Processing of GABABR1 in heterologous expression systems.
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Animals↗
Biomedical subjects
A Karschin
Publications and source records attributed to A Karschin.
Alternative splicing generates a novel isoform of the rat metabotropic GABA(B)R1 receptor.
Here we present a novel isoform of the metabotropic G-protein-coupled receptor for gamma-aminobutyric acid (GABA). The isoform, termed GABA(B)R1c (R1c), differs from the recently identified R1a and R1b receptors by an in-frame insertion of 31 amino acids between the second extracellular loop and the fifth transmembrane region. Analysis of the rat GABA(B)R1 gene demonstrates that the insertion is the result of an alternative splicing event within a 567-bp intron between exons 16 and 17. In situ hybridization in the rat brain shows a wide distribution of R1c transcripts and an overlap with the R1a and R1b transcripts. The highest mRNA levels are found in cerebellar Purkinje cells, cerebral cortex, thalamus and hippocampal CA1 and CA3 regions. Western blots and immunodetection of recombinant epitope-tagged receptors as well as [125I]CGP71872 photoaffinity labelling of cell membranes demonstrate that R1c is correctly expressed, although at a lower level than the previously identified isoforms. When coexpressed with the newly characterized GABA(B)R2, R1c functionally couples to G-protein-activated Kir3.1/3.2 channels in Xenopus oocytes and to PLC-activating chimeric G(alpha)qo subunits in HEK-293 cells with a similar EC50 for agonists. These data suggest that the R1c isoform represents a functional GABA(B)R in the rat brain.
Alternative Splicing↗
Acute suppression of inwardly rectifying Kir2.1 channels by direct tyrosine kinase phosphorylation.
Signaling via cytosolic and receptor tyrosine kinases is associated with cell growth and differentiation but also targets onto transmitter receptors and ion channels. Here, regulation by tyrosine kinase (TK) activity was investigated for inwardly rectifying K+ (Kir2.1) channels that control membrane excitability in many central neurons. In mammalian tsA-201 cells, the membrane-permeable protein tyrosine phosphatase inhibitor, perorthovanadate (100 microM), suppressed currents through recombinant Kir2.1 channels by 60 +/- 20%. Coapplication of the TK inhibitor genistein (100 microM) completely abolished this effect. Native Kir2.1 channels in rat basophilic leukocytes were affected by manipulation of the TK and protein tyrosine phosphatase activity in a qualitatively similar manner. Site mutation of a tyrosine consensus residue for TK phosphorylation in the C-terminal domain of Kir2.1 generated channel properties indistinguishable from wild-type Kir2.1 channels. However, Kir2.1Y242F channels were no longer suppressed following exposure to perorthovanadate, indicating that the channel is a direct substrate for TKs. After coexpression of nerve growth factor receptor with Kir2.1 channels in tsA-201 cells and Xenopus oocytes, the activity of Kir2.1 was rapidly suppressed by applied nerve growth factor (0.5 microgram/ml) by 31 +/- 10 and 21 +/- 15%, respectively. Acute inhibition was also evoked by epidermal growth factor and insulin via endogenous insulin receptors, indicating that Kir2.1 channels may serve as a general target for neurotrophic growth factors in the brain.
Amino Acid Sequence↗
GABA(B)-receptor subtypes assemble into functional heteromeric complexes.
