RESUMO
Progesterone (≥1 µM) is used in recovery of cerebral ischemia, an effect likely contributed to by cerebrovascular dilation. The targets of this progesterone action are unknown. We report that micromolar (µM) progesterone activates mouse cerebrovascular myocyte BK channels; this action is lost in ß1-/- mice myocytes and in lipid bilayers containing BK α subunit homomeric channels but sustained on ß1/ß4-containing heteromers. Progesterone binds to both regulatory subunits, involving two steroid binding sites conserved in ß1-ß4: high-affinity (sub-µM), which involves Trp87 in ß1 loop, and low-affinity (µM) defined by TM1 Tyr32 and TM2 Trp163. Thus progesterone, but not its oxime, bridges TM1-TM2. Mutation of the high-affinity site blunts channel activation by progesterone underscoring a permissive role of the high-affinity site: progesterone binding to this site enables steroid binding at the low-affinity site, which activates the channel. In support of our model, cerebrovascular dilation evoked by µM progesterone is lost by mutating Tyr32 or Trp163 in ß1 whereas these mutations do not affect alcohol-induced cerebrovascular constriction. Furthermore, this alcohol action is effectively counteracted both in vitro and in vivo by progesterone but not by its oxime.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta , Progesterona , Camundongos , Animais , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Progesterona/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Esteroides/farmacologia , Sítios de Ligação , Etanol/farmacologia , Oximas/farmacologiaRESUMO
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.
Assuntos
Estradiol/metabolismo , Ativação do Canal Iônico , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Miócitos de Músculo Liso/metabolismo , Cálcio/metabolismo , Células HEK293 , Humanos , Potenciais da Membrana , Técnicas de Patch-Clamp/métodos , Subunidades Proteicas , Receptores de Estradiol/metabolismoRESUMO
Large conductance voltage- and Ca2+-activated K+ (BKCa) channels are essential regulators of membrane excitability in a wide variety of cells and tissues. An important mechanism of modulation of BKCa channel activity is its association with auxiliary subunits. In smooth muscle cells, the most predominant regulatory subunit of BKCa channels is the ß1-subunit. We have previously described that BKCa channels with distinctive N-terminal ends (starting with the amino acid sequence MDAL, MSSN or MANG) are differentially modulated by the ß1-subunit, but not by the ß2. Here we extended our studies to understand how the distinct N-terminal regions differentially modulate channel activity by ß-subunits. We recorded inside-out single-channel currents from HEK293T cells co-expressing the BKCa containing three N-terminal sequences with two ß1-ß2 chimeric constructs containing the extracellular loop of ß1 or ß2, and the transmembrane and cytoplasmic domains of ß2 or ß1, respectively. Both ß chimeric constructs induced leftward shifts of voltage-activation curves of channels starting with MANG and MDAL, in the presence of 10 or 100 µM intracellular Ca2+. However, MSSN showed no shift of the voltage-activation, at the same Ca2+ concentrations. The presence of the extracellular loop of ß1 in the chimera resembled results seen with the full ß1 subunit, suggesting that the extracellular region of ß1 might be responsible for the lack of modulation observed in MSSN. We further studied a poly-serine stretch present in the N-terminal region of MSSN and observed that the voltage-activation curves of BKCa channels either containing or lacking this poly-serine stretch were leftward shifted by ß1-subunit in a similar way. Overall, our results provide further insights into the mechanism of modulation of the different N-terminal regions of the BKCa channel by ß-subunits and highlight the extension of this region of the channel as a form of modulation of channel activity.
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico , Cinética , Peptídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
Large-conductance Ca2+- and voltage-activated potassium (MaxiK or BK) channels are composed of a pore-forming α subunit (Slo) and 4 types of auxiliary ß subunits or just a pore-forming α subunit. Although multiple N-linked glycosylation sites in the extracellular loop of ß subunits have been identified, very little is known about how glycosylation influences the structure and function of BK channels. Using a combination of site-directed mutagenesis, western blot and patch-clamp recordings, we demonstrated that 3 sites in the extracellular loop of ß2 subunit are N-glycosylated (N-X-T/S at N88, N96 and N119). Glycosylation of these sites strongly and differentially regulate gating kinetics, outward rectification, toxin sensitivity and physical association between the α and ß2 subunits. We constructed a model and used molecular dynamics (MD) to simulate how the glycosylation facilitates the association of α/ß2 subunits and modulates the dimension of the extracellular cavum above the pore of the channel, ultimately to modify biophysical and pharmacological properties of BK channels. Our results suggest that N-glycosylation of ß2 subunits plays crucial roles in imparting functional heterogeneity of BK channels, and is potentially involved in the pathological phenotypes of carbohydrate metabolic diseases.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Sequência de Aminoácidos , Animais , Charibdotoxina/farmacologia , Glicosilação , Células HEK293 , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Camundongos , Modelos Biológicos , Simulação de Dinâmica Molecular , Fenótipo , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory ß-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or ß1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) ß1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the ß1-subunit within the α/ß1-subunit complex.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Animais , Transferência de Energia , Feminino , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Modelos Moleculares , Oócitos , Conformação Proteica , Domínios Proteicos , Xenopus laevisRESUMO
Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory ß-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary ß-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between ß1 and ß3 or ß2 auxiliary subunits, we were able to identify that the cytoplasmic regions of ß1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of ß1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of ß1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the ß1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.
Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Potássio/metabolismo , Animais , Sítios de Ligação/genética , Feminino , Humanos , Ativação do Canal Iônico/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Lisina/química , Lisina/genética , Lisina/fisiologia , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Modelos Moleculares , Mutação , Oócitos/metabolismo , Oócitos/fisiologia , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Xenopus laevisRESUMO
Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory ß1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm-10 µm to either leaflet of cell-free membranes at a wide range of [Ca(2+)]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50 = 1 nm; Emax reached at 10 nm), with physiological [Ca(2+)]i and voltages favoring this activation. Homomeric cbv1 or cbv1-ß2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK ß1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate ß1 subunit-containing channels when ß1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK ß1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of ß1-specific BK channel activators.
Assuntos
Ativação do Canal Iônico/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Leucotrieno B4/metabolismo , Animais , Cálcio/metabolismo , Artérias Cerebrais/citologia , Feminino , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Leucotrieno A4/química , Leucotrieno A4/metabolismo , Leucotrieno A4/farmacologia , Leucotrieno B4/química , Leucotrieno B4/farmacologia , Leucotrieno C4/química , Leucotrieno C4/metabolismo , Leucotrieno C4/farmacologia , Leucotrieno D4/química , Leucotrieno D4/metabolismo , Leucotrieno D4/farmacologia , Leucotrieno E4/química , Leucotrieno E4/metabolismo , Leucotrieno E4/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Microinjeções , Modelos Moleculares , Estrutura Molecular , Células Musculares/citologia , Células Musculares/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Terciária de Proteína , RNA Complementar/administração & dosagem , RNA Complementar/genética , Ratos , Xenopus laevisRESUMO
We demonstrated previously that BK (K(Ca)1.1) channel activity (NP(o)) increases in response to bisphenol A (BPA). Moreover, BK channels containing regulatory ß1 subunits were more sensitive to the stimulatory effect of BPA. How BPA increases BK channel NPo remains mostly unknown. Estradiol activates BK channels by binding to an extracellular site, but neither the existence nor location of a BPA binding site has been demonstrated. We tested the hypothesis that an extracellular binding site is responsible for activation of BK channels by BPA. We synthesized membrane-impermeant BPA-monosulfate (BPA-MS) and used patch clamp electrophysiology to study channels composed of α or α + ß1 subunits in cell-attached (C-A), whole-cell (W-C), and inside-out (I-O) patches. In C-A patches, bath application of BPA-MS (100 µM) had no effect on the NP(o) of BK channels, regardless of their subunit composition. Importantly, however, subsequent addition of membrane-permeant BPA (100 µM) increased the NP(o) of both α and α + ß1 channels in C-A patches. The C-A data indicate that in order to alter BK channel NP(o), BPA must interact with the channel itself (or some closely associated partner) and diffusible messengers are not involved. In W-C patches, 100 µM BPA-MS activated current in cells expressingα subunits, whereas cells expressing α + ß1 subunits responded similarly to a log-order lower concentration (10 µM). The W-C data suggest that an extracellular activation site exists, but do not eliminate the possibility that an intracellular site may also be present. In I-O patches, where the cytoplasmic face was exposed to the bath, BPA-MS had no effect on the NP(o) of BK α subunits, but BPA increased it. BPA-MS increased the NP(o) of α + ß1 channels in I-O patches, but not as much as BPA. We conclude that BPA activates BK α via an extracellular site and that BPA-sensitivity is increased by the ß1 subunit, which may also constitute part of an intracellular binding site.
