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1.
Proc Natl Acad Sci U S A ; 119(13): e2109431119, 2022 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-35333652

RESUMEN

SignificanceCholesterol is one of the main components found in plasma membranes and is involved in lipid-dependent signaling enabled by integral membrane proteins such as inwardly rectifying potassium (Kir) channels. Similar to other ion channels, most of the Kir channels are down-regulated by cholesterol. One of the very few notable exceptions is Kir3.4, which is up-regulated by this important lipid. Here, we discovered and characterized a molecular switch that controls the impact (up-regulation vs. down-regulation) of cholesterol on Kir3.4. Our results provide a detailed molecular mechanism of tunable cholesterol regulation of a potassium channel.


Asunto(s)
Colesterol , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Membrana Celular/metabolismo , Colesterol/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Potasio/metabolismo , Transducción de Señal
2.
Adv Exp Med Biol ; 1422: 169-191, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36988881

RESUMEN

Inwardly rectifying potassium (Kir) channels are integral membrane proteins that control the flux of potassium ions across cell membranes and regulate membrane permeability. All eukaryotic Kir channels require the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for activation. In recent years, it has become evident that the function of many members of this family of channels is also mediated by another essential lipid-cholesterol. Here, we focus on members of the Kir2 and Kir3 subfamilies and their modulation by these two key lipids. We discuss how PI(4,5)P2 and cholesterol bind to Kir2 and Kir3 channels and how they affect channel activity. We also discuss the accumulating evidence indicating that there is interplay between PI(4,5)P2 and cholesterol in the modulation of Kir2 and Kir3 channels. In particular, we review the crosstalk between PI(4,5)P2 and cholesterol in the modulation of the ubiquitously expressed Kir2.1 channel and the synergy between these two lipids in the modulation of the Kir3.4 channel, which is primarily expressed in the heart. Additionally, we demonstrate that there is also synergy in the modulation of Kir3.2 channels, which are expressed in the brain. These observations suggest that alterations in the relative levels PI(4,5)P2 and cholesterol may fine-tune Kir channel activity.


Asunto(s)
Canales de Potasio de Rectificación Interna , Membrana Celular/metabolismo , Colesterol/metabolismo , Potasio/metabolismo , Lípidos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G
3.
Adv Exp Med Biol ; 1422: 3-59, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36988876

RESUMEN

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is the most abundant membrane phosphoinositide and cholesterol is an essential component of the plasma membrane (PM). Both lipids play key roles in a variety of cellular functions including as signaling molecules and major regulators of protein function. This chapter provides an overview of these two important lipids. Starting from a brief description of their structure, synthesis, and regulation, the chapter continues to describe the primary functions and signaling processes in which PI(4,5)P2 and cholesterol are involved. While PI(4,5)P2 and cholesterol can act independently, they often act in concert or affect each other's impact. The chapters in this volume on "Cholesterol and PI(4,5)P2 in Vital Biological Functions: From Coexistence to Crosstalk" focus on the emerging relationship between cholesterol and PI(4,5)P2 in a variety of biological systems and processes. In this chapter, the next section provides examples from the ion channel field demonstrating that PI(4,5)P2 and cholesterol can act via common mechanisms. The chapter ends with a discussion of future directions.


Asunto(s)
Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositoles , Fosfatidilinositol 4,5-Difosfato/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas/metabolismo , Colesterol/metabolismo
4.
Adv Exp Med Biol ; 1422: 143-165, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36988880

RESUMEN

Lipid mistrafficking is a biochemical hallmark of Niemann-Pick Type C (NPC) disease and is classically characterized with endo/lysosomal accumulation of unesterified cholesterol due to genetic mutations in the cholesterol transporter proteins NPC1 and NPC2. Storage of this essential signaling lipid leads to a sequence of downstream events, including oxidative stress, calcium imbalance, neuroinflammation, and progressive neurodegeneration, another hallmark of NPC disease. These observations have been validated in a growing number of studies ranging from NPC cell cultures and animal models to patient specimens. In recent reports, alterations in the levels of another class of critical signaling lipids, namely phosphoinositides, have been described in NPC disease. Focusing on cholesterol and phosphoinositides, the chapter begins by reviewing the interactions of NPC proteins with cholesterol and their role in cholesterol transport. It then continues to describe the modulation of cholesterol efflux in NPC disease. The chapter concludes with a summary of findings related to the functional consequences of perturbations in phosphoinositides in this fatal disease.