B-type receptors for the neurotransmitter GABA (gamma-aminobutyric acid) inhibit neuronal activity through G-protein-coupled second-messenger systems, which regulate the release of neurotransmitters and the activity of ion channels and adenylyl cyclase. Physiological and biochemical studies show that there are differences in drug efficiencies at different GABA(B) receptors, so it is expected that GABA(B)-receptor (GABA(B)R) subtypes exist. Two GABA(B)-receptor splice variants have been cloned (GABA(B)R1a and GABA(B)R1b), but native GABA(B) receptors and recombinant receptors showed unexplained differences in agonist-binding potencies. Moreover, the activation of presumed effector ion channels in heterologous cells expressing the recombinant receptors proved difficult. Here we describe a new GABA(B) receptor subtype, GABA(B)R2, which does not bind available GABA(B) antagonists with measurable potency. GABA(B)R1a, GABA(B)R1b and GABA(B)R2 alone do not activate Kir3-type potassium channels efficiently, but co-expression of these receptors yields a robust coupling to activation of Kir3 channels. We provide evidence for the assembly of heteromeric GABA(B) receptors in vivo and show that GABA(B)R2 and GABA(B)R1a/b proteins immunoprecipitate and localize together at dendritic spines. The heteromeric receptor complexes exhibit a significant increase in agonist- and partial-agonist-binding potencies as compared with individual receptors and probably represent the predominant native GABA(B) receptor. Heteromeric assembly among G-protein-coupled receptors has not, to our knowledge, been described before.
Amino Acid Sequence↗
Partial gene structure and assignment to chromosome 2q37 of the human inwardly rectifying K+ channel (Kir7.1) gene (KCNJ13).
The novel weakly inward rectifying potassium channel Kir7.1 is a low-conductance channel that is predominantly expressed in epithelial cells. Here we describe a partial genomic characterization and the chromosomal assignment of the human Kir7.1 gene (KCNJ13). Analysis of the genomic structure using a PCR-based approach revealed a single 2088-bp intron in the coding region of KCNJ13. PCR analysis of monochromosomal and radiation hybrid panels assigns KCNJ13 to band 2q37 between markers D2S331 and D2S345. In addition, a single nucleotide polymorphism (C524-->T), leading to an exchange of a Thr with an Ile residue at amino acid position 175, was found.
Amino Acid Sequence↗
Human gamma-aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying K+ channels.
gamma-Aminobutyric acid type B receptors (GABABRs) are involved in the fine tuning of inhibitory synaptic transmission. Presynaptic GABABRs inhibit neurotransmitter release by down-regulating high-voltage activated Ca2+ channels, whereas postsynaptic GABABRs decrease neuronal excitability by activating a prominent inwardly rectifying K+ (Kir) conductance that underlies the late inhibitory postsynaptic potentials. Here we report the cloning and functional characterization of two human GABABRs, hGABABR1a (hR1a) and hGABABR1b (hR1b). These receptors closely match the pharmacological properties and molecular weights of the most abundant native GABABRs. We show that in transfected mammalian cells hR1a and hR1b can modulate heteromeric Kir3.1/3.2 and Kir3.1/3.4 channels. Heterologous expression therefore supports the notion that Kir3 channels are the postsynaptic effectors of GABABRs. Our data further demonstrate that in principle either of the cloned receptors could mediate inhibitory postsynaptic potentials. We find that in the cerebellum hR1a and hR1b transcripts are largely confined to granule and Purkinje cells, respectively. This finding supports a selective association of hR1b, and not hR1a, with postsynaptic Kir3 channels. The mapping of the GABABR1 gene to human chromosome 6p21.3, in the vicinity of a susceptibility locus (EJM1) for idiopathic generalized epilepsies, identifies a candidate gene for inherited forms of epilepsy.
Amino Acid Sequence↗
The epithelial inward rectifier channel Kir7.1 displays unusual K+ permeation properties.
