Regulation of axonal HCN1 trafficking in perforant path involves expression of specific TRIP8b isoforms

The functions of HCN channels in neurons depend critically on their subcellular localization, requiring fine-tuned machinery that regulates subcellular channel trafficking. Here we provide evidence that regulatory mechanisms governing axonal HCN channel trafficking involve association of the channels with specific isoforms of the auxiliary subunit TRIP8b. In the medial perforant path, which normally contains HCN1 channels in axon terminals in immature but not in adult rodents, we found axonal HCN1 significantly increased in adult mice lacking TRIP8b (TRIP8b(-/-)). Interestingly, adult mice harboring a mutation that results in expression of only the two most abundant TRIP8b isoforms (TRIP8b[1b/2](-/-)) exhibited an HCN1 expression pattern similar to wildtype mice, suggesting that presence of one or both of these isoforms (TRIP8b(1a), TRIP8b(1a-4)) prevents HCN1 from being transported to medial perforant path axons in adult mice. Concordantly, expression analyses demonstrated a strong increase of expression of both TRIP8b isoforms in rat entorhinal cortex with age. However, when overexpressed in cultured entorhinal neurons of rats, TRIP8b(1a), but not TRIP8b(1a-4), altered substantially the subcellular distribution of HCN1 by promoting somatodendritic and reducing axonal expression of the channels. Taken together, we conclude that TRIP8b isoforms are important regulators of HCN1 trafficking in entorhinal neurons and that the alternatively-spliced isoform TRIP8b(1a) could be responsible for the age-dependent redistribution of HCN channels out of perforant path axon terminals.     

Trafficking and gating of hyperpolarization-activated cyclic nucleotide-gated channels are regulated by interaction with tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) and cyclic AMP at distinct sites

Ion channel trafficking and gating are often influenced by interactions with auxiliary subunits. Tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) is an auxiliary subunit for neuronal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. TRIP8b interacts directly with two distinct sites of HCN channel pore-forming subunits to control channel trafficking and gating. Here we use mutagenesis combined with electrophysiological studies to define and distinguish the functional importance of the HCN/TRIP8b interaction sites. Interaction with the last three amino acids of the HCN1 C terminus governed the effect of TRIP8b on channel trafficking, whereas TRIP8b interaction with the HCN1 cyclic nucleotide binding domain (CNBD) affected trafficking and gating. Biochemical studies revealed that direct interaction between TRIP8b and the HCN1 CNBD was disrupted by cAMP and that TRIP8b binding to the CNBD required an arginine residue also necessary for cAMP binding. In accord, increasing cAMP levels in cells antagonized the up-regulation of HCN1 channels mediated by a TRIP8b construct binding the CNBD exclusively. These data illustrate the distinct roles of the two TRIP8b-HCN interaction domains and suggest that TRIP8b and cAMP may directly compete for binding the HCN CNBD to control HCN channel gating, kinetics, and trafficking.     

TRIP8b Is Required for Maximal Expression of HCN1 in the Mouse Retina

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are cation-selective channels present in retina, brain and heart. The activity of HCN channels contributes to signal integration, cell excitability and pacemaker activity. HCN1 channels expressed in photoreceptors participate in keeping light responses transient and are required for normal mesopic vision. The subcellular localization of HCN1 varies among cell types. In photoreceptors HCN1 is concentrated in the inner segments while in other retinal neurons, HCN1 is evenly distributed though the cell. This is in contrast to hippocampal neurons where HCN1 is concentrated in a subset of dendrites. A key regulator of HCN1 trafficking and activity is tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b). Multiple splice isoforms of TRIP8b are expressed throughout the brain and can differentially regulate the surface expression and activity of HCN1. The purpose of the present study was to determine which isoforms of TRIP8b are expressed in the retina and to test if loss of TRIP8b alters HCN1 expression or trafficking. We found that TRIP8b colocalizes with HCN1 in multiple retina neurons and all major splice isoforms of TRIP8b are expressed in the retina. Photoreceptors express three different isoforms. In TRIP8b knockout mice, the ability of HCN1 to traffic to the surface of retinal neurons is unaffected. However, there is a large decrease in the total amount of HCN1. We conclude that TRIP8b in the retina is needed to achieve maximal expression of HCN1.     