Asunto(s)
Colesterol , Enfermedad de Niemann-Pick Tipo C , Animales , Colesterol/metabolismo , Proteínas/metabolismo , Transporte Biológico , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/metabolismo , Mutación
5.
J Chem Educ ; 99(12): 4085-4093, 2022 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-37519308

RESUMEN

The goal of undergraduate chemistry laboratories is to allow students to learn about chemical systems and key laboratory skills. They should then apply this knowledge to solve problems and connect macroscopic observations in the laboratory with those occurring at the submicroscopic level. Unfortunately, these needs are not met through traditional confirmation labs. Therefore, many chemistry instructors are turning towards research-based labs course-based undergraduate research experiences (CUREs). There are also many cases where summer workshops, often with non-traditional pedagogy, are used for students. This article describes the STEM CoLab Program, a novel type of summer workshop that seeks to build student chemistry knowledge and skills in research and presentation at the beginning of their college work. This program uses the principles of CUREs for students who are just entering the university, mostly as freshmen. Several different phenomena have been investigated during the program. In this paper, we report the overall work of the program from 2016 through 2021 and provide additional details on the program's implementation in 2020 and 2021 when students conducted their work from home, using a combination of a take home research kit for studying salivary amylase "in vitro" and computer-based visualizations of amylase-inhibitor interactions "in silico" using PyMOL and online docking tools.

6.
J Lipid Res ; 61(7): 1004-1013, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32371566

RESUMEN

Niemann-Pick disease type C1 (NPC1) is a lipid storage disorder in which cholesterol and glycosphingolipids accumulate in late endosomal/lysosomal compartments because of mutations in the NPC1 gene. A hallmark of NPC1 is progressive neurodegeneration of the cerebellum as well as visceral organ damage; however, the mechanisms driving this disease pathology are not fully understood. Phosphoinositides are phospholipids that play distinct roles in signal transduction and vesicle trafficking. Here, we utilized a consensus spectra analysis of MS imaging data sets and orthogonal LC/MS analyses to evaluate the spatial distribution of phosphoinositides and quantify them in cerebellar tissue from Npc1-null mice. Our results suggest significant depletion of multiple phosphoinositide species, including PI, PIP, and PIP2, in the cerebellum of the Npc1-null mice in both whole-tissue lysates and myelin-enriched fractions. Additionally, we observed altered levels of the regulatory enzyme phosphatidylinositol 4-kinase type 2α in Npc1-null mice. In contrast, the levels of related kinases, phosphatases, and transfer proteins were unaltered in the Npc1-null mouse model, as observed by Western blot analysis. Our discovery of phosphoinositide lipid biomarkers for NPC1 opens new perspectives on the pathophysiology underlying this fatal neurodegenerative disease.


Asunto(s)
Cerebelo/diagnóstico por imagen , Cerebelo/metabolismo , Imagen Molecular , Enfermedad de Niemann-Pick Tipo C/diagnóstico por imagen , Enfermedad de Niemann-Pick Tipo C/metabolismo , Fosfatidilinositoles/metabolismo , Animales , Cromatografía Liquida , Espectrometría de Masas , Ratones , Ratones Noqueados
7.
J Lipid Res ; 60(1): 19-29, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30420402

RESUMEN

Cholesterol, a critical component of the cellular plasma membrane, is essential for normal neuronal function. Cholesterol content is highest in the brain, where most cholesterol is synthesized de novo; HMG-CoA reductase controls the synthesis rate. Despite strict control, elevated blood cholesterol levels are common and are associated with various neurological disorders. G protein-gated inwardly rectifying potassium (GIRK) channels mediate the actions of inhibitory brain neurotransmitters. Loss of GIRK function enhances neuron excitability; gain of function reduces neuronal activity. However, the effect of dietary cholesterol or HMG-CoA reductase inhibition (i.e., statin therapy) on GIRK function remains unknown. Using a rat model, we compared the effects of a high-cholesterol versus normal diet both with and without atorvastatin, a widely prescribed HMG-CoA reductase inhibitor, on neuronal GIRK currents. The high-cholesterol diet increased hippocampal CA1 region cholesterol levels and correspondingly increased neuronal GIRK currents. Both phenomena were reversed by cholesterol depletion in vitro. Atorvastatin countered the high-cholesterol diet effects on neuronal cholesterol content and GIRK currents; these effects were reversed by cholesterol enrichment in vitro. Our findings suggest that high-cholesterol diet and atorvastatin therapy affect ion channel function in the brain by modulating neuronal cholesterol levels.


