Biomedical subjects

C Chavkin

Publications and source records attributed to C Chavkin.

At least 37 records · Page 2Linked to original sources

PubMed1998

Administered and endogenously released kappa opioids decrease pilocarpine-induced seizures and seizure-induced histopathology.

The effects of kappa opioids on seizures and seizure-induced histopathology were investigated with the pilocarpine model of temporal lobe epilepsy. Rats treated with the kappa opioid receptor agonist U50488h before pilocarpine showed: 1) increased seizure latency; 2) decreased seizure duration; 3) decreased mossy fiber sprouting; and 4) increased hilar neuron survival when compared with rats pretreated with saline. Behavioral effects of U50488h were blocked by the kappa opioid receptor antagonist norbinaltorphimine (nBNI), whereas the changes caused by U50488h in the histological response to pilocarpine were not blocked by nBNI. Rats treated with nBNI before pilocarpine exhibited: 1) increased incidence of seizures; 2) increased mossy fiber sprouting; and 3) increased hilar neuron loss when compared with rats treated with pilocarpine alone. These changes suggest a protective role of endogenously released kappa opioids in this seizure model. The location of functional kappa opioid receptors in the rat dentate gyrus was documented electrophysiologically to enable correlation with kappa opioid effects on histopathology. The kappa selective agonist, U69593, reversibly decreased the amplitude of excitatory postsynaptic potentials in the middle molecular layer of the dentate gyrus from the ventral but not the more dorsal portion of the hippocampal formation. Thus, kappa opioids decreased the severity and incidence of behavioral seizures and secondarily decreased seizure-induced histopathology via the decreased incidence of seizures.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1998

Agonist induced homologous desensitization of mu-opioid receptors mediated by G protein-coupled receptor kinases is dependent on agonist efficacy.

Using Xenopus laevis oocytes coexpressing mammalian mu-opioid receptors (MORs), beta-adrenergic receptor kinase 2 (beta-ARK2) [also called G protein-coupled receptor kinase (GRK3)], and beta-arrestin 2 (beta-arr 2), we compared the rates of beta-ARK2 (GRK3)- and beta-arr 2-mediated homologous receptor desensitization produced by treatment with opioid agonists of different efficacies. The response to MOR activation was measured using two-electrode voltage clamp as an increase in the conductance of the coexpressed G protein-coupled inwardly rectifying potassium (heteromultimer of KIR3.1 and KIR3.4) channels. Treatment with opioids of high efficacy, either [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin, fentanyl, or sufentanyl, produced a GRK3- and beta-arr 2-dependent reduction in response in <20 min, whereas treatment with the partial agonist morphine produced receptor desensitization at a significantly slower rate. Because GRK3 requires activation and membrane targeting by free G protein betagamma subunits released after agonist-mediated activation of G proteins, a low efficacy agonist such as morphine may produce weak receptor desensitization as a consequence of poor GRK3 activation. To address this hypothesis, we substituted GRK5, a GRK that does not require activation by G protein betagamma. In oocytes expressing GRK5 instead of GRK3, both [D-Ala2,N-MePhe4, Gly-ol5]enkephalin and fentanyl, but not morphine, produced desensitization of MOR-activated potassium conductance. Thus, mu-opioid agonists produced significant receptor desensitization, mediated by either GRK3 or GRK5, at a rate dependent on agonist efficacy.

Analgesics, Opioid↗

PubMed1997

Mu and delta opioid receptors are differentially desensitized by the coexpression of beta-adrenergic receptor kinase 2 and beta-arrestin 2 in xenopus oocytes.

