RESUMO
The olfactory tubercle (OT) is found in the brains of mammals that are highly dependent on their sense of smell. Its human analogue is the poorly understood anterior perforated substance. Previous work on rat brain slices identified two types of field potential responses from the OT. The association fibre (AF) pathway was sensitive to muscarinic modulation, whereas the lateral olfactory tract (LOT) fibre pathway was not. Here, we establish that serotonin (5-hydroxytryptamine; 5-HT) also inhibits field potential excitatory postsynaptic potentials (EPSPs) in the AF, but not in the LOT fibre, pathway. Parallel experiments with adenosine (ADO) excluded ADO mediation of the 5-HT effect. Exogenous 5-HT at 30 microm caused a long-lasting approximately 40% reduction in the amplitude of AF postsynaptic responses, without affecting the time-course of EPSP decline, indicating a fairly restricted disposition of the 5-HT receptors responsible. The 5-HT(1)-preferring, 5-HT(5)-preferring and 5-HT(7)-preferring agonist 5-carboxamidotryptamine caused similar inhibition at approximately 100 nm. The 5-HT(1A)-preferring ligand 8-hydroxy-di-n-propylamino-tetralin at 10 microm, and the 5-HT uptake inhibitor citalopram at 3 microm, caused inhibition of AF-stimulated field potential responses in the 5-10% range. Order-of-potency information suggested a receptor of the 5-HT(1B) or 5-HT(1D) subtype. The 5-HT(1D) agonist L-694,247 (1 microm) suppressed the AF response by approximately 10% when used on its own. After washing out of L-694,427, inhibition by 30 microm 5-HT was reduced to negligible levels. Allowing for a partial agonist action of L-694,427 and complex interactions of 5-HT receptors within the OT, these results support the presence of active 5-HT(1D)-type receptors in the principal cell layer of the OT.
Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Condutos Olfatórios/metabolismo , Serotonina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Citalopram/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Técnicas In Vitro , Masculino , Condutos Olfatórios/efeitos dos fármacos , Oxidiazóis/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Serotonina/análogos & derivados , Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Triptaminas/farmacologiaRESUMO
M-currents are K+ currents generated by members of the KCNQ family of K+ channels (Wang et al., 1998). However, in some cells, M-like currents may be contaminated by members of other K+ channel gene families, such as the erg family (Meves et al., 1999; Selyanko et al., 1999). In the present experiments, we have used the acute expression of pore-defective mutants of KCNQ3 (DN-KCNQ3) and Merg1a (DN-Merg1a) as dominant negatives to separate the contributions of these two families to M-like currents in NG108-15 neuroblastoma hybrid cells and rat sympathetic neurons. Two kinetically and pharmacologically separable components of M-like current could be recorded from NG108-15 cells that were individually suppressed by DN-Merg1a and DN-KCNQ3, respectively. In contrast, only DN-KCNQ3, and not DN-Merg1a, reduced currents recorded from sympathetic neurons. Pharmacological tests suggested that the residual current in DN-KCNQ3-treated sympathetic neurons was carried by residual KCNQ channels. Ineffectiveness of DN-Merg1a in sympathetic neurons was not caused by lack of expression, as judged by confocal microscopy of Flag-tagged DN-Merg1a. These results accord with previous inferences regarding the roles of erg and KCNQ channels in generating M-like currents. This experimental approach should therefore be useful in delineating the contributions of members of these two gene families to K+ currents in other cells.
Assuntos
Genes Dominantes , Família Multigênica , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Canais de Potássio/metabolismo , Subunidades Proteicas , Animais , Células Cultivadas , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Expressão Gênica , Células Híbridas/metabolismo , Canal de Potássio KCNQ3 , Camundongos , Neuroblastoma/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio/biossíntese , Ratos , Ratos Sprague-Dawley , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/metabolismo , TransfecçãoRESUMO
1. The single channel properties of KCNQ2/KCNQ3 channels underlying neuronal voltage-dependent M-type potassium currents were studied in cell-attached patches from transfected Chinese hamster ovary (CHO) cells. Macroscopic currents produced by homo- and heteromeric KCNQ2/KCNQ3 channels were measured using the perforated-patch whole-cell technique. 2. Compared with heteromeric KCNQ2 + KCNQ3 channels, homomeric KCNQ2 channels had lower slope conductance (9.0 +/- 0.3 and 5.8 +/- 0.3 pS, respectively) and open probability at 0 mV (0.30 +/- 0.07 and 0.15 +/- 0.03, respectively), consistent with their 3.8-fold smaller macroscopic currents. By contrast, homomeric KCNQ3 channels had the same slope conductance (9.0 +/- 1.1 pS) as KCNQ2 + KCNQ3 channels, and higher open probability (0.59 +/- 0.11), inconsistent with their 12.7-fold smaller macroscopic currents. Thus, KCNQ2 and KCNQ3 subunits may play different roles in the expression of M-type currents, with KCNQ2 ensuring surface expression of underlying channels and KCNQ3 modifying their function. 3. Both in homo- and heteromeric KCNQ2/KCNQ3 channels the shut time distributions were fitted with three, and the open time distributions with two, exponential components. By measuring these and other parameters (e.g. conductance and open probability) KCNQ2/ KCNQ3 channels can be shown to resemble previously characterised neuronal M-type channels.
