RESUMEN
The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.
Asunto(s)
Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Subunidades de Proteína/metabolismo , Secuencia de Aminoácidos , Animales , Cisteína/química , Electrofisiología , Humanos , Activación del Canal Iónico , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Oocitos , Técnicas de Placa-Clamp , Subunidades de Proteína/química , Canales de Potasio de la Superfamilia Shaker/metabolismo , Xenopus laevisRESUMEN
Large conductance voltage- and Ca(2+)-activated K(+) (BK(Ca)) channels regulate important physiological processes such as neurotransmitter release and vascular tone. BK(Ca) channels possess a voltage sensor mainly represented by the S4 transmembrane domain. Changes in membrane potential displace the voltage sensor, producing a conformational change that leads to channel opening. By site-directed fluorescent labeling of residues in the S3-S4 region and by using voltage clamp fluorometry, we have resolved the conformational changes the channel undergoes during activation. The voltage dependence of these conformational changes (detected as changes in fluorescence emission, fluorescence vs. voltage curves) always preceded the channel activation curves, as expected for protein rearrangements associated to the movement of the voltage sensor. Extremely slow conformational changes were revealed by fluorescent labeling of position 202, elicited by a mutual interaction of the fluorophore with the adjacent tryptophan 203.
Asunto(s)
Fluorometría/métodos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Técnicas de Placa-Clamp , Conformación Proteica , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Humanos , Activación del Canal Iónico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Datos de Secuencia Molecular , Estructura Molecular , Mutación Puntual , Canales de Potasio , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Alineación de Secuencia , Triptófano/metabolismo , Xenopus laevisRESUMEN
The cytoplasmic side of the voltage-dependent Na+ channel pore is putatively formed by the S6 segments of domains I to IV. The role of amino acid residues of I-S6 and II-S6 in channel gating and local anesthetic (LA) block was investigated using the cysteine scanning mutagenesis of the rat skeletal muscle Na+ channel (Nav1.4). G428C uniquely reduced sensitivity to rested state or first-pulse block by lidocaine without alterations in the voltage dependence or kinetics of gating that would otherwise account for the increase in the IC50 for block. Mutations in I-S6 (N434C and I436C) and in II-S6 (L785C and V787C) increased sensitivity to first-pulse block by lidocaine. Enhanced inactivation accounted for the increased sensitivity of N434C to lidocaine and hastening of inactivation of I436C in the absence of drug could account for higher affinity first-pulse block. Mutations in I-S6 (I424C, I425C, and F430C) and in II-S6 (I782C and V786C) reduced the use-dependent lidocaine block. The reduction in use-dependent block of F430C was consistent with alterations in inactivation gating; the other mutants did not exhibit gating changes that could explain the reduced sensitivity to lidocaine. Therefore, several amino acids (I424, I425, G428, I782, and V786), in addition to those previously identified (Yarov-Yarovoy et al., 2002), alter the sensitivity of Nav1.4 to lidocaine, independent of mutation-induced changes in gating. The magnitude of the change in the IC50 values, the isoform, and LA dependence of the changes in affinity suggest that the determinants of binding in I-S6 and II-S6 are subsidiary to those in IV-S6.
Asunto(s)
Aminoácidos/fisiología , Anestésicos Locales/farmacología , Activación del Canal Iónico/fisiología , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Canales de Sodio/química , Animales , Relación Dosis-Respuesta a Droga , Humanos , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas Musculares/fisiología , Mutación , Fragmentos de Péptidos/antagonistas & inhibidores , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Ratas , Canales de Sodio/fisiologíaRESUMEN
BmK 11(2) is a 7216Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch. Nanomolar concentrations of the toxin prolong amphibian nerve action potentials without attenuation of the amplitude. The pharmacological action of the toxin and its sequence similarity to other alpha-scorpion toxins suggest that BmK 11(2) selectively alters voltage-gated Na channels. In order to test whether BmK 11(2) preferentially modulates the gating or kinetics of certain channel isoforms, we applied BmK 11(2) to muscle, heart and neuronal Na channels. 100nM BmK 11(2) increased the peak current amplitude of skeletal muscle (micro1) and neuronal (N1E-115) Na currents by 40 and 20%, respectively, and reduced the cardiac Na (hH1) current by 15%. The toxin slowed current decay of all isoforms, most prominently in N1E-115 (tau(BmK)/tau(Control)=12), micro1 (11), and less so for hH1 (1.3). BmK 11(2) shifted the voltage dependence of activation of micro1 and N1E-115 currents. BmK 11(2) had no effect on steady-state inactivation, use-dependent availability, and the kinetics of entry into slowly recovering inactivated states.
Asunto(s)
Venenos de Escorpión/farmacología , Escorpiones , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/efectos de los fármacos , Animales , Células Cultivadas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Corazón/efectos de los fármacos , Humanos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Isoformas de Proteínas , RatasRESUMEN
A polypeptide that extensively prolongs action potentials (APs) in frog nerve has been isolated and purified from the venom of the scorpion Buthus martensii Karsch (BMK). The polypeptide was purified using gel filtration, ion exchange, FPLC, and HPLC chromatography. APs recorded in the presence of nanomolar concentrations of the polypeptide were extensively prolonged without much attenuation in their heights. The N-terminal sequence of BMK 11(2) was found to be: VRDGYIADDKD-AYF-GRDAYYDDDEKKKD. Sequence similarity comparisons to other alpha-scorpion toxins suggest that the two blanks in the sequences are cysteines. The molecular weight (M.W.) of BMK 11(2) was determined by LC/MS/MS to be 7216 Da. Voltage-clamp experiments conducted on plasmid-transfected human kidney cells expressing the alpha and beta subunits of the rat sodium channel showed that BMK 11(2) acted to prolong Na channel inactivation. Also, in the presence of 100-200 nM BMK 11(2), a persistent non-activating Na current was induced when the membrane was depolarized from a -120 mV holding potential. BMK 11(2) caused Na channel fast inactivation to be further slowed when the holding potential was increased, suggesting that BMK 11(2) effects are voltage dependent. Na channel slow inactivation and return from slow inactivation were unaffected by the presence of BMK 11(2). Since the polypeptide prolongs APs when both K+ and Ca+ channels were blocked and shows sequence similarity to other alpha-neurotoxins, it appears likely that BMK 11(2) acts to selectively alter Na channel inactivation to produce its effect.