TRIP8b splice forms act in concert to regulate the localization and expression of HCN1 channels in CA1 pyramidal neurons

HCN1 channel subunits, which contribute to the hyperpolarization-activated cation current (Ih), are selectively targeted to distal apical dendrites of hippocampal CA1 pyramidal neurons. Here, we addressed the importance of the brain-specific auxiliary subunit of HCN1, TRIP8b, in regulating HCN1 expression and localization. More than ten N-terminal splice variants of TRIP8b exist in brain and exert distinct effects on HCN1 trafficking when overexpressed. We found that isoform-wide disruption of the TRIP8b/HCN1 interaction caused HCN1 to be mistargeted throughout CA1 somatodendritic compartments. In contrast, HCN1 was targeted normally to CA1 distal dendrites in a TRIP8b knockout mouse that selectively lacked exons 1b and 2. Of the two remaining hippocampal TRIP8b isoforms, TRIP8b(1a-4) promoted HCN1 surface expression in dendrites, whereas TRIP8b(1a) suppressed HCN1 misexpression in axons. Thus, proper subcellular localization of HCN1 depends on its differential additive and subtractive sculpting by two isoforms of a single auxiliary subunit.     

Discovery of a small-molecule inhibitor of the TRIP8b–HCN interaction with efficacy in neurons

Major depressive disorder is a critical public health problem with a lifetime prevalence of nearly 17% in the United States. One potential therapeutic target is the interaction between hyperpolarization-activated cyclic nucleotide–gated (HCN) channels and an auxiliary subunit of the channel named tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b). HCN channels regulate neuronal excitability in the mammalian hippocampus, and recent work has established that antagonizing HCN function rescues cognitive impairment caused by chronic stress. Here, we utilize a high-throughput virtual screen to find small molecules capable of disrupting the TRIP8b–HCN interaction. We found that the hit compound NUCC-0200590 disrupts the TRIP8b–HCN interaction in vitro and in vivo. These results provide a compelling strategy for developing new small molecules capable of disrupting the TRIP8b–HCN interaction.     

Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP

Hyperpolarization-activated cyclic nucleotide–regulated cation (HCN) channels generate the hyperpolarization-activated cation current Ih present in many neurons. These channels are directly regulated by the binding of cAMP, which both shifts the voltage dependence of HCN channel opening to more positive potentials and increases maximal Ih at extreme negative voltages where voltage gating is complete. Here we report that the HCN channel brain-specific auxiliary subunit TRIP8b produces opposing actions on these two effects of cAMP. In the first action, TRIP8b inhibits the effect of cAMP to shift voltage gating, decreasing both the sensitivity of the channel to cAMP (K_1/2) and the efficacy of cAMP (maximal voltage shift); conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic actions are well described by a cyclic allosteric mechanism in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state. In a second apparently independent action, TRIP8b enhances the action of cAMP to increase maximal Ih. This latter effect cannot be explained by the cyclic allosteric model but results from a previously uncharacterized action of TRIP8b to reduce maximal current through the channel in the absence of cAMP. Because the binding of cAMP also antagonizes this second effect of TRIP8b, application of cAMP produces a larger increase in maximal Ih in the presence of TRIP8b than in its absence. These findings may provide a mechanistic explanation for the wide variability in the effects of modulatory transmitters on the voltage gating and maximal amplitude of Ih reported for different neurons in the brain.     

Association of adaptor protein TRIP8b with clathrin

TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.     

