RESUMO
GIRK2 is a major contributor to G protein-activated inward rectifier potassium channels in the mammalian brain. How GIRK channels open upon contact with Gbetagamma remains unknown. Using a yeast genetic screen to select constitutively active mutants from a randomly mutagenized GIRK2 library, we identified five gating mutations at four residues in the transmembrane domain. Further mutagenesis indicates that GIRK channel opening involves a rotation of the transmembrane segments, bringing one of these residues (V188) to a pore-lining position in the open conformation. Combined with double-mutant studies, these findings suggest that GIRK channels gate by moving from the open conformation inferred from our yeast study of Kir2.1 to a closed conformation perhaps resembling the known KcsA structure.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Mutagênese , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Condutividade Elétrica , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Expressão Gênica , Ativação do Canal Iônico/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/metabolismo , Canais de Potássio/química , Canais de Potássio/fisiologia , Conformação Proteica , Alinhamento de Sequência , Relação Estrutura-Atividade , XenopusRESUMO
PDZ domains are multifunctional protein-interaction motifs that often bind to the C-terminus of protein targets. Nitric oxide (NO), an endogenous signaling molecule, plays critical roles in nervous, immune, and cardiovascular function. Although there are numerous physiological functions for neuron-derived NO, produced primarily by the neuronal NO synthase (nNOS), excess nNOS activity mediates brain injury in cerebral ischemia and in animal models of Parkinson's disease. Subcellular localization of nNOS activity must therefore be tightly regulated. To determine ligands for the PDZ domain of nNOS, we screened 13 billion distinct peptides and found that the nNOS-PDZ domain binds tightly to peptides ending Asp-X-Val. This differs from the only known (Thr/Ser)-X-Val consensus that interacts with PDZ domains from PSD-95. Preference for Asp at the -2 peptide position is mediated by Tyr-77 of nNOS. A Y77D78 to H77E78 substitution changes the binding specificity from Asp-X-Val to Thr-X-Val. Guided by the Asp-X-Val consensus, candidate nNOS interacting proteins have been identified including glutamate and melatonin receptors. Our results demonstrate that PDZ domains have distinct peptide binding specificity.
Assuntos
Neurônios/enzimologia , Óxido Nítrico Sintase/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biotecnologia , Encéfalo/metabolismo , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Estrutura Molecular , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The C. elegans sax-3 gene encodes a predicted transmembrane protein with five immunoglobulin domains and three fibronectin type III repeats that is closely related to Drosophila Robo. Mutations in sax-3 lead to repeated midline crossing by ventral cord axons that normally do not cross the midline after they join the ventral cord, a phenotype similar to that of robo mutants. sax-3 is also required for guidance of some axons to the ventral cord, implicating this gene in two different types of guidance events. A sax-3::GFP fusion gene is expressed in developing neurons during axon outgrowth, and sax-3 function is required at the time of axon guidance, suggesting that this gene mediates cell interactions during guidance decisions.
Assuntos
Axônios/fisiologia , Caenorhabditis elegans/embriologia , Sequência Conservada/genética , Imunoglobulinas/genética , Receptores Imunológicos/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/crescimento & desenvolvimento , Fibronectinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Helmintos/genética , Dados de Sequência Molecular , Neurônios Motores/química , Mutação , Proteínas do Tecido Nervoso , Receptores Imunológicos/química , Receptores Imunológicos/fisiologia , Proteínas Recombinantes de Fusão , Proteínas RoundaboutRESUMO
Neural signaling is based on the regulated timing and extent of channel opening; therefore, it is important to understand how ion channels open and close in response to neurotransmitters and intracellular messengers. Here, we examine this question for potassium channels, an extraordinarily diverse group of ion channels. Voltage-gated potassium (Kv) channels control action-potential waveforms and neuronal firing patterns by opening and closing in response to membrane-potential changes. These effects can be strongly modulated by cytoplasmic factors such as kinases, phosphatases, and small GTPases. A Kv alpha subunit contains six transmembrane segments, including an intrinsic voltage sensor. In contrast, inwardly rectifying potassium (Kir) channels have just two transmembrane segments in each of its four pore-lining alpha subunits. A variety of intracellular second messengers mediate transmitter and metabolic regulation of Kir channels. For example, Kir3 (GIRK) channels open on binding to the G protein betagamma subunits, thereby mediating slow inhibitory postsynaptic potentials in the brain. Our structure-based functional analysis on the cytoplasmic N-terminal tetramerization domain T1 of the voltage-gated channel, Kv1.2, uncovered a new function for this domain, modulation of voltage gating, and suggested a possible means of communication between second messenger pathways and Kv channels. A yeast screen for active Kir3.2 channels subjected to random mutagenesis has identified residues in the transmembrane segments that are crucial for controlling the opening of Kir3.2 channels. The identification of structural elements involved in potassium channel gating in these systems highlights principles that may be important in the regulation of other types of channels.