Asunto(s)
Atorvastatina/farmacología , Colesterol en la Dieta/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/fisiología , Suplementos Dietéticos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Fenómenos Electrofisiológicos/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley
8.
Adv Exp Med Biol ; 1135: 119-138, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31098814

RESUMEN

Inwardly rectifying potassium (Kir) channels play a variety of critical cellular roles including modulating membrane excitability in neurons, cardiomyocytes and muscle cells, and setting the resting membrane potential, heart rate, vascular tone, insulin release, and salt flow across epithelia. These processes are regulated by a variegated list of modulators. In particular, in recent years, cholesterol has been shown to modulate a growing number of Kir channels. Subsequent to the discovery that members of the Kir2 subfamily were down-regulated by cholesterol, we have shown that members of several other Kir subfamilies were also modulated by cholesterol. However, not all cholesterol sensitive Kir channels were down-regulated by cholesterol. Our recent studies focused on three Kir channels: Kir2.1 (IRK1), Kir3.2^ (GIRK2^) and Kir3.4* (GIRK4*). Among these, Kir2.1 was down-regulated by cholesterol whereas Kir3.2^ and Kir3.4* were both up-regulated by cholesterol. Despite the opposite impact of cholesterol on these Kir3 channels compared to Kir2.1, putative cholesterol binding sites in all three channels were identified in equivalent transmembrane domains. Interestingly, however, there are intriguing differences in the specific residues that interact with the cholesterol molecule in these Kir channels. Here we compare and contrast the molecular characteristics of the putative cholesterol binding sites in the three channels, and discuss the potential implications of the differences for the impact of cholesterol on ion channels.


Asunto(s)
Colesterol/química , Canales de Potasio de Rectificación Interna/química , Sitios de Unión , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Humanos , Potenciales de la Membrana
9.
Adv Exp Med Biol ; 1135: 139-160, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31098815

RESUMEN

In recent years, a growing number of studies have implicated the coordinated action of NPC1 and NPC2 in intralysosomal transport and efflux of cholesterol. Our current understanding of this process developed with just over two decades of research. Since the cloning of the genes encoding the NPC1 and NPC2 proteins, studies of the biochemical defects observed when either gene is mutated along with computational and structural studies have unraveled key steps in the underlying mechanism. Here, we summarize the major contributions to our understanding of the proposed cholesterol transport controlled by NPC1 and NPC2, and briefly discuss recent findings of cholesterol binding and transport proteins beyond NPC1 and NPC2. We conclude with key questions and major challenges for future research on cholesterol transport by the NPC1 and NPC2 proteins.


Asunto(s)
Proteínas Portadoras/química , Colesterol/química , Glicoproteínas/química , Glicoproteínas de Membrana/química , Transporte Biológico , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína Niemann-Pick C1 , Proteínas de Transporte Vesicular
10.
Adv Exp Med Biol ; 1135: 47-66, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31098810

RESUMEN

Cholesterol-protein interactions play a critical role in lipid metabolism and maintenance of cell integrity. To elucidate the molecular mechanisms underlying these interactions, a growing number of studies have focused on determining the crystal structures of a variety of proteins complexed with cholesterol. These include structures in which cholesterol binds to transmembrane domains, and structures in which cholesterol interacts with soluble ones. However, it remains unknown whether there are differences in the prerequisites for cholesterol binding to these two types of domains. Thus, to define the molecular determinants that characterize the binding of cholesterol to these two distinct protein domains, we employed the database of crystal structures of proteins complexed with cholesterol. Our analysis suggests that cholesterol may bind more strongly to soluble domains than to transmembrane domains. The interactions between cholesterol and the protein in both cases critically depends on hydrophobic and aromatic residues. In addition, cholesterol binding sites in both types of domains involve polar and/or charged residues. However, the percentage of appearance of the different types of polar/charged residues in cholesterol binding sites differs between soluble and transmembrane domains. No differences were observed in the conformational characteristics of the cholesterol molecules bound to soluble versus transmembrane protein domains suggesting that cholesterol is insensitive to the environment provided by the different protein domains.