The Xenopus oocyte expression system was used to test the hypothesis that homologous opioid receptor desensitization results from receptor phosphorylation by G protein-coupled receptor kinases. Activation of delta (DOR), mu (MOR) opioid, or beta2-adrenergic receptors increased K+ conductance in oocytes coexpressing the G protein-gated inwardly rectifying K+ channel subunits GIRK1 and GIRK4, and the intrinsic rate of desensitization was small. Coexpression of beta-adrenergic receptor kinase 2 (beta-ARK2) and beta-arrestin 2 (beta-arr2) synergistically produced a rapid desensitization of both DOR and beta2-adrenergic receptor signaling with a t1/2 < 4 min. beta-ARK2 and beta-arr2 more slowly desensitized MOR responses; a similar synergistic effect on MOR required 2-3 h of agonist treatment. DOR mutants lacking serine and threonine residues at the end of the cytoplasmic tail coupled effectively to GIRK channels but were insensitive to beta-ARK2 and beta-arr2. However, a DOR mutant having serine residues mutated to alanine in the third cytoplasmic loop was indistinguishable in coupling and desensitization from the wild type DOR. These studies establish that opioid receptors can be regulated by beta-ARK2 and beta-arr2 and that a portion of the COOH terminus of DOR enhances sensitivity to this modulation.

Amino Acid Sequence↗

PubMed1997

GIRK1 immunoreactivity is present predominantly in dendrites, dendritic spines, and somata in the CA1 region of the hippocampus.

Electron microscopic analysis of the CA1 region of the rat hippocampus revealed that specific immunoreactivity (IR) for a G protein-gated, inwardly rectifying potassium channel (GIRK1) was present exclusively in neurons and predominantly located in spiny dendrites of pyramidal cells. Within stratum lacunosum-moleculare and the superficial stratum radiatum, GIRK1-IR was often present immediately adjacent to asymmetric (excitatory-type) postsynaptic densities in dendritic spines. The subcellular localization of GIRK1-IR in the Golgi apparatus of pyramidal cell somata and in the plasma membrane of dendrites and dendritic spines confirms the hypothesis that GIRK1 is synthesized by pyramidal cells and transported to the more distal dendritic processes. G protein-coupled receptor activation of a dendritic potassium conductance would attenuate the propagation of excitatory synaptic inputs and thereby produce postsynaptic inhibition. Thus, these results show that the GIRK family of channels joins the list of voltage-sensitive channels now known to be expressed in dendritic spines.

Amino Acid Sequence↗

PubMed1997

Changes in hippocampal circuitry after pilocarpine-induced seizures as revealed by opioid receptor distribution and activation.

The pilocarpine model of temporal lobe epilepsy was used to study the time-dependent changes in dentate gyrus circuitry after seizures. Seizures caused a decrease in mu- and delta-opioid receptor immunoreactive (MOR-IR and DOR-IR, respectively) neurons in the hilus and MOR-IR neurons in the granule cell layer. Additionally, diffuse DOR-IR, MOR-IR, and GABA immunoreactivities (GABA-IR) were increased in the inner molecular layer. Using the in vitro hippocampal slice preparation to study the physiological consequences of the anatomical changes, we found that the disinhibitory effects of the mu-opioid receptor agonist [D-Ala2, MePhe4,Gly-(ol)5]-enkephalin (DAMGO) and the GABAA receptor antagonist bicuculline were greatly depressed 5-13 d after pilocarpine injection but returned to control levels within 6 weeks. The amplitudes of monosynaptic evoked IPSCs and the effects of DAMGO on this parameter were also slightly decreased 5-13 d after pilocarpine injection but significantly increased at 6 weeks. DAMGO significantly decreased the mean amplitude of spontaneous IPSCs (sIPSCs) at 6 weeks after pilocarpine injection but not in controls. The delta-opioid receptor agonist [D-Pen2,5]-enkephalin (DPDPE) principally inhibited excitatory transmission in saline-treated animals without affecting either sIPSCs or evoked IPSCs. The DPDPE-induced inhibition of excitatory transmission became more pronounced at 6 weeks after pilocarpine injection. These results illustrate the anatomical reorganization and functional changes in dentate gyrus circuitry evident in an animal model of temporal lobe epilepsy and provide evidence of compensatory changes after trauma to the hippocampal formation.

Animals↗

PubMed1997

Kappa opioid receptor-like immunoreactivity is present in substance P-containing subcortical afferents in guinea pig dentate gyrus.