Assuntos
Canais de Potássio/fisiologia , Animais , Células CHO , Cricetinae , Condutividade Elétrica , Canais Iônicos/fisiologia , Canal de Potássio KCNQ2 , Canal de Potássio KCNQ3 , Canais de Potássio de Abertura Dependente da Tensão da Membrana , TransfecçãoRESUMO
The region of alternative splicing in the KCNQ2 potassium channel gene was determined by RNase protection analysis of KCNQ2 mRNA transcripts. Systematic analysis of KCNQ2 alternative splice variant expression in rat superior cervical ganglia revealed multiple variant isoforms. One class of KCNQ2 splice variants, those that contained exon 15a, was found to have significantly different kinetics to those of the other isoforms. These transcripts encoded channel subunits that, when co-expressed with the KCNQ3 subunit, activated and deactivated approximately 2.5 times more slowly than other isoforms. Deletion of exon 15a in these isoforms produced a reversion to the faster kinetics. Comparison of the kinetic properties of the cloned channel splice variants with those of the native M-current suggests that alternative splicing of the KCNQ2 gene may contribute to the variation in M-current kinetics seen in vivo.
Assuntos
Processamento Alternativo , Canais de Potássio/genética , Canais de Potássio/fisiologia , Transcrição Gênica , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Células CHO , Cricetinae , DNA Complementar/genética , Éxons/genética , Expressão Gênica , Canal de Potássio KCNQ2 , Cinética , Dados de Sequência Molecular , Neurônios/fisiologia , Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Receptores Colinérgicos/fisiologia , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/fisiologiaRESUMO
The isolation of the peptide inhibitor of M-type K(+) current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K(+) channels was also studied. BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.
Assuntos
Proteínas de Transporte de Cátions , Proteínas de Ligação a DNA , Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Venenos de Escorpião/biossíntese , Venenos de Escorpião/química , Venenos de Escorpião/genética , Transativadores , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Canal de Potássio ERG1 , Eletrofisiologia , Escherichia coli/metabolismo , Canais de Potássio Éter-A-Go-Go , Humanos , Concentração Inibidora 50 , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Cinética , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Canais de Potássio/metabolismo , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Escorpiões , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Fatores de Tempo , Regulador Transcricional ERG , Células Tumorais CultivadasRESUMO
1. KCNQ1-4 potassium channels were expressed in mammalian Chinese hamster ovary (CHO) cells stably transfected with M1 muscarinic acetylcholine receptors and currents were recorded using the whole-cell perforated patch technique and cell-attached patch recording. 2. Stimulation of M1 receptors by 10 microM oxotremorine-M (Oxo-M) strongly reduced (to 0-10%) currents produced by KCNQ1-4 subunits expressed individually and also those produced by KCNQ2 + KCNQ3 and KCNQ1 + KCNE1 heteromers, which are thought to generate neuronal M-currents (IK,M) and cardiac slow delayed rectifier currents (IK,s), respectively. 3. The activity of KCNQ2 + KCNQ3, KCNQ2 and KCNQ3 channels recorded with cell-attached pipettes was strongly and reversibly reduced by Oxo-M applied to the extra-patch membrane. 4. It is concluded that M1 receptors couple to all known KCNQ subunits and that inhibition of KCNQ2 + KCNQ3 channels, like that of native M-channels, requires a diffusible second messenger.
Assuntos
Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Receptores Muscarínicos/metabolismo , Animais , Células CHO , Cricetinae , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Canal de Potássio KCNQ2 , Canal de Potássio KCNQ3 , Agonistas Muscarínicos/farmacologia , Oxotremorina/análogos & derivados , Oxotremorina/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio/genética , Receptor Muscarínico M1 , Receptores Muscarínicos/efeitos dos fármacos , Estimulação Química , TransfecçãoRESUMO
In Shaker-group potassium channels the presence of a tyrosine residue, just downstream of the pore signature sequence GYG, determines sensitivity to tetraethylammonium (TEA). The KCNQ family of channels has a variety of amino acid residues in the equivalent position. We studied the effect of TEA on currents generated by KCNQ homomers and heteromers expressed in CHO cells. We used wild-type KCNQ1-4 channels and heteromeric KCNQ2/3 channels incorporating either wild-type KCNQ3 subunits or a mutated KCNQ3 in which tyrosine replaced threonine at position 323 (mutant T323Y). IC50 values were (mM): KCNQ1, 5.0; KCNQ2, 0.3; KCNQ3, > 30; KCNQ4, 3.0; KCNQ2 + KCNQ3, 3.8; and KCNQ2 + KCNQ3(T323Y), 0.5. While the high TEA sensitivity of KCNQ2 may be conferred by a tyrosine residue lacking in the other channels, the intermediate TEA sensitivity of KCNQ1 and KCNQ4 implies that other residues are also important in determining TEA block of the KCNQ channels.
Assuntos
Bloqueadores dos Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Tetraetilamônio/farmacologia , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Canal de Potássio KCNQ2 , Canal de Potássio KCNQ3 , Técnicas de Patch-Clamp , Canais de Potássio/genética , Treonina/fisiologia , Transfecção , Tirosina/fisiologiaRESUMO
The potassium M current was originally identified in sympathetic ganglion cells, and analogous currents have been reported in some central neurons and also in some neural cell lines. It has recently been suggested that the M channel in sympathetic neurons comprises a heteromultimer of KCNQ2 and KCNQ3 (Wang et al., 1998) but it is unclear whether all other M-like currents are generated by these channels. Here we report that the M-like current previously described in NG108-15 mouse neuroblastoma x rat glioma cells has two components, "fast" and "slow", that may be differentiated kinetically and pharmacologically. We provide evidence from PCR analysis and expression studies to indicate that these two components are mediated by two distinct molecular species of K(+) channel: the fast component resembles that in sympathetic ganglia and is probably carried by KCNQ2/3 channels, whereas the slow component appears to be carried by merg1a channels. Thus, the channels generating M-like currents in different cells may be heterogeneous in molecular composition.