Rat and human cDNAs were isolated that both encoded a 360 amino acid polypeptide with a tertiary structure typical of inwardly rectifying K+ channel (Kir) subunits. The new proteins, termed Kir7.1, were <37% identical to other Kir subunits and showed various unique residues at conserved sites, particularly near the pore region. High levels of Kir7.1 transcripts were detected in rat brain, lung, kidney, and testis. In situ hybridization of rat brain sections demonstrated that Kir7.1 mRNA was absent from neurons and glia but strongly expressed in the secretory epithelial cells of the choroid plexus (as confirmed by in situ patch-clamp measurements). In cRNA-injected Xenopus oocytes Kir7.1 generated macroscopic Kir currents that showed a very shallow dependence on external K+ ([K+]e), which is in marked contrast to all other Kir channels. At a holding potential of -100 mV, the inward current through Kir7.1 averaged -3.8 +/- 1.04 microA with 2 mM [K+]e and -4.82 +/- 1.87 microA with 96 mM [K+]e. Kir7.1 has a methionine at position 125 in the pore region where other Kir channels have an arginine. When this residue was replaced by the conserved arginine in mutant Kir7.1 channels, the pronounced dependence of K+ permeability on [K+]e, characteristic for other Kir channels, was restored and the Ba2+ sensitivity was increased by a factor of approximately 25 (Ki = 27 microM). These findings support the important role of this site in the regulation of K+ permeability in Kir channels by extracellular cations.
Amino Acid Sequence↗
Review: Evolutionary link between prokaryotic and eukaryotic K+ channels.
Considering the importance of K+ channels in controlling the crucial K+ gradient across the plasma membranes of all living cells, it comes as no surprise that, besides being present in every eukaryotic cell, these integral membrane proteins have recently also been identified in prokaryotes. Today, approximately a dozen successfully completed and many more ongoing sequencing projects permit a search for genes related to K+ channels in the genomes of both eubacteria and archaea. The coding regions of homologues show a remarkable variety in primary structure. They predict membrane proteins with one, two, three and six hydrophobic segments surrounding a putative K+-selective pore (H5) and the presence or absence of a cytosolic putative NAD+-binding domain (PNBD) that probably senses the reducing power of the cell. The analysis of kinships on the basis of phylogenetic algorithms identifies sequences closely related to eukaryotic voltage-dependent Kv channels, but also defines members of a primordial class of prokaryotic K+ channel (containing the 2TMS/PNBD motif). Considering the unique mechanisms that may account for the assembly of modern proteins from different ancestral genes, and with more primary sequence data soon to appear, a scheme for the evolutionary origin of K+ channels comes within reach.
Journal Article↗
A hyperprostaglandin E syndrome mutation in Kir1.1 (renal outer medullary potassium) channels reveals a crucial residue for channel function in Kir1.3 channels.
Loss of function mutations in kidney Kir1.1 (renal outer medullary potassium channel, KCNJ1) inwardly rectifying potassium channels can be found in patients suffering from hyperprostaglandin E syndrome (HPS), the antenatal form of Bartter syndrome. A novel mutation found in a sporadic case substitutes an asparagine by a positively charged lysine residue at amino acid position 124 in the extracellular M1-H5 linker region. When heterologously expressed in Xenopus oocytes and mammalian cells, current amplitudes from mutant Kir1.1a[N124K] channels were reduced by a factor of approximately 12 as compared with wild type. A lysine at the equivalent position is present in only one of the known Kir subunits, the newly identified Kir1.3, which is also poorly expressed in the recombinant system. When the lysine residue in guinea pig Kir1.3 (gpKir1.3) isolated from a genomic library was changed to an asparagine (reverse HPS mutation), mutant channels yielded macroscopic currents with amplitudes increased 6-fold. From single channel analysis it became apparent that the decrease in mutant Kir1.1 channels and the increase in mutant gpKir1.3 macroscopic currents were mainly due to the number of expressed functional channels. Coexpression experiments revealed a dominant-negative effect of Kir1.1a[N124K] and gpKir1.3 on macroscopic current amplitudes when coexpressed with wild type Kir1.1a and gpKir[K110N], respectively. Thus we postulate that in Kir1.3 channels the extracellular positively charged lysine is of crucial functional importance. The HPS phenotype in man can be explained by the lower expression of functional channels by the Kir1. 1a[N124K] mutant.
Amino Acid Sequence↗
KATP channel formation by the sulphonylurea receptors SUR1 with Kir6.2 subunits in rat dorsal vagal neurons in situ.