Assuntos
Compostos Benzidrílicos/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Fenóis/metabolismo , Linhagem Celular , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana , Técnicas de Patch-Clamp , Estrutura Terciária de ProteínaRESUMO
Tungstate, a compound with antidiabetic, antiobesity, and antihypertensive properties, activates the large-conductance voltage- and Ca(2+)-dependent K(+) (BK) channel containing either ß1 or ß4 subunits. The BK activation by tungstate is Mg(2+)-dependent and promotes arterial vasodilation, but only in precontracted mouse arteries expressing ß1. In this study, we further explored how the ß1 subunit participates in tungstate activation of BK channels. Activation of heterologously expressed human BKαß1 channels in inside-out patches is fully dependent on the Mg(2+) sensitivity of the BK α channel subunit even at high (10 µM) cytosolic Ca(2+) concentration. Alanine mutagenesis of ß1 extracellular residues Y74 or S104, which destabilize the active voltage sensor, greatly decreased the tungstate-induced left-shift of the BKαß1 G-V curves in either the absence or presence of physiologically relevant cytosolic Ca(2+) levels (10 µM). The weakened tungstate activation of the BKαß1Y74A and BKαß1S104A mutant channels was not related to decreased Mg(2+) sensitivity. These results, together with previously published reports, support the idea that the putative binding site for tungstate-mediated BK channel activation is located in the pore-forming α channel subunit, around the Mg(2+) binding site. The role of ß1 in tungstate-induced channel activation seems to rely on its interaction with the BK α subunit to modulate channel activity. Loop residues that are essential for the regulation of voltage sensor activation and gating of the BK channel are also relevant for BK activation by tungstate.
Assuntos
Ativação do Canal Iônico , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Compostos de Tungstênio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Alanina/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Cálcio/metabolismo , Células HEK293 , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Magnésio/farmacologia , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Estrutura Terciária de ProteínaRESUMO
Large-conductance, voltage-, and Ca²âº-dependent K⺠(BK) channels are broadly expressed in various tissues to modulate neuronal activity, smooth muscle contraction, and secretion. BK channel activation depends on the interactions among the voltage sensing domain (VSD), the cytosolic domain (CTD), and the pore gate domain (PGD) of the Slo1 α-subunit, and is further regulated by accessory ß subunits (ß1-ß4). How ß subunits fine-tune BK channel activation is critical to understand the tissue-specific functions of BK channels. Multiple sites in both Slo1 and the ß subunits have been identified to contribute to the interaction between Slo1 and the ß subunits. However, it is unclear whether and how the interdomain interactions among the VSD, CTD, and PGD are altered by the ß subunits to affect channel activation. Here we show that human ß1 and ß2 subunits alter interactions between bound Mg²âº and gating charge R213 and disrupt the disulfide bond formation at the VSD-CTD interface of mouse Slo1, indicating that the ß subunits alter the VSD-CTD interface. Reciprocally, mutations in the Slo1 that alter the VSD-CTD interaction can specifically change the effects of the ß1 subunit on the Ca²âº activation and of the ß2 subunit on the voltage activation. Together, our data suggest a novel mechanism by which the ß subunits modulated BK channel activation such that a ß subunit may interact with the VSD or the CTD and alter the VSD-CTD interface of the Slo1, which enables the ß subunit to have effects broadly on both voltage and Ca²âº-dependent activation.
Assuntos
Membrana Celular/metabolismo , Citosol/metabolismo , Ativação do Canal Iônico/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Animais , Membrana Celular/química , Citosol/química , Feminino , Humanos , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Camundongos , Estrutura Terciária de Proteína , Xenopus laevisRESUMO
The Ca(2+)/voltage-gated K(+) large conductance (BK) channel ß1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK ß1 allows the BK channels to reduce myogenic tone, facilitating vasodilation. The endogenous steroid lithocholic acid (LCA) dilates cerebral arteries via BK channel activation, which requires recognition by a BK ß1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate ß1-containing BK channels and evoke vasodilation remain unknown. We performed a chemical structure database similarity search using LCA as a template, along with a two-step reaction to generate sodium 3-hydroxyolean-12-en-30-oate (HENA). HENA activated the BK (cbv1 + ß1) channels cloned from rat cerebral artery myocytes with a potency (EC50 = 53 µM) similar to and an efficacy (×2.5 potentiation) significantly greater than that of LCA. This HENA action was replicated on native channels in rat cerebral artery myocytes. HENA failed to activate the channels made of cbv1 + ß2, ß3, ß4, or ß1T169A, indicating that this drug selectively targets ß1-containing BK channels via the BK ß1 steroid-sensing site. HENA (3-45 µM) dilated the rat and C57BL/6 mouse pressurized cerebral arteries. Consistent with the electrophysiologic results, this effect was larger than that of LCA. HENA failed to dilate the arteries from the KCNMB1 knockout mouse, underscoring BK ß1's role in HENA action. Finally, carotid artery-infusion of HENA (45 µM) dilated the pial cerebral arterioles via selective BK-channel targeting. In conclusion, we have identified for the first time a nonsteroidal agent that selectively activates ß1-containing BK channels by targeting the steroid-sensing site in BK ß1, rendering vasodilation.
