RESUMO
Voltage-gated ion channels open and close in response to changes in membrane potential, thereby enabling electrical signaling in excitable cells. The voltage sensitivity is conferred through four voltage-sensor domains (VSDs) where positively charged residues in the fourth transmembrane segment (S4) sense the potential. While an open state is known from the Kv1.2/2.1 X-ray structure, the conformational changes underlying voltage sensing have not been resolved. We present 20 additional interactions in one open and four different closed conformations based on metal-ion bridges between all four segments of the VSD in the voltage-gated Shaker K channel. A subset of the experimental constraints was used to generate Rosetta models of the conformations that were subjected to molecular simulation and tested against the remaining constraints. This achieves a detailed model of intermediate conformations during VSD gating. The results provide molecular insight into the transition, suggesting that S4 slides at least 12 Å along its axis to open the channel with a 3(10) helix region present that moves in sequence in S4 in order to occupy the same position in space opposite F290 from open through the three first closed states.
Assuntos
Proteínas de Drosophila/metabolismo , Ativação do Canal Iônico/fisiologia , Metais/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , Sítios de Ligação/genética , Cádmio/química , Cádmio/metabolismo , Quelantes/farmacologia , Simulação por Computador , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ácido Egtázico/farmacologia , Feminino , Ativação do Canal Iônico/genética , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Metais/química , Modelos Moleculares , Mutação , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Terciária de Proteína , Superfamília Shaker de Canais de Potássio/química , Superfamília Shaker de Canais de Potássio/genética , Xenopus laevisRESUMO
Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd(2+) bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K(+) coordination, a hallmark for C-type inactivation. An engineered Cd(2+) bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.
Assuntos
Ativação do Canal Iônico , Superfamília Shaker de Canais de Potássio/química , Animais , Cádmio/química , Deleção de Genes , Potenciais da Membrana/fisiologia , Simulação de Dinâmica Molecular , Mutação , Potássio/química , Domínios Proteicos , Eletricidade Estática , Xenopus laevisRESUMO
The expression of neuropeptides is often extremely restricted in the nervous system, making them powerful markers for addressing cell specification . In the developing Drosophila ventral nerve cord, only six cells, the Ap4 neurons, of some 10,000 neurons, express the neuropeptide FMRFamide (FMRFa). Each Ap4/FMRFa neuron is the last-born cell generated by an identifiable and well-studied progenitor cell, neuroblast 5-6 (NB5-6T). The restricted expression of FMRFa and the wealth of information regarding its gene regulation and Ap4 neuron specification makes FMRFa a valuable readout for addressing many aspects of neural development, i.e., spatial and temporal patterning cues, cell cycle control, cell specification, axon transport, and retrograde signaling. To this end, we have conducted a forward genetic screen utilizing an Ap4-specific FMRFa-eGFP transgenic reporter as our readout. A total of 9781 EMS-mutated chromosomes were screened for perturbations in FMRFa-eGFP expression, and 611 mutants were identified. Seventy-nine of the strongest mutants were mapped down to the affected gene by deficiency mapping or whole-genome sequencing. We isolated novel alleles for previously known FMRFa regulators, confirming the validity of the screen. In addition, we identified novel essential genes, including several with previously undefined functions in neural development. Our identification of genes affecting most major steps required for successful terminal differentiation of Ap4 neurons provides a comprehensive view of the genetic flow controlling the generation of highly unique neuronal cell types in the developing nervous system.