Asunto(s)
Colesterol/química , Dominios Proteicos , Proteínas/química , Sitios de Unión , Transporte Biológico , Unión Proteica
11.
Adv Exp Med Biol ; 1135: 67-86, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31098811

RESUMEN

Cholesterol is a highly asymmetric lipid molecule. As an essential constituent of the cell membrane, cholesterol plays important structural and signaling roles in various biological processes. The first high-resolution crystal structure of a transmembrane protein in complex with cholesterol was a human ß2-adrenergic receptor structure deposited to the Protein Data Bank in 2007. Since then, the number of the cholesterol-bound crystal structures has grown considerably providing an invaluable resource for obtaining insights into the structural characteristics of cholesterol binding. In this work, we examine the spatial and orientation distributions of cholesterol relative to the protein framework in a collection of 73 crystal structures of membrane proteins. To characterize the cholesterol-protein interactions, we apply singular value decomposition to an array of interatomic distances, which allows us to systematically assess the flexibility and variability of cholesterols in transmembrane proteins. Together, this joint analysis reveals the common characteristics among the observed cholesterol structures, thereby offering important guidelines for prediction and modification of potential cholesterol binding sites in transmembrane proteins.


Asunto(s)
Colesterol/química , Proteínas de la Membrana/química , Sitios de Unión , Membrana Celular , Humanos , Unión Proteica , Receptores Adrenérgicos beta 2/química
12.
J Biol Chem ; 292(15): 6135-6147, 2017 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-28213520

RESUMEN

Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease.


Asunto(s)
Región CA1 Hipocampal/metabolismo , Colesterol/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Activación del Canal Iónico/fisiología , Células Piramidales/metabolismo , Regulación hacia Arriba/fisiología , Animales , Región CA1 Hipocampal/química , Región CA1 Hipocampal/citología , Colesterol/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Membrana Dobles de Lípidos/química , Masculino , Oocitos , Dominios Proteicos , Células Piramidales/química , Células Piramidales/citología , Ratas , Ratas Sprague-Dawley , Xenopus
13.
Biochim Biophys Acta Biomembr ; 1859(7): 1233-1241, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28377218

RESUMEN

G-protein gated inwardly rectifying potassium (GIRK or Kir3) channels play a major role in the control of the heart rate, and require the membrane phospholipid phosphatidylinositol-bis-phosphate (PI(4,5)P2) for activation. Recently, we have shown that the activity of the heterotetrameric Kir3.1/Kir3.4 channel that underlies atrial KACh currents was enhanced by cholesterol. Similarly, the activities of both the Kir3.4 homomer and its active pore mutant Kir3.4* (Kir3.4_S143T) were also enhanced by cholesterol. Here we employ planar lipid bilayers to investigate the crosstalk between PI(4,5)P2 and cholesterol, and demonstrate that these two lipids act synergistically to activate Kir3.4* currents. Further studies using the Xenopus oocytes heterologous expression system suggest that PI(4,5)P2 and cholesterol act via distinct binding sites. Whereas PI(4,5)P2 binds to the cytosolic domain of the channel, the putative binding region of cholesterol is located at the center of the transmembrane domain overlapping the central glycine hinge region of the channel. Together, our data suggest that changes in the levels of two key membrane lipids - cholesterol and PI(4,5)P2 - could act in concert to provide fine-tuning of Kir3 channel function.


