAR., We reveal that the two auxiliary subunits require a shared surface on the transmembrane domain of the AMPAR for their function, but the gating is influenced by this surface in opposing directions for each auxiliary subunit., Our results provide new insights into the mechanistic difference of AMPAR modulation by auxiliary subunits and a conceptual framework for functional engineering of the complex., Abstract During excitatory synaptic transmission, various structurally unrelated transmembrane auxiliary subunits control the function of AMPA receptors (AMPARs), but the underlying mechanisms remain unclear. We identified lipid-exposed residues in the transmembrane domain (TMD) of the GluA2 subunit of AMPARs that are critical for the function of AMPAR auxiliary subunits, stargazin (Stg) and cornichon 3 (CNIH3). These residues are essential for stabilizing the AMPAR-CNIH3 complex in detergents and overlap with the contacts made between GluA2 TMD and Stg in the cryoEM structures. Mutating these residues had opposite effects on gating modulation and complex stability when Stg- and CNIH3-bound AMPARs were compared. Specifically, in detergent the GluA2-A793F formed an unstable complex with CNIIH3 but in the membrane the GluA2-A793F-CNIH3 complex expressed a gain of function. In contrast, the GluA2-A793F-Stg complex was stable, but had diminished gating modulation. GluA2-C528L destabilized the AMPAR-CNIH3 complex but stabilized the AMPAR-Stg complex, with overall loss of function in gating modulation. Furthermore, loss-of-function mutations in this TMD region cancelled the effects of a gain-of-function Stg carrying mutation in its extracellular loop, demonstrating that both the extracellular and the TMD elements contribute independently to gating modulation. The elements of AMPAR functionally recruited by auxiliary subunits are, therefore, located not only in the extracellular domains but also in the lipid accessible surface of the AMPAR. The TMD surface we defined is a potential target for auxiliary subunit-specific compounds, because engineering of this hotspot induces opposing functional outcomes by Stg and CNIH3. The collection of mutant-phenotype mapping provides a framework for engineering AMPAR gating using auxiliary subunits. [ABSTRACT FROM AUTHOR]

AMPA receptors (AMPARs) are tetrameric ion channels that together with other ionotropic glutamate receptors (iGluRs), the NMDA and kainate receptors, mediate a majority of excitatory neurotransmission in the central nervous system. Whereas NMDA receptors gate channels with slow kinetics, responsible primarily for generating long-term synaptic potentiation and depression, AMPARs are the main fast transduction elements at synapses and are critical for the expression of plasticity. The kinetic and conductance properties of AMPARs are laid down during their biogenesis and are regulated by post-transcriptional RNA editing, splice variation, post-translational modification, and subunit composition. Furthermore, AMPAR assembly, trafficking, and functional heterogeneity depends on a large repertoire of auxiliary subunits—a feature that is particularly striking for this type of iGluR. Here, we discuss how the subunit structure, stoichiometry, and auxiliary subunits generate a heterogeneous plethora of receptors, each tailored to fulfill a vital role in fast synaptic signaling and plasticity. [ABSTRACT FROM AUTHOR]

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia,which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2-/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth. [ABSTRACT FROM AUTHOR]

Evolution has acted to shape the action potential in different regions of the heart in order to produce a maximally stable and efficient pump. This has been achieved by creating regional differences in ion channel expression levels within the heart as well as differences between equivalent cardiac tissues in different species. These region- and species-dependent differences in channel expression are established by regulatory evolution, evolution of the regulatory mechanisms that control channel expression levels. Ion channel auxiliary subunits are obvious targets for regulatory evolution, in order to change channel expression levels and/or modify channel function. This review focuses on the transmural gradients of ion channel expression in the heart and the role that regulation of auxiliary subunit expression plays in generating and shaping these gradients. [ABSTRACT FROM AUTHOR]