Assuntos
Artérias Cerebrais/efeitos dos fármacos , Colanos/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Esteroides/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Artérias Cerebrais/metabolismo , Colanos/química , Feminino , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Ácido Litocólico/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Ratos , Ratos Sprague-Dawley , Esteroides/química , Xenopus laevisRESUMO
Large-conductance voltage- and Ca(2+)-gated K(+) channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory ß subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an â¼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in ß2 and ß3, as in ß1 and ß4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse ß3 (mß3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0-αS6. We identified by elimination mß3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in ß1. These are conserved in ß2-ß4, which have four additional Cys (eight in total), except that mß3 has one fewer. In ß1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mß3, the free Cys is at position 7; position 2 lacks a Cys present in all other ß2-ß4; and the disulfide pattern is 1-8, 3-4, and 5-6. Presumably, Cys 2 cross-links to Cys 7 in all other ß2-ß4. Cross-linking of mß3 Cys152 to Cys substituted in the flanks of αS0-S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mß3 perturbed. Although the mß3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/análise , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/análise , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Subunidades Proteicas/análise , Subunidades Proteicas/química , Sequência de Aminoácidos , Animais , Cisteína/análise , Cisteína/química , Dissulfetos/análise , Dissulfetos/química , Eletrofisiologia , Células HEK293 , Humanos , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Camundongos , Modelos Animais , Dados de Sequência Molecular , Peptídeos/farmacologia , Estrutura Terciária de Proteína/efeitos dos fármacos , Subunidades Proteicas/fisiologiaRESUMO
The large conductance voltage- and Ca(2+)-activated K(+) channel (MaxiK, BK(Ca), BK) is composed of four pore-forming α-subunits and can be associated with regulatory ß-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A(2) prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK ß1-subunit in TP-MaxiK association. We found that the ß1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that ß1 and TP are closely associated at the cell periphery. The molecular mechanism of ß1-TP interaction involves predominantly the ß1 extracellular loop. As reported previously, TP activation by the thromboxane A(2) analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP(o)) as a function of voltage. However, the positive shift of the FP(o) versus voltage curve by U46619 relative to the control was less prominent when ß1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, ß1 gene ablation reduced the EC(50) of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the ß1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Tromboxano A2/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Vasoconstrição/efeitos dos fármacosRESUMO
A family of tissue-specific auxiliary ß subunits modulates large conductance voltage- and calcium-activated potassium (BK) channel gating properties to suit their diverse functions. Paradoxically, ß subunits both promote BK channel activation through a stabilization of voltage sensor activation and reduce BK channel openings through an increased energetic barrier of the closed-to-open transition. The molecular determinants underlying ß subunit function, including the dual gating effects, remain unknown. In this study, we report the first identification of a ß1 functional domain consisting of Y74, S104, Y105, and I106 residues located in the extracellular loop of ß1. These amino acids reside within two regions of highest conservation among related ß1, ß2, and ß4 subunits. Analysis in the context of the Horrigan-Aldrich gating model revealed that this domain functions to both promote voltage sensor activation and also reduce intrinsic gating. Free energy calculations suggest that the dual effects of the ß1 Y74 and S104-I106 domains can be largely accounted for by a relative destabilization of channels in open states that have few voltage sensors activated. These results suggest a unique and novel mechanism for ß subunit modulation of voltage-gated potassium channels wherein interactions between extracellular ß subunit residues with the external portions of the gate and voltage sensor regulate channel opening.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , TransfecçãoRESUMO
Large conductance, voltage- and calcium-gated potassium (BK) channels regulate several physiological processes, including myogenic tone and thus, artery diameter. Nongenomic modulation of BK activity by steroids is increasingly recognized, but the precise location of steroid action remains unknown. We have shown that artery dilation by lithocholate (LC) and related cholane steroids is caused by a 2× increase in vascular myocyte BK activity (EC(50) = 45 µM), an action that requires ß1 but not other (ß2-ß4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK α- (cbv1) and ß1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions in BK ß1 transmembrane domain 2 proposed by computational dynamics: the outer site includes L157, L158, and T165, whereas the inner site includes T169, L172, and L173. As expected from computational modeling, cbv1+rß1T165A,T169A channels were LC-unresponsive. However, cbv1 + rß1T165A and cbv1 + rß1T165A,L157A,L158A were fully sensitive to LC. Data indicate that the transmembrane domain 2 outer site does not contribute to steroid action. Cbv1 + rß1T169A was LC-insensitive, with rß1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + rß1L172A, and cbv1 + rß1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid α-hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for LC action. Therefore, ß1 TM2 T169,L172,L173 provides the interaction area for cholane steroid activation of BK channels. Because this amino acid triplet is unique to BK ß1, our study provides a structural basis for advancing ß1 subunit-specific pharmacology of BK channels.