Asunto(s)
Colesterol/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Activación del Canal Iónico , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animales , Sitios de Unión , Células HEK293 , Humanos , Xenopus
14.
Curr Top Membr ; 80: 187-208, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28863816

RESUMEN

The concept that cholesterol binds to proteins via specific binding motifs, and thereby modulates their function, has emerged two decades ago. When we recently embarked on studies to uncover the putative binding region(s) of cholesterol in the Kir2.1 channel, we carried out an unbiased approach that combines computational and experimental methods. This approach resulted in the identification of novel cholesterol-binding regions distinct from known cholesterol-binding motifs. In recent years, a plethora of structures of proteins complexed with cholesterol have been determined revealing variegated cholesterol-binding regions that can provide invaluable insights into the prerequisites for cholesterol binding. Thus, using this database of structures, the goal of this chapter is to present a comprehensive analysis of representative cholesterol-binding regions, and thereby determine the molecular requirements for cholesterol binding. The analysis demonstrates that the primary requirement for cholesterol binding is a highly hydrophobic environment, and that the interaction with the cholesterol molecule can be stabilized by stacking interactions between its ring structure and hydrophobic aromatic residues, and by hydrogen bonding between its hydroxyl group and a variety of protein residues. This general requirement suggests that the known cholesterol-binding motifs describe a subset of cholesterol-binding regions, and provides a framework for expanding the search for novel cholesterol-binding regions in ion channels.


Asunto(s)
Colesterol/metabolismo , Canales Iónicos/metabolismo , Secuencias de Aminoácidos , Animales , Humanos , Canales Iónicos/química , Unión Proteica
15.
Biochim Biophys Acta ; 1848(10 Pt A): 2406-13, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26196595

RESUMEN

Cholesterol is one of the major lipid components of membranes in mammalian cells. In recent years, cholesterol has emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels in general and on inwardly rectifying potassium (Kir) channels in particular is a decrease in activity. In contrast, we have recently shown that native G-protein gated Kir (GIRK or Kir3) channels that underlie atrial KACh currents are up-regulated by cholesterol. Here we unveil the biophysical basis of cholesterol-induced increase in KACh activity. Using planar lipid bilayers we show that cholesterol significantly enhances the channel open frequency of the Kir3.1/Kir3.4 channels, which underlie KACh currents. In contrast, our data indicate that cholesterol does not affect their unitary conductance. Furthermore, using fluorescent and TIRF microscopy as well as surface protein biotinylation, we also show that cholesterol enrichment in vitro has no effect on surface expression of GFP-tagged channels expressed in Xenopus oocytes or transfected into HEK293 cells. Together, these data demonstrate for the first time that cholesterol enhances Kir3-mediated current by increasing the channel open probability.


Asunto(s)
Colesterol/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Activación del Canal Iónico/fisiología , Modelos Biológicos , Modelos Estadísticos , Potasio/metabolismo , Animales , Simulación por Computador , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Células HEK293 , Humanos , Modelos Químicos , Oocitos/química , Oocitos/fisiología , Xenopus laevis
16.
Biophys J ; 108(1): 62-75, 2015 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-25564853

RESUMEN

The slow delayed rectifier (IKs) channel is composed of the KCNQ1 channel and KCNE1 auxiliary subunit, and functions to repolarize action potentials in the human heart. IKs activators may provide therapeutic efficacy for treating long QT syndromes. Here, we show that a new KCNQ1 activator, ML277, can enhance IKs amplitude in adult guinea pig and canine ventricular myocytes. We probe its binding site and mechanism of action by computational analysis based on our recently reported KCNQ1 and KCNQ1/KCNE1 3D models, followed by experimental validation. Results from a pocket analysis and docking exercise suggest that ML277 binds to a side pocket in KCNQ1 and the KCNE1-free side pocket of KCNQ1/KCNE1. Molecular-dynamics (MD) simulations based on the most favorable channel/ML277 docking configurations reveal a well-defined ML277 binding space surrounded by the S2-S3 loop and S4-S5 helix on the intracellular side, and by S4-S6 transmembrane helices on the lateral sides. A detailed analysis of MD trajectories suggests two mechanisms of ML277 action. First, ML277 restricts the conformational dynamics of the KCNQ1 pore, optimizing K(+) ion coordination in the selectivity filter and increasing current amplitudes. Second, ML277 binding induces global motions in the channel, including regions critical for KCNQ1 gating transitions. We conclude that ML277 activates IKs by binding to an intersubunit space and allosterically influencing pore conductance and gating transitions. KCNE1 association protects KCNQ1 from an arrhythmogenic (constitutive current-inducing) effect of ML277, but does not preclude its current-enhancing effect.