We have previously shown that kappa opioid receptor-like immunoreactivity (KT-LI) is present in axons and terminals in the granule cell layer and inner molecular layer of the guinea pig dentate gyrus. The distribution and ultrastructural appearance of processes with KT-LI were similar to those of the substance P (SP)-containing afferents which arise from the supramammillary region of the hypothalamus (SUM) and enter the hippocampal formation through the fimbria-fornix. The objective of the present study was to determine whether the terminals with KT-LI are likely to be SUM afferents. To accomplish this we 1) compared the intensity of KT- and SP-immunolabeling in the dentate gyrus ipsilateral and contralateral to a unilateral fornix transection and 2) used dual-labeling electron microscopy to determine whether terminals with KT-LI colocalize SP-LI in the dentate gyrus. Light microscopic examination of the dentate gyrus demonstrated that KT-LI and SP-LI were in thin processes with overlapping distributions in strata granulosum and moleculare. Following fornix transection, both KT-LI and SP-LI were dramatically reduced in these regions of the dentate gyrus ipsilateral to the transection, consistent with an SUM origin. By electron microscopy, most (71%) terminals with KT-LI also contained detectable SP-LI in single-section analysis. Many dual-labeled terminals formed thick asymmetric synaptic contacts with large dendritic shafts (2-5 microns) or granule cell perikarya, and a smaller proportion contacted dendritic spines; these characteristics resembled those of identified SUM afferents in other species. The demonstrations that 1) KT-LI colocalizes with SP-LI in a morphologically distinctive population of axon terminals and 2) most of the processes with KT-LI enter through the fimbria-fornix suggest that kappa opioid receptors are present in the SUM projection to the dentate gyrus.

Afferent Pathways↗

PubMed1997

Agonist-induced phosphorylation of the kappa-opioid receptor.

Antipeptide antibodies against the kappa-opioid receptor were used to test whether acute or chronic exposure to kappa agonists altered the phosphorylation state of the kappa-opioid receptor. Immunoprecipitation of the kappa receptor from guinea pig hippocampal slices preincubated in [32P]orthophosphoric acid revealed a basal phosphorylation of the kappa-opioid receptor. The amount of 32P incorporation into the receptor was increased following a 75-min treatment with the kappa agonist U50,488H. This effect was blocked by the selective kappa receptor antagonist norbinaltorphimine. The time course of this change in the phosphorylation state of the receptor correlated with a desensitization of the electrophysiological response to kappa agonists measured in the dentate gyrus of hippocampal slices. The phosphorylation state of the kappa-opioid receptor was also elevated in brain slices from guinea pigs made tolerant to U50,488H by 5 days of continuous exposure and then maintained in kappa agonist to avoid acute opiate withdrawal. The results of this study show that the kappa-opioid receptor was phosphorylated in an agonist-dependent manner in brain slices taken from untreated and U50,488H-tolerant animals.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1997

Spontaneous excitatory currents and kappa-opioid receptor inhibition in dentate gyrus are increased in the rat pilocarpine model of temporal lobe epilepsy.

Temporal lobe epilepsy is associated with a characteristic pattern of synaptic reorganization in the hippocampal formation, consisting of neuronal loss and aberrant growth of mossy fiber collaterals into the dentate gyrus inner molecular layer. We have used the rat pilocarpine model of temporal lobe epilepsy to study the functional consequences of mossy fiber sprouting on excitatory activity and kappa-opioid receptor-mediated inhibition. Using the whole cell voltage-clamp technique, we found that abnormal excitatory activity was evident in granule cells of the dentate gyrus from pilocarpine-treated rats. The frequency of spontaneous excitatory postsynaptic currents (EPSCs) was increased greatly in cells from tissue in which significant mossy fiber sprouting had developed. In the presence of bicuculline, giant spontaneous EPSCs, with large amplitudes and long durations, were seen only in association with mossy fiber sprouting. Giant EPSCs also could be evoked by low-intensity stimulation of the perforant path. Mossy fibers release not only excitatory amino acids, but also opioid peptides. kappa-Opioid receptor-mediated inhibition in normal Sprague-Dawley rats was seen only in hippocampal sections from the ventral pole. In pilocarpine-treated rats, however, kappa receptor-mediated effects were seen in both ventral and more dorsal sections. Thus in this model of temporal lobe epilepsy, several types of abnormal excitatory activity were observed, thereby supporting the idea that mossy fiber sprouting leads to recurrent excitatory connections. At the same time, inhibition of excitatory activity by kappa-opioid receptors was increased, perhaps representing an endogenous anticonvulsant mechanism.