Activation of GABAB Receptors Ameliorates Cognitive Impairment via Restoring the Balance of HCN1/HCN2 Surface Expression in the Hippocampal CA1 Area in Rats With Chronic Cerebral Hypoperfusion

Hyperpolarization-activated cyclic-nucleotide-gated cation nonselective (HCN) channels are involved in the pathology of nervous system diseases. HCN channels and γ-aminobutyric acid (GABA) receptors can mutually co-regulate the function of neurons in many brain areas. However, little is known about the co-regulation of HCN channels and GABA receptors in the chronic ischemic rats with possible features of vascular dementia. Protein kinase A (PKA) and TPR containing Rab8b interacting protein (TRIP8b) can modulate GABA_B receptors cell surface stability and HCN channel trafficking, respectively, and adaptor-associated kinase 1 (AAK1) inhibits the function of the major TRIP8b-interacting protein adaptor protein 2 (AP2) via phosphorylating the AP2 μ2 subunit. Until now, the role of these regulatory factors in chronic cerebral hypoperfusion is unclear. In the present study, we evaluated whether and how HCN channels and GABA_B receptors were pathologically altered and investigated neuroprotective effects of GABA_B receptors activation and cross-talk networks between GABA_B receptors and HCN channels in the hippocampal CA1 area in chronic cerebral hypoperfusion rat model. We found that cerebral hypoperfusion for 5 weeks by permanent occlusion of bilateral common carotid arteries (two-vessel occlusion, 2VO) induced marked spatial and nonspatial learning and memory deficits, significant neuronal loss and decrease in dendritic spine density, impairment of long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses, and reduction of surface expression of GABA_B R1, GABA_B R2, and HCN1, but increase in HCN2 surface expression. Meanwhile, the protein expression of TRIP8b (1a-4), TRIP8b (1b-2), and AAK1 was significantly decreased. Baclofen, a GABA_B receptor agonist, markedly improved the memory impairment and alleviated neuronal damage. Besides, baclofen attenuated the decrease of surface expression of GABA_B R1, GABA_B R2, and HCN1, but downregulated HCN2 surface expression. Furthermore, baclofen could restore expression of AAK1 protein and significantly increase p-PKA, TRIP8b (1a-4), TRIP8b (1b-2), and p-AP2 μ2 expression. Those findings suggested that, under chronic cerebral hypoperfusion, activation of PKA could attenuate baclofen-induced decrease in surface expression of GABA_B R1 and GABA_B R2, and activation of GABA_B receptors not only increased the expression of TRIP8b (1a-4) and TRIP8b (1b-2) but also regulated the function of TRIP8b via AAK1 and p-AP2 μ2, which restored the balance of HCN1/HCN2 surface expression in rat hippocampal CA1 area, and thus ameliorated cognitive impairment.     

Up-regulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 (HCN3) by Specific Interaction with K+ Channel Tetramerization Domain-containing Protein 3 (KCTD3)

Most ion channels consist of the principal ion-permeating core subunit(s) and accessory proteins that are assembled with the channel core. The biological functions of the latter proteins are diverse and include the regulation of the biophysical properties of the ion channel, its connection to signaling pathways and the control of its cell surface expression. There is recent evidence that native hyperpolarization-activated cyclic nucleotide-gated channel complexes (HCN1–4) also contain accessory subunits, among which TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) has been most extensively studied. Here, we identify KCTD3, a so far uncharacterized member of the potassium channel tetramerization-domain containing (KCTD) protein family as an HCN3-interacting protein. KCTD3 is widely expressed in brain and some non-neuronal tissues and colocalizes with HCN3 in specific regions of the brain including hypothalamus. Within the HCN channel family, KCTD3 specifically binds to HCN3 and leads to a profound up-regulation of cell surface expression and current density of this channel. HCN3 can also functionally interact with TRIP8b; however, we found no evidence for channel complexes containing both TRIP8b and KCTD3. The C terminus of HCN3 is crucially required for functional interaction with KCTD3. Replacement of the cytosolic C terminus of HCN2 by the corresponding domain of HCN3 renders HCN2 sensitive to regulation by KCTD3. The C-terminal-half of KCTD3 is sufficient for binding to HCN3. However, the complete protein including the N-terminal tetramerization domain is needed for HCN3 current up-regulation. Together, our experiments indicate that KCTD3 is an accessory subunit of native HCN3 complexes.     

