RESUMO
Cyclic nucleotide-modulated ion channels play crucial roles in signal transduction in eukaryotes. The molecular mechanism by which ligand binding leads to channel opening remains poorly understood, due in part to the lack of a robust method for preparing sufficient amounts of purified, stable protein required for structural and biochemical characterization. To overcome this limitation, we designed a stable, highly expressed chimeric ion channel consisting of the transmembrane domains of the well characterized potassium channel KcsA and the cyclic nucleotide-binding domains of the prokaryotic cyclic nucleotide-modulated channel MloK1. This chimera demonstrates KcsA-like pH-sensitive activity which is modulated by cAMP, reminiscent of the dual modulation in hyperpolarization-activated and cyclic nucleotide-gated channels that display voltage-dependent activity that is also modulated by cAMP. Using this chimeric construct, we were able to measure for the first time the binding thermodynamics of cAMP to an intact cyclic nucleotide-modulated ion channel using isothermal titration calorimetry. The energetics of ligand binding to channels reconstituted in lipid bilayers are substantially different from those observed in detergent micelles, suggesting that the conformation of the chimera's transmembrane domain is sensitive to its (lipid or lipid-mimetic) environment and that ligand binding induces conformational changes in the transmembrane domain. Nevertheless, because cAMP on its own does not activate these chimeric channels, cAMP binding likely has a smaller energetic contribution to gating than proton binding suggesting that there is only a small difference in cAMP binding energy between the open and closed states of the channel.
Assuntos
Proteínas de Bactérias/metabolismo , AMP Cíclico/metabolismo , Ativação do Canal Iônico , Lipídeos de Membrana/metabolismo , Mesorhizobium/metabolismo , Canais de Potássio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , AMP Cíclico/química , AMP Cíclico/genética , Concentração de Íons de Hidrogênio , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Mesorhizobium/química , Mesorhizobium/genética , Canais de Potássio/química , Canais de Potássio/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genéticaRESUMO
Structures of the prokaryotic K(+) channel, KcsA, highlight the role of the selectivity filter carbonyls from the GYG signature sequence in determining a highly selective pore, but channels displaying this sequence vary widely in their cation selectivity. Furthermore, variable selectivity can be found within the same channel during a process called C-type inactivation. We investigated the mechanism for changes in selectivity associated with inactivation in a model K(+) channel, KcsA. We found that E71A, a noninactivating KcsA mutant in which a hydrogen-bond behind the selectivity filter is disrupted, also displays decreased K(+) selectivity. In E71A channels, Na(+) permeates at higher rates as seen with and flux measurements and analysis of intracellular Na(+) block. Crystal structures of E71A reveal that the selectivity filter no longer assumes the "collapsed," presumed inactivated, conformation in low K(+), but a "flipped" conformation, that is also observed in high K(+), high Na(+), and even Na(+) only conditions. The data reveal the importance of the E71-D80 interaction in both favoring inactivation and maintaining high K(+) selectivity. We propose a molecular mechanism by which inactivation and K(+) selectivity are linked, a mechanism that may also be at work in other channels containing the canonical GYG signature sequence.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico , Canais de Potássio/química , Canais de Potássio/metabolismo , Conformação Proteica , Proteínas de Bactérias/genética , Modelos Moleculares , Dados de Sequência Molecular , Potássio/metabolismo , Canais de Potássio/genética , Radioisótopos de Rubídio/química , Radioisótopos de Rubídio/metabolismo , Radioisótopos de Sódio , Difração de Raios XRESUMO
The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Despite prior attempts to determine the mechanism responsible for pH gating, the proton sensor has remained elusive. We have constructed a KcsA channel mutant that remains open up to pH 9.0 by replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge. A series of individual and combined mutations were investigated by using single-channel recordings in lipid bilayers. We propose that these residues are the proton-binding sites and at neutral pH they form a complex network of inter- and intrasubunit salt bridges and hydrogen bonds near the bundle crossing that greatly stabilize the closed state. In our model, these residues change their ionization state at acidic pH, thereby disrupting this network, modifying the electrostatic landscape near the channel gate, and favoring channel opening.
Assuntos
Técnicas Biossensoriais , Proteínas de Escherichia coli/química , Modelos Moleculares , Canais de Potássio/química , Proteínas de Bactérias , Ácido Glutâmico/química , Histidina/química , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Mutação/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The bacterial potassium channel KcsA is gated open by the binding of protons to amino acids on the intracellular side of the channel. We have identified, via channel mutagenesis and x-ray crystallography, two pH-sensing amino acids and a set of nearby residues involved in molecular interactions that influence gating. We found that the minimal mutation of one histidine (H25) and one glutamate (E118) near the cytoplasmic gate completely abolished pH-dependent gating. Mutation of nearby residues either alone or in pairs altered the channel's response to pH. In addition, mutations of certain pairs of residues dramatically increased the energy barriers between the closed and open states. We proposed a Monod-Wyman-Changeux model for proton binding and pH-dependent gating in KcsA, where H25 is a "strong" sensor displaying a large shift in pKa between closed and open states, and E118 is a "weak" pH sensor. Modifying model parameters that are involved in either the intrinsic gating equilibrium or the pKa values of the pH-sensing residues was sufficient to capture the effects of all mutations.
Assuntos
Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico , Simulação de Dinâmica Molecular , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Prótons , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dados de Sequência Molecular , Mutação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genéticaRESUMO
Potassium channels allow K(+) ions to diffuse through their pores while preventing smaller Na(+) ions from permeating. Discrimination between these similar, abundant ions enables these proteins to control electrical and chemical activity in all organisms. Selection occurs at the narrow selectivity filter containing structurally identified K(+) binding sites. Selectivity is thought to arise because smaller ions such as Na(+) do not bind to these K(+) sites in a thermodynamically favorable way. Using the model K(+) channel KcsA, we examined how intracellular Na(+) and Li(+) interact with the pore and the permeant ions using electrophysiology, molecular dynamics simulations and X-ray crystallography. Our results suggest that these small cations have a separate binding site within the K(+) selectivity filter. We propose that selective permeation from the intracellular side primarily results from a large energy barrier blocking filter entry for Na(+) and Li(+) in the presence of K(+), not from a difference of binding affinity between ions.