Assuntos
Membrana Celular/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Ácido Litocólico/farmacologia , Alanina/genética , Substituição de Aminoácidos/efeitos dos fármacos , Animais , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Colanos/farmacologia , Ácido Litocólico/química , Modelos Moleculares , Estrutura Terciária de Proteína , Ratos , Relação Estrutura-AtividadeRESUMO
Large-conductance, Ca(2+)- and voltage-sensitive K(+) (BK) channels regulate neuronal functions such as spike frequency adaptation and transmitter release. BK channels are composed of four Slo1 subunits, which contain the voltage-sensing and pore-gate domains in the membrane and Ca(2+) binding sites in the cytoplasmic domain, and accessory ß subunits. Four types of BK channel ß subunits (ß1-ß4) show differential tissue distribution and unique functional modulation, resulting in diverse phenotypes of BK channels. Previous studies show that both the ß1 and ß2 subunits increase Ca(2+) sensitivity, but different mechanisms may underline these modulations. However, the structural domains in Slo1 that are critical for Ca(2+)-dependent activation and targeted by these ß subunits are not known. Here, we report that the N termini of both the transmembrane (including S0) and cytoplasmic domains of Slo1 are critical for ß2 modulation based on the study of differential effects of the ß2 subunit on two orthologs, mouse Slo1 and Drosophila Slo1. The N terminus of the cytoplasmic domain of Slo1, including the AC region (ßA-αC) of the RCK1 (regulator of K(+) conductance) domain and the peptide linking it to S6, both of which have been shown previously to mediate the coupling between Ca(2+) binding and channel opening, is specifically required for the ß2 but not for the ß1 modulation. These results suggest that the ß2 subunit modulates the coupling between Ca(2+) binding and channel opening, and, although sharing structural homology, the BK channel ß subunits interact with structural domains in the Slo1 subunit differently to enhance channel activity.
Assuntos
Membrana Celular/química , Membrana Celular/fisiologia , Citoplasma/fisiologia , Ativação do Canal Iônico/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Sequência de Aminoácidos , Animais , Membrana Celular/genética , Citoplasma/química , Drosophila , Feminino , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/fisiologia , Estrutura Terciária de Proteína/genética , Xenopus laevisRESUMO
BACKGROUND: The pH and voltage-regulated Slo3 K(+) channel, a homologue of the Ca(2+)- and voltage-regulated Slo1 K(+) channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels. METHODOLOGY/PRINCIPAL FINDINGS: To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm. CONCLUSIONS/SIGNIFICANCE: These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Western Blotting , Linhagem Celular , Humanos , Imunoprecipitação , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Masculino , Camundongos , Proteínas do Tecido Nervoso/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatogônias/metabolismo , Xenopus laevisRESUMO
N termini of auxiliary beta subunits that produce inactivation of large-conductance Ca(2+)-activated K(+) (BK) channels reach their pore-blocking position by first passing through side portals into an antechamber separating the BK pore module and the large C-terminal cytosolic domain. Previous work indicated that the beta2 subunit inactivation domain is protected from digestion by trypsin when bound in the inactivated conformation. Other results suggest that, even when channels are closed, an inactivation domain can also be protected from digestion by trypsin when bound within the antechamber. Here, we provide additional tests of this model and examine its applicability to other beta subunit N termini. First, we show that specific mutations in the beta2 inactivation segment can speed up digestion by trypsin under closed-channel conditions, supporting the idea that the beta2 N terminus is protected by binding within the antechamber. Second, we show that cytosolic channel blockers distinguish between protection mediated by inactivation and protection under closed-channel conditions, implicating two distinct sites of protection. Together, these results confirm the idea that beta2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity. In contrast, the beta 3a N terminus is digested over 10-fold more quickly than the beta2 N terminus. Analysis of factors that contribute to differences in digestion rates suggests that binding of an N terminus within the antechamber constrains the trypsin accessibility of digestible basic residues, even when such residues are positioned outside the antechamber. Our analysis indicates that up to two N termini may simultaneously be protected from digestion. These results indicate that inactivation domains have sites of binding in addition to those directly involved in inactivation.