Enhancement of Dorsal Hippocampal Activity by Knockdown of HCN1 Channels Leads to Anxiolytic- and Antidepressant-like Behaviors

The hippocampus is an integral brain region for affective disorders. TRIP8b knockout mice lacking functional HCN channels as well as both HCN1 and HCN2 knockout mice have been shown to display antidepressant-like behaviors. The mechanisms or?brain regions involved in these alterations in behavior, however, are not clear. We developed a lentiviral shRNA system to examine whether knockdown of HCN1 protein in the dorsal hippocampal CA1 region is sufficient to produce antidepressant-like effects. We found that knockdown of HCN1 channels increased cellular excitability and resulted in physiological changes consistent with a reduction of I(h). Rats infused with lentiviral shRNA-HCN1 in the dorsal hippocampal CA1 region displayed antidepressant- and anxiolytic-like behaviors associated with widespread enhancement of hippocampal activity and upregulation of BDNF-mTOR signaling pathways. Our results suggest that HCN1 protein could be a potential target for treatment of anxiety and depression disorders.     

Rescue of a Trafficking Defective Human Pacemaker Channel via a Novel Mechanism: ROLES OF Src, Fyn, AND Yes TYROSINE KINASES*

Therapeutic strategies such as using channel blockers and reducing culture temperature have been used to rescue some long QT-associated voltage-gated potassium Kv trafficking defective mutant channels. A hyperpolarization-activated cyclic nucleotide-gated HCN4 pacemaker channel mutant (D553N) has been recently found in a patient associated with cardiac arrhythmias including long QT. D553N showed the defective trafficking to the cell surface, leading to little ionic current expression (loss-of-function). We show in this report that enhanced tyrosine phosphorylation mediated by Src, Fyn, and Yes kinases was able to restore the surface expression of D553N for normal current expression. Src or Yes, but not Fyn, significantly increased the current density and surface expression of D553N. Fyn accelerated the activation kinetics of the rescued D553N. Co-expression of D553N with Yes exhibited the slowest activation kinetics of D553N. Src, Fyn, and Yes significantly enhanced the tyrosine phosphorylation of D553N. A combination of Src, Fyn, and Yes rescued the current expression and the gating of D553N comparable with those of wild-type HCN4. In conclusion, we demonstrate a novel mechanism using three endogenous Src kinases to rescue a trafficking defective HCN4 mutant channel (D553N) by enhancing the tyrosine phosphorylation of the mutant channel protein.Defective trafficking leading to the reduced surface expression of ion channels is one of the mechanisms responsible for a loss-of-function of the ion channel on the plasma membrane (1). Several methods have been developed to rescue the voltage-gated potassium Kv trafficking defective channels: reducing the culture temperature, applying the channel blockers, altering the molar ratio of glycerol, and using the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (2–6).Hyperpolarizing-activated cyclic nucleotide-gated (HCN)³ pacemaker channels generate time- and voltage-dependent inward currents, named I_h in neurons or I_f in the heart (7). They are important in various cell functions including excitability, synapse transmission, and rhythmic activity (7). The most well studied regulation of I_f is its response to autonomic stimulation. β-Adrenergic receptor activation increases and acetylcholine receptor activation decreases the intracellular cAMP levels, which in turn increases/decreases I_f by binding to the cyclic nucleotide-binding domain of the HCN channels, respectively (7). Other important mechanisms for the modulation of I_f/HCN channels have recently been found including β-subunit (8), lipids (9, 10), and p38 mitogen-activated protein kinase (11).Accumulating evidence has revealed tyrosine phosphorylation as an important mechanism for modulation of HCN channel properties (12–16). An acute increase in tyrosine phosphorylation of I_f or HCN channels increases the channel activity, including an increase in the current amplitude, a positive shift of the voltage-dependent activation, an acceleration of activation kinetics, and an increase in whole cell conductance (12–15). Recently, we discovered that the cell surface expression of HCN2 channels can be remarkably inhibited by tyrosine dephosphorylation mediated by receptor-like protein tyrosine phosphatase α (RPTPα) and increased by tyrosine phosphorylation via Src kinase after long term treatment (17).D553N, a missense HCN4 mutant, was recently identified in a patient with cardiac arrhythmia associated with depressed HCN gating properties (18). Functional and structural assays revealed that D553N expresses little ionic currents, which is possibly due to the defective channel trafficking so that the channels cannot reach the plasma membrane for normal functions (18).The Src kinase family has nine members (19). They are closely related and share the same regulatory function. Three of them, Src, Fyn, and Yes, are ubiquitously expressed in a variety of tissues including neurons and myocytes (19, 20). Without stimulation, they are inactive. However, mutation of key tyrosine residue results in the constitutively active form of the kinase, SrcY529F, FynY531F, and YesY537F, respectively (15, 21, 22). Using these Src kinases, we show in this report a novel approach that can restore the surface expression of D553N for normal current expression via tyrosine phosphorylation.     

