RESUMEN
Activation gating in KcsA is elicited by changes in intracellular proton concentration. Thompson et al. identified a charge cluster around the inner gate that plays a key role in defining proton activation in KcsA. Here, through functional and spectroscopic approaches, we confirmed the role of this charge cluster and now provide a mechanism of pH-dependent gating. Channel opening is driven by a set of electrostatic interactions that include R117, E120 and E118 at the bottom of TM2 and H25 at the end of TM1. We propose that electrostatic compensation in this charge cluster stabilizes the closed conformation at neutral pH and that its disruption at low pH facilitates the transition to the open conformation by means of helix-helix repulsion.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Activación del Canal Iónico , Canales de Potasio/química , Canales de Potasio/metabolismo , Protones , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Activación del Canal Iónico/genética , Activación del Canal Iónico/fisiología , Modelos Biológicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Concentración Osmolar , Canales de Potasio/genética , Estructura Terciaria de Proteína/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Electricidad EstáticaRESUMEN
The molecular nature of the structure responsible for proton sensitivity in KcsA has been identified as a charge cluster that surrounds the inner helical bundle gate. Here, we show that this proton sensor can be modified to engineer a constitutively open form of KcsA, amenable to functional, spectroscopic and structural analyses. By combining charge neutralizations for all acidic and basic residues in the cluster at positions 25, 117-122 and 124 (but not E118), a mutant KcsA is generated that displays constitutively open channel activity up to pH 9. The structure of this mutant revealed that full opening appears to be inhibited by lattice forces since the activation gate seems to be only on the early stages of opening.