Animals↗

PubMed1997

Kappa opioid receptor tolerance in the guinea pig hippocampus.

We investigated whether chronic, in vivo administration of U50,488H, a kappa-1 opioid agonist, caused the development of tolerance to both the electrophysiological effects of applied kappa opioids and endogenously released dynorphins. In hippocampal slices from drug-naive guinea pigs, application of U69,593, a kappa-1 agonist, produced a concentration-dependent inhibition (EC50 = 20 nM) of the amplitude of the granule cell population response in the dentate gyrus. In slices from chronically U50,488H-treated animals, the concentration-response curve for U69,593 was shifted 3-fold to the right (EC50 = 59 nM), with a significant decrease in the maximal effect of U69,593. We also found that the effects of endogenously released dynorphins were significantly attenuated by chronic U50,488H treatment. There was no cross-tolerance between kappa and mu opioid receptor agonists as measured with the in vitro electrophysiological assay, and the noncompetitive N-methyl-D-aspartate receptor antagonist MK801 did not prevent the development of tolerance to either the electrophysiological effects or the hypothermic effects of kappa opioids. Our study demonstrates that receptor-selective tolerance to the kappa opioid actions in the guinea pig hippocampus does develop after chronic U50,488H treatment; but, unlike the mechanisms reported to underlie tolerance to kappa opioid analgesia, the inhibitory effects in the hippocampus did not depend on activation of N-methyl-D-aspartate receptors.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1996

Vicia villosa agglutinin labels a subset of neurons coexpressing both the mu opioid receptor and parvalbumin in the developing rat subiculum.

Vicia villosa agglutinin (VVA), anti-parvalbumin antiserum and an affinity-purified anti-mu opioid receptor antibody were used to triple-label neurons in the postnatal rat subiculum. VVA labeled a subset of mu opioid receptor-positive neurons that were also immunoreactive for parvalbumin. The morphology of the triple-labeled neurons was heterogeneous, and included multipolar, ovoid and pyramidal-shaped neurons. Neurons single-labeled for the mu opioid receptor, VVA or parvalbumin were also morphologically heterogeneous. The postnatal development of mu opioid receptor immunoreactivity (IR), parvalbumin-IR and VVA binding was investigated using triple-labeling immunocytochemistry. Mu opioid receptor-IR appeared first and was present at postnatal day 1 (P1). Parvalbumin-IR was first observed in somata at P10, followed by proximal and distal dendrites at P15 and P20 respectively. Faint VVA labeling was seen first at P10 and surrounded a limited number of neurons. The intensity of labeling and the number of neurons labeled with VVA increased between P10 and P20; however, both measures remained below adult levels at P20. This study further illustrates the neurochemical heterogeneity of interneurons in the hippocampal formation and shows the developmentally early appearance of mu opioid receptor-IR compared to the late appearance of VVA binding and parvalbumin-IR.

Animals↗

PubMed1996

Kappa opioid receptor-like immunoreactivity in guinea pig brain: ultrastructural localization in presynaptic terminals in hippocampal formation.