1. Functional and molecular properties of ATP-sensitive K+ (KATP) channels were studied in dorsal vagal neurons (DVNs) of rat brainstem slices using patch-clamp and single-cell antisense RNA amplification-polymerase chain reaction (PCR) techniques. 2. In the cell-attached configuration, 1 mM cyanide resulted in block of spontaneous firing and concomitant opening of single channels with a mean single open time of 2-3 ms and a burst duration of up to several hundred milliseconds. Inhibition of such single-channel activity with 200 microM tolbutamide led to the reappearance of spontaneous discharge. 3. Whole-cell recordings during anoxia revealed a hyperpolarization of the DVNs. Harvesting of cytoplasm, antisense RNA amplification and subsequent PCR showed coexpression for single DVNs of mRNA for the sulphonylurea receptor SUR1 isoform and for the inwardly rectifying K+ channel subunit Kir6.2, but not for the SUR2 or Kir6.1 isoforms of these channel/receptor subclasses. 4. Upon anoxia, a stable depolarization by less than 10 mV was observed in non-excitable cells in the dorsal vagal nucleus. These cells, which expressed glial fibrillary acidic protein (GFAP), showed a high level of mRNA for Kir6.2, a weak signal for SUR1, whereas SUR2 or Kir6.1 were not detected. 5. The results suggest that functional KATP channels in DVNs are constituted by the formation of Kir6.2 subunits with SUR1 receptors.
ATP-Binding Cassette Transporters↗
Kir2.4: a novel K+ inward rectifier channel associated with motoneurons of cranial nerve nuclei.
Members of the Kir2 subfamily of inwardly rectifying K+ channels characterized by their strong current rectification are widely expressed both in the periphery and in the CNS in mammals. We have cloned from rat brain a fourth subfamily member, designated Kir2.4 (IRK4), which shares 53-63% similarity to Kir2.1, Kir2.2, or Kir2.3 on the amino acid level. In situ hybridization analysis identifies Kir2.4 as the most restricted of all Kir subunits in the brain. Kir2. 4 transcripts are expressed predominantly in motoneurons of cranial nerve motor nuclei within the general somatic and special visceral motor cell column and thus are uniquely related to a functional system. Heterologous expression of Kir2.4 in Xenopus oocytes and mammalian cells gives rise to low-conductance channels (15 pS), with an affinity to the channel blockers Ba2+ (Ki = 390 microM) and Cs+ (Ki = 8.06 mM) 30-50-fold lower than in other Kir channels. Low Ba2+ sensitivity allows dissection of Kir2.4 currents from other Kir conductances in hypoglossal motoneurons (HMs) in rat brainstem slices. The finding that Ba2+-mediated block of Kir2.4 in HMs evokes tonic activity and increases the frequency of induced spike discharge indicates that Kir2.4 channels are of major importance in controlling excitability of motoneurons in situ.
Animals↗
Evolutionary link between prokaryotic and eukaryotic K+ channels.
Considering the importance of K+ channels in controlling the crucial K+ gradient across the plasma membranes of all living cells, it comes as no surprise that, besides being present in every eukaryotic cell, these integral membrane proteins have recently also been identified in prokaryotes. Today, approximately a dozen successfully completed and many more ongoing sequencing projects permit a search for genes related to K+ channels in the genomes of both eubacteria and archaea. The coding regions of homologues show a remarkable variety in primary structure. They predict membrane proteins with one, two, three and six hydrophobic segments surrounding a putative K(+)-selective pore (H5) and the presence or absence of a cytosolic putative NAD(+)-binding domain (PNBD) that probably senses the reducing power of the cell. The analysis of kinships on the basis of phylogenetic algorithms identifies sequences closely related to eukaryotic voltage-dependent Kv channels, but also defines members of a primordial class of prokaryotic K+ channel (containing the 2TMS/PNBD motif). Considering the unique mechanisms that may account for the assembly of modern proteins from different ancestral genes, and with more primary sequence data soon to appear, a scheme for the evolutionary origin of K+ channels comes within reach.