Comparison of the PTS1- and Rab8b-binding properties of Pex5p and Pex5Rp/TRIP8b

Tetratricopeptide (TPR)-domain proteins are involved in various cellular processes. The TPR domain is known to be responsible for interaction with other proteins commonly recognizing sequence motifs at the C-termini. One such TPR-protein, TRIP8b, was originally identified in rat as an interaction partner of Rab8b, and its human orthologue as a protein related to the peroxisomal targeting signal 1 (PTS1) receptor Pex5p (Pex5Rp). Somewhat later, the mouse orthologue was reported to bind the hyperpolarization-activated, cyclic nucleotide-regulated HCN channels, and, very recently, the rat orthologue was shown to interact with latrophilin 1, the calcium-independent receptor of alpha-latrotoxin. Here we employed various methodological approaches to investigate and compare the binding specificities of the human PTS1 receptor Pex5p and the related protein Pex5Rp/TRIP8b towards a subset of targets, including Rab8b and various C-termini resembling PTS1. The results show that the TPR domains of Pex5p and Pex5Rp/TRIP8b have distinct but overlapping substrate specificities. This suggests that selectivity in the recognition of substrates by the TPR domains of Pex5p and Pex5Rp/TRIP8b is a matter of considerable complexity, and that no single determinant appears to be sufficient in unambiguously defining a binding target for either protein. This idea is further corroborated by our observations that changes in the surrounding residues or the conformational state of one of the binding partners can profoundly alter their binding activities. The implications of these findings for the possible peroxisome-related functions of Pex5Rp/TRIP8b are discussed.     

P-Loop Residues Critical for Selectivity in K+ Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

HCN channels are thought to be structurally similar to K_v channels, but show much lower selectivity for K⁺. The ∼3.3 Å selectivity filter of K⁺ channels is formed by the pore-lining sequence XT(V/I)GYG, with X usually T, and is held stable by key residues in the P-loop. Differences in the P-loop sequence of HCN channels (eg. the pore-lining sequence L₄₇₈C₄₇₉IGYG) suggest these residues could account for differences in selectivity between these channel families. Despite being expressed, L478T/C479T HCN4 channels did not produce current. Since threonine in the second position is highly conserved in K⁺ channels, we also studied C479T channels. Based on permeability ratios (P_X/P_K), C479T HCN4 channels (K⁺(1)>Rb⁺(0.85)>Cs⁺(0.59)>Li⁺(0.50)≥Na⁺(0.49)) were less selective than WT rabbit HCN4 (K⁺(1)>Rb⁺(0.48)>Cs⁺(0.31)≥Na⁺(0.29)>Li⁺(0.03)), indicating that the TIGYG sequence is insufficient to confer K⁺ selectivity to HCN channels. C479T HCN4 channels had an increased permeability to large organic cations than WT HCN4 channels, as well as increased unitary K⁺ conductance, and altered channel gating. Collectively, these results suggest that HCN4 channels have larger pores than K⁺ channels and replacement of the cysteine at position 479 with threonine further increases pore size. Furthermore, selected mutations in other regions linked previously to pore stability in K⁺ channels (ie. S475D, S475E and F471W/K472W) were also unable to confer K⁺ selectivity to C479T HCN4 channels. Our findings establish the presence of the TIGYG pore-lining sequence does not confer K⁺ selectivity to rabbit HCN4 channels, and suggests that differences in selectivity of HCN4 versus K⁺ channels originate from differences outside the P-loop region.     