Physiological and pharmacological studies have suggested that kappa opioid receptors (KORs) may be located presynaptically in the guinea pig hippocampal formation. In the present study, KOR-like immunoreactivity (-LI) was examined by using a rabbit antibody raised against a synthetic peptide from the carboxyl terminus of a cloned rat kappa receptor (KT). The specificity of affinity-purified KT antibody was confirmed by Western blotting, enzyme-linked immunosorbent assay, immunolabeling of KORs expressed in Xenopus oocytes, and immunocytochemical preadsorption controls. Specificity also was demonstrated by the light microscopic distribution of KT-LI in sections through the forebrain and the pons, which was largely consistent with the distribution of KORs previously reported, and resembled that of immunoreactivity for dynorphin B, an endogenous ligand for KORs. Detailed analysis of the hippocampal formation revealed that KT-LI was located predominantly in thin processes in the granule cell and inner molecular layers of the dentate gyrus. A few KT-labeled processes were also present in stratum lacunosum-moleculare of the CA1 region and all layers of the CA3 region of the hippocampus. By electron microscopy, KT-LI was restricted to unmyelinated axons and axon terminals, and was associated with plasma membranes, large dense-core vesicles, and cytoplasmic surfaces of small vesicles. In the dentate gyrus, immunolabeled terminals formed asymmetric synapses with granule cell perikarya and large unlabeled dendrites. In the CA3 region of hippocampus, KT-LI was present in small unmyelinated axons. The results of this study 1) demonstrate the specificity of the KT antibody, 2) show that the distribution of KT labeling corresponds well with previous KOR and dynorphin localization in many regions, and 3) provide ultrastructural evidence that KORs are located presynaptically in the guinea pig hippocampal formation.

Animals↗

PubMed1996

N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.

Animals↗

PubMed1996

Endogenous opioid regulation of hippocampal function.

Endogenous opioid peptides modulate neural transmission in the hippocampus. Procnkephalin-derived peptides have been demonstrated to act at mu and delta opioid receptors to inhibit GABA release from inhibitory interneurons, resulting in increased excitability of hippocampal pyramidal cells and dentate gyrus granule cells. Prodynorphin-derived peptides primarily act at presynaptic kappa opioid receptors to inhibit excitatory amino acid release from perforant path and mossy fiber terminals. Opioid receptors reduce membrane excitability by modulating ion conductances, and in this way they may decrease voltage-dependent calcium influx and transmitter release. Synaptic plasticity in the hippocampus also is modulated by endogenous opioids. Enkephalins facilitate long-term potentiation, whereas dynorphins inhibit the induction of this type of neuroplasticity. Further, opioids may play important roles in hippocampal epilepsy. Recurrent seizures induce changes in the expression of opioid peptides and receptors. Also, enkephalins have proconvulsant effects in the epileptic hippocampus, whereas dynorphins may function as endogenous anticonvulsants.

Animals↗

PubMed1996

Induction of CRE-mediated gene expression by stimuli that generate long-lasting LTP in area CA1 of the hippocampus.

Gene expression regulated by the cAMP response element (CRE) has been implicated in synaptic plasticity and long-term memory. It has been proposed that CRE-mediated gene expression is stimulated by signals that induce long-term potentiation (LTP). To test this hypothesis, we made mice transgenic for a CRE-regulated reporter construct. We focused on long-lasting long-term potentiation (L-LTP), because it depends on cAMP-dependent protein kinase activity (PKA) and de novo gene expression. CRE-mediated gene expression was markedly increased after L-LTP, but not after decremental UP (D-LTP). Furthermore, inhibitors of PKA blocked L-LTP and associated increases in CRE-mediated gene expression. These data demonstrate that the signaling required for the generation of L-LTP but not D-LTP is sufficient to stimulate CRE-mediated transcription in the hippocampus.

Animals↗

PubMed1996

The conserved aspartate residue in the third putative transmembrane domain of the delta-opioid receptor is not the anionic counterpart for cationic opiate binding but is a constituent of the receptor binding site.