Animals↗
A novel slow hyperpolarization-activated potassium current (IK(SHA)) from a mouse hippocampal cell line.
1. A slow hyperpolarization-activated inwardly rectifying K+ current (IK(SHA)) with novel characteristics was identified from the mouse embryonic hippocampus x neuroblastoma cell line HN9.10e. 2. The non-inactivating current activated negative to a membrane potential of -80 mV with slow and complex activation kinetics (tau act approximately 1-7 s) and a characteristic delay of 1-10 s (-80 to -140 mV) that was linearly dependent on the membrane potential. 3. Tail currents and instantaneous open channel currents determined through fast voltage ramps reversed at the K+ equilibrium potential (EK) indicating that primarily K+, but not Na+, permeated the channels. 4. IK(SHA) was unaffected by altering the intracellular Ca2+ concentration between approximately 0 and 10 microM, but was susceptible to block by 5 mM extracellular Ca2+, Ba2+ (Ki = 0.42 mM), and Cs+ (Ki = 2.77 mM) 5. In cells stably transformed with M2 muscarinic receptors, IK(SHA) was rapidly, but reversibly, suppressed by application of micromolar concentrations of muscarine. 6. At the single channel level K(SHA) channel openings were observed with the characteristic delay upon membrane hyperpolarization. Analysis of unitary currents revealed an inwardly rectifying I-V profile and a channel slope conductance of 7 pS. Channel activity persisted in the inside-out configuration for many minutes. 7. It is concluded that IK(SHA) in HN9.10e cells represents a novel K+ current, which is activated upon membrane hyperpolarization. It is functionally different from both classic inwardly rectifying IKir currents and other cationic hyperpolarization-activated IH currents that have been previously described in neuronal or glial cells.
Animals↗
Expression cloning of rat cerebellar adenosine A1 receptor by coupling to Kir channels.
G protein activation of inwardly rectifying K+ (Kir) channels by heptahelical receptors is an important signaling motif in slow synaptic transmission in the mammalian brain. To isolate candidate receptors responsive to the purine nucleoside adenosine, a cerebellar cDNA library was constructed in the vector pSGEM and transcripts were injected into Xenopus Laevis oocytes co-expressing Kir3.1 and/or Kir3.2 subunits. Stepwise fractionation and functional characterization of the library using two-electrode voltage clamp measurements resulted in the identification of a single unique cDNA clone with an open reading frame of 326 amino acids. The pharmacological properties as determined from the responses to cyclopentyl-adenosine (CPA, EC50 = 7 nM) and CGS21680 (EC50 = 2.6 microM) were typical of adenosine A1 receptors. The differential receptor coupling to heteromeric Kir channels composed of Kir3.1-4 subunits provides a useful technique to isolate novel heptahelical receptors.
Adenosine↗
Mutations in the ROMK gene in antenatal Bartter syndrome are associated with impaired K+ channel function.
Children with the antenatal variant of Bartter syndrome present the typical pattern of impaired salt reabsorption in the thick ascending limb of Henle's loop (TALH) resulting in marked ante- and postnatal salt wasting. In some of these patients mutations in the renal potassium channel ROMK (KCNJ1) have been found. We analyzed the electrophysiological function of five recently described ROMK channel mutations (V72E, D108H, P110L, A198T and V315G). In whole cell patch clamp recordings wildtype rat ROMK1 exhibited K+ currents of >1 nA at a membrane potential of 100 mV when transfected into COS-7 kidney cells. These currents were sensitive to external Ba2+ and internal Mg2+, which are typical features of the inwardly rectifying KIR channel. In contrast mutated ROMK1 cDNAs expressed either no or only infrequently small currents (<200 pA). Loss of tubular K+ channel function probably prevents apical membrane potassium recycling with secondary inhibition of Na-K-2Cl-cotransport in the TALH. We conclude that mutations in the potassium channel ROMK are the primary events causing renal salt wasting in a subset of patients with the antenatal variant of Bartter syndrome.