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Tripsina/metabolismo , Motivos de Aminoácidos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Eletrofisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Camundongos , Mutação , Oócitos , Ligação Proteica/fisiologia , Conformação Proteica , Estrutura Terciária de Proteína , Tripsina/química , Xenopus laevisRESUMO
The large-conductance Ca(2+)-activated potassium (BK(Ca)) channel of smooth muscle is unusually sensitive to Ca(2+) as compared with the BK(Ca) channels of brain and skeletal muscle. This is due to the tissue-specific expression of the BK(Ca) auxiliary subunit beta1, whose presence dramatically increases both the potency and efficacy of Ca(2+) in promoting channel opening. beta1 contains no Ca(2+) binding sites of its own, and thus the mechanism by which it increases the BK(Ca) channel's Ca(2+) sensitivity has been of some interest. Previously, we demonstrated that beta1 stabilizes voltage sensor activation, such that activation occurs at more negative voltages with beta1 present. This decreases the work that Ca(2+) must do to open the channel and thereby increases the channel's apparent Ca(2+) affinity without altering the real affinities of the channel's Ca(2+) binding sites. To explain the full effect of beta1 on the channel's Ca(2+) sensitivity, however, we also proposed that there must be effects of beta1 on Ca(2+) binding. Here, to test this hypothesis, we have used high-resolution Ca(2+) dose-response curves together with binding site-specific mutations to measure the effects of beta1 on Ca(2+) binding. We find that coexpression of beta1 alters Ca(2+) binding at both of the BK(Ca) channel's two types of high-affinity Ca(2+) binding sites, primarily increasing the affinity of the RCK1 sites when the channel is open and decreasing the affinity of the Ca(2+) bowl sites when the channel is closed. Both of these modifications increase the difference in affinity between open and closed, such that Ca(2+) binding at either site has a larger effect on channel opening when beta1 is present.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Animais , Sítios de Ligação/genética , Cálcio/química , Linhagem Celular Transformada , Transferência de Energia/fisiologia , Humanos , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica/fisiologia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Lithocholate (LC) (10-300 microM) in physiological solution is sensed by vascular myocyte large conductance, calcium- and voltage-gated potassium (BK) channel beta(1) accessory subunits, leading to channel activation and arterial dilation. However, the structural features in steroid and target that determine LC action are unknown. We tested LC and close analogs on BK channel (pore-forming cbv1+beta(1) subunits) activity using the product of the number of functional ion channels in the membrane patch (N) and the open channel probability (Po). LC (5beta-cholanic acid-3alpha-ol), 5alpha-cholanic acid-3alpha-ol, and 5beta-cholanic acid-3beta-ol increased NPo (EC(50) approximately 45 microM). At maximal increase in NPo, LC increased NPo by 180%, whereas 5alpha-cholanic acid-3alpha-ol and 5beta-cholanic acid-3beta-ol raised NPo by 40%. Thus, the alpha-hydroxyl and the cis A-B ring junction are both required for robust channel potentiation. Lacking both features, 5alpha-cholanic acid-3beta-ol and 5-cholenic acid-3beta-ol were inactive. Three-dimensional structures show that only LC displays a bean shape with clear-cut convex and concave hemispheres; 5alpha-cholanic acid-3alpha-ol and 5beta-cholanic acid-3beta-ol partially matched LC shape, and 5alpha-cholanic acid-3beta-ol and 5-cholenic acid-3beta-ol did not. Increasing polarity in steroid rings (5beta-cholanic acid-3alpha-sulfate) or reducing polarity in lateral chain (5beta-cholanic acid 3alpha-ol methyl ester) rendered poorly active compounds, consistent with steroid insertion between beta(1) and bilayer lipids, with the steroid-charged tail near the aqueous phase. Molecular dynamics identified two regions in beta(1) transmembrane domain 2 that meet unique requirements for bonding with the LC concave hemisphere, where the steroid functional groups are located.