Opioids are cationic compounds that mediate their biological action through three highly homologous receptors (mu, delta, and kappa) known to belong to the G protein-coupled receptor (GPR) family. The third putative transmembrane domain of opioid receptors contains a conserved aspartate residue that is typically found in biogenic amine binding GPRs and is generally believed to form an ion pair with the cationic neurotransmitters. Using site-directed mutagenesis, we investigated the possibility of an identical role for this residue (Asp128) in the mouse delta-opioid receptor. Removal of the carboxylate group via an aspartate-to-alanine mutation did not modify binding affinity of a representative set of opioid compounds, including bremazocine, diprenorphine, naloxone, Tyr-D-Thr-Gly-Phe-Leu-Thr, [D-Ala2,D-Leu5]enkephalin, cyclic[D-penicillamine2,D-penicillamine5]enkephalin, deltorphin II, (+/-)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl-2,5-di-methyl-1- piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide, and naltrindole. It nevertheless decreased receptor expression level and affected the binding of three agonists ([D-Ala2,D-Leu5]enkephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr, and (+/-)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl-2,5-di- methyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide) when the receptor was under Na(+)-induced low affinity state. On the other hand, the aspartate-to-asparagine mutation strongly impaired the binding of all of the above ligands and highlighted differential modes of interaction for alkaloids and peptides. Finally, removal of the homologous carboxylate group in the mouse mu receptor had distinct effects because it dramatically reduced the binding potency of some, but not all, tested ligands. Taken together, these results demonstrate that (i) the direct ligand/receptor interaction previously demonstrated for the beta-adrenergic receptor does not take place in the delta receptor, (ii) Asp128 nevertheless contributes to stabilization of the spatial conformation of the binding pocket, and (iii) these conclusions cannot be extended to the closely related mu receptor.

Amino Acid Sequence↗

PubMed1996

k-Opioid receptor activation of a dendrotoxin-sensitive potassium channel mediates presynaptic inhibition of mossy fiber neurotransmitter release.

Activation of kappa-opioid receptors on mossy fiber terminals in the hippocampus inhibits excitatory amino acid release. The mechanism of presynaptic inhibition at the mossy fiber synapse was investigated through whole-cell voltage-clamp of CA3 pyramidal cells. The application of a kappa-opioid agonist, U69593, reduced the amplitude of the excitatory postsynaptic current response, and this effect was reversed with a k receptor antagonist. Presynaptic potassium channels were blocked by bath application of channel toxins, and the effect of kappa receptor activation was tested. The inhibition caused by U69593 was blocked by low doses of 4-aminopyridine (30 microM) and the selective peptide toxins dendrotoxin and mast cell degranulating peptide. The inhibition was not blocked by low doses of tetraethylammonium chloride (1 mM), barium, or glibenclamide. Thus, we conclude that presynaptic kappa-opioid receptors are coupled to a Shaker-type voltage-dependent potassium channel that is sensitive to dendrotoxin and mast cell degranulating peptide. An increase in presynaptic potassium conductance would enhance the rate of repolarization after action potential invasion, thereby limiting calcium influx and neurotransmitter release. This is the first physiological demonstration of the involvement of a dendrotoxin-sensitive potassium current in presynaptic inhibition mediated by a G protein-coupled receptor.

4-Aminopyridine↗

PubMed1995

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Amino Acid Sequence↗

PubMed1995

Phosphorylation of presynaptic and postsynaptic calcium channels by cAMP-dependent protein kinase in hippocampal neurons.

Phosphorylation by cAMP-dependent protein kinase (PKA) and other second messenger-activated protein kinases modulates the activity of a variety of effector proteins including ion channels. Anti-peptide antibodies specific for the alpha 1 subunits of the class B, C or E calcium channels from rat brain specifically recognize a pair of polypeptides of 220 and 240 kDa, 200 and 220 kDa, and 240 and 250 kDa, respectively, in hippocampal slices in vitro. These calcium channels are localized predominantly on presynaptic and dendritic, somatic and dendritic, and somatic sites, respectively, in hippocampal neurons. Both size forms of alpha 1B and alpha 1E and the full-length form of alpha 1C are phosphorylated by PKA after solubilization and immunoprecipitation. Stimulation of PKA in intact hippocampal slices also induced phosphorylation of 25-50% of the PKA sites on class B N-type calcium channels, class C L-type calcium channels and class E calcium channels, as assessed by a back-phosphorylation method. Tetraethylammonium ion (TEA), which causes neuronal depolarization and promotes repetitive action potentials and neurotransmitter release by blocking potassium channels, also stimulated phosphorylation of class B, C and E alpha 1 subunits, suggesting that these three classes of channels are phosphorylated by PKA in response to endogenous electrical activity in the hippocampus. Regulation of calcium influx through these calcium channels by PKA may influence calcium-dependent processes within hippocampal neurons, including neurotransmitter release, calcium-activated enzymes and gene expression.

Animals↗