Adolescent↗
Overlapping distribution of K(ATP) channel-forming Kir6.2 subunit and the sulfonylurea receptor SUR1 in rodent brain.
ATP-sensitive K+ channels comprise a complex of at least two proteins: a member of the inwardly rectifying Kir6 family (e.g. Kir6.2) and a sulphonylurea receptor (e.g. SUR1) which belongs to the ATP-binding cassette (ABC) superfamily. Using specific radiolabeled antisense oligonucleotides, the cellular localization of both mRNAs was investigated in the rodent brain by in situ hybridization. The distribution of both transcripts was widespread throughout the brain and showed a high degree of overlap with peak expression levels in the hippocampus, neocortex, olfactory bulb, cerebellum, and several distinct nuclei of the midbrain and brainstem, indicating their important role in vital brain function.
ATP-Binding Cassette Transporters↗
Subunit interactions in the assembly of neuronal Kir3.0 inwardly rectifying K+ channels.
Cardiac G protein-activated Kir (GIRK) channels may assemble as heterotetrameric polypeptides from two subunits, Kir3.1 and Kir3.4. For a functional comparison with native channels in the CNS we investigated all possible combinations of heteromeric channel formation from brain Kir3.1, Kir3.2, Kir3.3, and Kir3.4 subunits in mRNA-injected Xenopus oocytes. Analysis of macroscopic current amplitudes and channel gating kinetics indicated that individual subunits or combinations of Kir3.2, Kir3.3, and Kir3.4 formed functional channels ineffectively. Each of these subunits gave rise to prominent currents with distinct characteristics only in the presence of Kir3.1 subunits. Functional expression of concatemeric constructs between Kir3.1 and Kir3.2/3.4 subunits as well as coimmunoprecipitations with subunit-specific antibodies confirmed heteromeric channel formation. Mutational swapping between subunits of a single pore loop residue (Kir3.1F137S; Kir3.3S114F; a phenylalanine confers slow channel gating in Kir3.1 subunits) revealed that Kir3.1 subunits are an important constituent for native heteromeric channels and dominate their functional properties. However, homomeric channels from Kir3.1 subunits in vivo may not exist due to the spatial conflict of bulky phenylalanines in the pore structure.
Amino Acid Sequence↗
Ontogeny of gene expression of Kir channel subunits in the rat.
We report the detailed gene expression of all subunits within the Kir2 and Kir3 inwardly rectifying K+ channel subfamilies in the developing rat. Using in situ hybridization, onset of expression and cellular distribution of transcripts in embryonic and postnatal rat brains as well as in peripheral tissues is evaluated. Beginning at embryonic day 13 (E13), except "forebrain" Kir2.3 subunits which are absent from the body and brain until E21, all subunits appear with distinct and mainly nonoverlapping expression patterns. During ontogenic development, expression in the CNS becomes more widespread, leading to widely overlapping mRNA patterns as observed in the adult rat. Subunits are mainly found in regions of the developing brain that are also positive in the adult. Most subunits, in particular Kir3.2 and Kir3.4, are expressed transiently in distinct brain nuclei during ontogeny. Appearance of Kir transcripts is not generally related to the progressive and recessive phases during neurogenesis, but rather regulated differentially for each subunit and any specific group of neurons. It is demonstrated for the first time that several subunits, and most abundantly Kir2.2, are present early in the peripheral nervous system, i.e., in dorsal root-, sensory cranial-, and sympathetic ganglia. Also, of all subunits Kir3.3 is ubiquitously expressed in the entire embryonic nervous system and throughout the body. In summary, analysis of ontogenic Kir channel expression helps deciphering the importance of Kir channels (as exemplified for the defective weaver Kir3.2 gene) during proliferation, differentiation, and synaptogenesis in the CNS.
Animals↗