Filamin A Promotes Dynamin-dependent Internalization of Hyperpolarization-activated Cyclic Nucleotide-gated Type 1 (HCN1) Channels and Restricts Ih in Hippocampal Neurons

The actin-binding protein filamin A (FLNa) regulates neuronal migration during development, yet its roles in the mature brain remain largely obscure. Here, we probed the effects of FLNa on the regulation of ion channels that influence neuronal properties. We focused on the HCN1 channels that conduct I_h, a hyperpolarization-activated current crucial for shaping intrinsic neuronal properties. Whereas regulation of HCN1 channels by FLNa has been observed in melanoma cell lines, its physiological relevance to neuronal function and the underlying cellular pathways that govern this regulation remain unknown. Using a combination of mutational, pharmacological, and imaging approaches, we find here that FLNa facilitates a selective and reversible dynamin-dependent internalization of HCN1 channels in HEK293 cells. This internalization is accompanied by a redistribution of HCN1 channels on the cell surface, by accumulation of the channels in endosomal compartments, and by reduced I_h density. In hippocampal neurons, expression of a truncated dominant-negative FLNa enhances the expression of native HCN1. Furthermore, acute abrogation of HCN1-FLNa interaction in neurons, with the use of decoy peptides that mimic the FLNa-binding domain of HCN1, abolishes the punctate distribution of HCN1 channels in neuronal cell bodies, augments endogenous I_h, and enhances the rebound-response (“voltage-sag”) of the neuronal membrane to transient hyperpolarizing events. Together, these results support a major function of FLNa in modulating ion channel abundance and membrane trafficking in neurons, thereby shaping their biophysical properties and function.     

Up-regulation of GABAB Receptor Signaling by Constitutive Assembly with the K+ Channel Tetramerization Domain-containing Protein 12 (KCTD12)

GABA_B receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA_B receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA_B1a, GABA_B1b, and GABA_B2 form fully functional heteromeric GABA_B(1a,2) and GABA_B(1b,2) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA_B(1a,2) and GABA_B(1b,2) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA_B receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA_B receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA_B receptor-mediated K⁺ current response. In summary, our experiments support that the up-regulation of functional GABA_B receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.     

Effector Contributions to Gβγ-mediated Signaling as Revealed by Muscarinic Potassium Channel Gating

Receptor-mediated activation of heterotrimeric G proteins leading to dissociation of the Gα subunit from Gβγ is a highly conserved signaling strategy used by numerous extracellular stimuli. Although Gβγ subunits regulate a variety of effectors, including kinases, cyclases, phospholipases, and ion channels (Clapham, D.E., and E.J. Neer. 1993. Nature (Lond.). 365:403–406), few tools exist for probing instantaneous Gβγ-effector interactions, and little is known about the kinetic contributions of effectors to the signaling process. In this study, we used the atrial muscarinic K⁺ channel, which is activated by direct interactions with Gβγ subunits (Logothetis, D.E., Y. Kurachi, J. Galper, E.J. Neer, and D.E. Clap. 1987. Nature (Lond.). 325:321–326; Wickman, K., J.A. Iniguez-Liuhi, P.A. Davenport, R. Taussig, G.B. Krapivinsky, M.E. Linder, A.G. Gilman, and D.E. Clapham. 1994. Nature (Lond.). 366: 654–663; Huang, C.-L., P.A. Slesinger, P.J. Casey, Y.N. Jan, and L.Y. Jan. 1995. Neuron. 15:1133–1143), as a sensitive reporter of the dynamics of Gβγ-effector interactions. Muscarinic K⁺ channels exhibit bursting behavior upon G protein activation, shifting between three distinct functional modes, characterized by the frequency of channel openings during individual bursts. Acetylcholine concentration (and by inference, the concentration of activated Gβγ) controls the fraction of time spent in each mode without changing either the burst duration or channel gating within individual modes. The picture which emerges is of a Gβγ effector with allosteric regulation and an intrinsic “off” switch which serves to limit its own activation. These two features combine to establish exquisite channel sensitivity to changes in Gβγ concentration, and may be indicative of the factors regulating other Gβγ-modulated effectors.     

