RESUMEN
In neurosecretion, allosteric communication between voltage sensors and Ca2+ binding in BK channels is crucially involved in damping excitatory stimuli. Nevertheless, the voltage-sensing mechanism of BK channels is still under debate. Here, based on gating current measurements, we demonstrate that two arginines in the transmembrane segment S4 (R210 and R213) function as the BK gating charges. Significantly, the energy landscape of the gating particles is electrostatically tuned by a network of salt bridges contained in the voltage sensor domain (VSD). Molecular dynamics simulations and proton transport experiments in the hyperpolarization-activated R210H mutant suggest that the electric field drops off within a narrow septum whose boundaries are defined by the gating charges. Unlike Kv channels, the charge movement in BK appears to be limited to a small displacement of the guanidinium moieties of R210 and R213, without significant movement of the S4.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio de Gran Conductancia Activados por el Calcio , Arginina/metabolismo , Activación del Canal Iónico/genética , Simulación de Dinámica Molecular , MutaciónRESUMEN
Allosteric interactions between the voltage-sensing domain (VSD), the Ca2+-binding sites, and the pore domain govern the mammalian Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the crosstalk between the Ca2+- and voltage-sensing mechanisms on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation by investigating the effects of internal Ca2+ on BK channel gating currents. Our results indicate that Ca2+ sensor occupancy has a strong impact on VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca2+ sensors and VSDs.
Asunto(s)
Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Cationes Bivalentes/metabolismo , Humanos , Unión Proteica , Dominios ProteicosRESUMEN
The accessory ß1 subunit modulates the Ca2+- and voltage-activated K+ (BK) channel gating properties mainly by increasing its apparent Ca2+ sensitivity. ß1 plays an important role in the modulation of arterial tone and blood pressure by vascular smooth muscle cells (SMCs). 17ß-estradiol (E2) increases the BK channel open probability (Po) in SMCs, through a ß1 subunit-dependent modulatory effect. Here, using molecular modeling, bioinformatics, mutagenesis, and electrophysiology, we identify a cluster of hydrophobic residues in the second transmembrane domain of the ß1 subunit, including the residues W163 and F166, as the binding site for E2. We further show that the increase in Po induced by E2 is associated with a stabilization of the voltage sensor in its active configuration and an increase in the coupling between the voltage sensor activation and pore opening. Since ß1 is a key molecular player in vasoregulation, the findings reported here are of importance in the design of novel drugs able to modulate BK channels.
Asunto(s)
Estradiol/metabolismo , Activación del Canal Iónico , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Miocitos del Músculo Liso/metabolismo , Calcio/metabolismo , Células HEK293 , Humanos , Potenciales de la Membrana , Técnicas de Placa-Clamp/métodos , Subunidades de Proteína , Receptores de Estradiol/metabolismoRESUMEN
This article reviews the contributions of the Cuban Neuroscience Center to the evolution of the statistical parametric mapping (SPM) of quantitative Multimodal Neuroimages (qMN), from its inception to more recent work. Attention is limited to methods that compare individual qMN to normative databases (n/qMN). This evolution is described in three successive stages: (a) the development of one variant of normative topographical quantitative EEG (n/qEEG-top) which carries out statistical comparison of individual EEG spectral topographies with regard to a normative database--as part of the now popular SPM of brain descriptive parameters; (b) the development of n/qEEG tomography (n/qEEG-TOM), which employs brain electrical tomography (BET) to calculate voxelwise SPM maps of source spectral features with respect to a norm; (c) the development of a more general n/qMN by substituting EEG parameters with other neuroimaging descriptive parameters to obtain SPM maps. The study also describes the creation of Cuban normative databases, starting with the Cuban EEG database obtained in the early 90s, and more recently, the Cuban Human Brain Mapping Project (CHBMP). This project has created a 240 subject database of the normal Cuban population, obtained from a population-based random sample, comprising clinical, neuropsychological, EEG, MRI and SPECT data for the same subjects. Examples of clinical studies using qMN are given and, more importantly, receiver operator characteristics (ROC) analyses of the different developments document a sustained effort to assess the clinical usefulness of the techniques.