Gene deletion of the voltage-gated calciumchannel auxiliary subunit α2δ-1 has been shown previously to have a cardiovascular phenotype, and a reduction in mechano- and cold sensitivity, coupled with delayed development of neuropathic allodynia. We have also previously shown that dorsal root ganglion (DRG) neuron calcium channel currents were significantly reduced in α2δ-1 knockout mice. To extend our findings in these sensory neurons, we have examined here the properties of action potentials (APs) in DRG neurons from α2δ-1 knockout mice in comparison to their wild-type (WT) littermates, in order to dissect how the calcium channels that are affected by α2δ-1 knockout are involved in setting the duration of individual APs and their firing frequency. Our main findings are that there is reduced Ca2+ entry on single AP stimulation, particularly in the axon proximal segment, reduced AP duration and reduced firing frequency to a 400 ms stimulation in α2δ-1 knockout neurons, consistent with the expected role of voltage-gated calcium channels in these events. Furthermore, lower intracellular Ca2+ buffering also resulted in reduced AP duration, and a lower frequency of AP firing in WT neurons, mimicking the effect of α2δ-1 knockout. By contrast, we did not obtain any consistent evidence for the involvement of Ca2+-activation of large conductance calcium-activated potassium (BK) and small conductance calcium-activated potassium (SK) channels in these events. In conclusion, the reduced Ca2+ elevation as a result of single AP stimulation is likely to result from the reduced duration of the AP in α2δ-1 knockout sensory neurons. This article is part of the themed issue 'Evolution brings Ca2+ and ATP together to control life and death'. [ABSTRACT FROM AUTHOR]

In Drosophila melanogaster , the functions of voltage-gated sodium (Na v ) channels are modulated by TipE and its orthologs. Here, we describe a novel TipE homolog of the American cockroach, Periplaneta americana , called PaTipE. Like DmTipE, PaTipE mRNAs are ubiquitously expressed. Surprisingly, PaTipE mRNA was undetectable in neurosecretory cells identified as dorsal unpaired median neurons. Phylogenetic analysis placed this new sequence in TipE clade, indicating an independent evolution from a common ancestor. Contrary to previous reports, our data indicate that the auxiliary subunits of insect Na v channels are very distant from the mammalian BKCa auxiliary subunits. To decipher the functional roles of PaTipE, we characterized the gating properties of DmNa v 1-1 channels co-expressed with DmTipE or PaTipE, in Xenopus oocytes. Compared to DmTipE, PaTipE increased Na + currents by a 4.2-fold. The voltage-dependence of steady-state fast inactivation of DmNa v 1-1/PaTipE channels was shifted by 5.8 mV to more negative potentials than that of DmNa v 1-1/DmTipE channels. DmNa v 1-1/PaTipE channels recovered 3.2-fold slower from the fast-inactivated state than DmNa v 1-1/DmTipE channels. In conclusion, this study supports that the insect Na v auxiliary subunits share functional features with their mammalian counterparts, although structurally and phylogenetically distant. [ABSTRACT FROM AUTHOR]

The large-conductance, calcium- and voltage-activated potassium (BK) channel has the largest single-channel conductance among potassium channels and can be activated by both membrane depolarization and increases in intracellular calcium concentration. BK channels consist of pore-forming, voltage- and calcium-sensing α subunits, either alone or in association with regulatory subunits. BK channels are widely expressed in various tissues and cells including both excitable and non-excitable cells and display diverse biophysical and pharmacological characteristics. This diversity can be explained in part by posttranslational modifications and alternative splicing of the α subunit, which is encoded by a single gene, KCNMA1, as well as by tissue-specific β subunit modulation. Recently, a leucine-rich repeat-containing membrane protein, LRRC26, was found to interact with BK channels and cause an unprecedented large negative shift (~-140 mV) in the voltage dependence of the BK channel activation. LRRC26 allows BK channels to open even at near-physiological calcium concentration and membrane voltage in non-excitable cells. Three LRRC26-related proteins, LRRC52, LRRC55, and LRRC38, were subsequently identified as BK channel modulators. These LRRC proteins are structurally and functionally distinct from the BK channel β subunits and were designated as γ subunits. The discovery of the γ subunits adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. Unlike BK channel β subunits, which have been intensively investigated both mechanistically and physiologically, our understanding of the γ subunits is very limited at this stage. This article reviews the structure, modulatory mechanisms, physiological relevance, and potential therapeutic implications of γ subunits as they are currently understood.

Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders.

Neto2 is a transmembrane protein that interacts with the neuron-specific KC-Cl- cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl- gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2-/- neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2-/- neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2-null mice would be prone to seizure activity. We found that Neto2-null mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity. [ABSTRACT FROM AUTHOR]