RESUMEN
Magnesium (Mg(2+)) plays a central role in biology, regulating the activity of many enzymes and stabilizing the structure of key macromolecules. In bacteria, CorA is the primary source of Mg(2+) uptake and is self-regulated by intracellular Mg(2+). Using a gating mutant at the divalent ion binding site, we were able to characterize CorA selectivity and permeation properties to both monovalent and divalent cations under perfused two-electrode voltage clamp. The present data demonstrate that under physiological conditions, CorA is a multioccupancy Mg(2+)-selective channel, fully excluding monovalent cations, and Ca(2+), whereas in absence of Mg(2+), CorA is essentially nonselective, displaying only mild preference against other divalents (Ca(2+) > Mn(2+) > Co(2+) > Mg(2+) > Ni(2)(+)). Selectivity against monovalent cations takes place via Mg(2+) binding at a high-affinity site, formed by the Gly-Met-Asn signature sequence (Gly312 and Asn314) at the extracellular side of the pore. This mechanism is reminiscent of repulsion models proposed for Ca(2+) channel selectivity despite differences in sequence and overall structure.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Magnesio/metabolismo , Modelos Moleculares , Thermotoga maritima/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Biología Computacional , Cartilla de ADN/genética , Vectores Genéticos , Datos de Secuencia Molecular , Oocitos/metabolismo , Técnicas de Placa-Clamp , Alineación de Secuencia , Electricidad Estática , Thermotoga maritima/química , Thermotoga maritima/metabolismo , Xenopus laevisRESUMEN
In voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P(2). In the chimera, enzymatic activity is controlled by membrane potential via hVSP1's endogenous phosphoinositide binding motif. These findings suggest that the endogenous VSD of hVSP1 is a control module that initiates signaling through the phosphatase domain and indicate a role for VSP-mediated phosphoinositide signaling in mammals.
Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Células CHO , Cricetinae , Electrofisiología , Humanos , Microscopía Fluorescente , Oocitos/metabolismo , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Transducción de Señal , XenopusRESUMEN
Most action potentials are produced by the sequential activation of voltage-gated sodium (Nav) and potassium (Kv) channels. This is mainly achieved by the rapid conformational rearrangement of voltage-sensor (VS) modules in Nav channels, with activation kinetics up to 6-fold faster than Shaker-type Kv channels. Here, using mutagenesis and gating current measurements, we show that a 3-fold acceleration of the VS kinetics in Nav versus Shaker Kv channels is produced by the hydrophilicity of two "speed-control" residues located in the S2 and S4 segments in Nav domains I-III. An additional 2-fold acceleration of the Nav VS kinetics is provided by the coexpression of the ß1 subunit, ubiquitously found in mammal tissues. This study uncovers the molecular bases responsible for the differential activation of Nav versus Kv channels, a fundamental prerequisite for the genesis of action potentials.
Asunto(s)
Potenciales de Acción/genética , Activación del Canal Iónico/genética , Canales de Potasio/genética , Canales de Potasio de la Superfamilia Shaker/genética , Secuencia de Aminoácidos/fisiología , Animales , Fenómenos Biofísicos/genética , Estimulación Eléctrica , Cinética , Microinyecciones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación/genética , Oocitos , Técnicas de Placa-Clamp , Conformación Proteica , Xenopus laevisRESUMEN
Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.