A new species of Maladera Mulsant & Rey, 1871 from Iran (Coleoptera: Scarabaeidae: Sericini) and a review of the distribution of the genus in Iran

A new species of Maladera Mulsant & Rey, 1871 is described from Iran: M. kermanensis sp. n. The habitus and genitalia of the new species are illustrated. Additional records of Maladera species and a checklist of the species occurring in Iran including a map of their distribution are given.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:812B8D62-2DD5-42CF-8BCD-F576CF3D2A7E     

Items from the Literature

Conn, H. J. Biological Stains. 7th ed., 355 pages, 27 figures, 9 × 6 inches, hard covers. The Williams &;: Wilkins Company, Baltimore 2, Maryland. $9.00. 1961.

Electron Microscopy Reimer, L., and GADACZ, H. Zur Schichtdickenkontrolle bei der elektronenmikroskopischen Schrägbeschattungsmethode. Z. wiss. Mikr., 65, 105-17. 1961.

Dyes and Their Biological Uses Macconaill, M. A., and GURR, E. The faviolic acid group of stains in histology. Irish J. Med. Sci., 1962, 1-12. 1962.

Animal Microtechnic Berres, H. H. Zur Darstellung peripherer Nervenfasem in der Haut. Z. wiss. Mikr., 65, 63-9. 1961.

Elias H., Henning A., and ELIAS P. M. Contributions to the geometry of sectioning. V. Some methods for the study of kidney structure. Z. wiss. Mikr., 65, 70-82. 1961.

Ewen, A. B. An improved aldehyde-fuchsin staining technique for neurosecretary products in insects. Tr. Amer. Micr. Soc, 81, 94-6. 1962.

Movat, Henry Z., STEINER, JAN W., and HUHN, DIETER. The fine structure of the glomerulus in acute glomerulonephritis. Lab. Investig., 11, 117-35. 1962.

Shaver, Evelyn L. The chromosomes of the opossum, Didelphis virginiana. Canad. J. Genet. Cytol. 4, 62-8. 1962.

Microorganisms Laird, Marshall. Trichodinids and other parasitic protozoa from the intertidal zone at Nanaimo, Vancouver Island. Canad. J. Zool., 39, 833-44. 1961.

Histochemistry Burstone, M. S. New histochemical techniques for the demonstration of tissue oxidase (cytochrome oxidase). I. Hisiochem. Cytochem., 7, 112-22. 1959.

Glenner, G. G., and LILLIE, R. D. Observations on the diazotization-coupling reaction for the histochemical demonstration of tyrosine: metal chelation and formazan variants. J. Histochem. Cytochem., 7, 416-22. 1959.

Hess, R., and Pearse, A. G. E. Histochemical demonstration of uridine diphosphate glucose dehydrogenase. Experientia, 15, 317-8. 1961.

LOVE, R., and LILES, R. H. Differentiation of nucleoproteins by inactivation of protein-bound amino groups and staining with toluidine blue and ammonium molybdate. J. Histochem. Cytochem., 7, 164-81. 1959.

Zimmerman, H., and Pearse, A. G. E. Limitations in the histochemical demonstration of pyridine nucleotide-linked dehydrogenases (“nothing dehydrogenase”). J. Histochem. Cytochem. 7, 271-5. 1959.