Asunto(s)
Canales de Potasio de Tipo Rectificador Tardío/metabolismo , Neurotransmisores/farmacología , Animales , Sitios de Unión , Células COS , Células Cultivadas , Chlorocebus aethiops , Canales de Potasio de Tipo Rectificador Tardío/genética , Perros , Cobayas , Iones/metabolismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Potasio/metabolismo , Estructura Secundaria de Proteína , Transfección
17.
Proc Natl Acad Sci U S A ; 109(36): E2399-408, 2012 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-22891352

RESUMEN

Voltage-gated K(+) (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4-S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP(2) depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP(2). Gating current measurements revealed that PIP(2) constrains the movement of the sensor through interactions with the S4-S5 linker. Thus, PIP(2) controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them.


Asunto(s)
Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Animales , Cristalografía por Rayos X , Cinética , Simulación de Dinámica Molecular , Mutagénesis , Oocitos/metabolismo , Técnicas de Placa-Clamp , Fosfolípidos/metabolismo , Subunidades de Proteína/metabolismo , Xenopus
18.
J Physiol ; 592(18): 4025-38, 2014 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-25038242

RESUMEN

A growing number of studies show that different types of ion channels localize in caveolae and are regulated by the level of membrane cholesterol. Furthermore, it has been proposed that cholesterol-induced regulation of ion channels might be attributed to partitioning into caveolae and association with caveolin-1 (Cav-1). We tested, therefore, whether Cav-1 regulates the function of inwardly rectifying potassium channels Kir2.1 that play major roles in the regulation of membrane potentials of numerous mammalian cells. Our earlier studies demonstrated that Kir2.1 channels are cholesterol sensitive. In this study, we show that Kir2.1 channels co-immunoprecipitate with Cav-1 and that co-expression of Kir2.1 channels with Cav-1 in HEK293 cells results in suppression of Kir2 current indicating that Cav-1 is a negative regulator of Kir2 function. These observations are confirmed by comparing Kir currents in bone marrow-derived macrophages isolated from Cav-1(-/-) and wild-type animals. We also show, however, that Kir2 channels maintain their sensitivity to cholesterol in HEK293 cells that have very low levels of endogenous Cav-1 and in bone marrow-derived macrophages isolated from Cav-1(-/-) knockout mice. Thus, these studies indicate that Cav-1 and/or intact caveolae are not required for cholesterol sensitivity of Kir channels. Moreover, a single point mutation of Kir2.1, L222I that abrogates the sensitivity of the channels to cholesterol also abolishes their sensitivity to Cav-1 suggesting that the two modulators regulate Kir2 channels via a common mechanism.


Asunto(s)
Caveolina 1/metabolismo , Colesterol/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Potenciales de Acción , Animales , Células Cultivadas , Células HEK293 , Humanos , Macrófagos/metabolismo , Macrófagos/fisiología , Ratones , Mutación Puntual , Canales de Potasio de Rectificación Interna/genética
19.
J Biol Chem ; 288(43): 31154-64, 2013 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-24019518

RESUMEN

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. We have recently shown that cholesterol regulates representative members of the Kir family and that in the majority of the cases, cholesterol suppresses channel function. Furthermore, recent data indicate that cholesterol regulates Kir channels by specific sterol-protein interactions, yet the location of the cholesterol binding site in Kir channels is unknown. Using a combined computational-experimental approach, we show that cholesterol may bind to two nonanular hydrophobic regions in the transmembrane domain of Kir2.1 located between adjacent subunits of the channel. The location of the binding regions suggests that cholesterol modulates channel function by affecting the hinging motion at the center of the pore-lining transmembrane helix that underlies channel gating either directly or through the interface between the N and C termini of the channel.


Asunto(s)
Colesterol/química , Simulación del Acoplamiento Molecular , Canales de Potasio de Rectificación Interna/química , Secuencias de Aminoácidos , Animales , Colesterol/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Oocitos , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Xenopus laevis
20.
J Biol Chem ; 287(7): 4925-35, 2012 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-22174416

RESUMEN

Hypercholesterolemia is a well-known risk factor for cardiovascular disease. In the heart, activation of K(ACh) mediates the vagal (parasympathetic) negative chronotropic effect on heart rate. Yet, the effect of cholesterol on K(ACh) is unknown. Here we show that cholesterol plays a critical role in modulating K(ACh) currents (I(K,ACh)) in atrial cardiomyocytes. Specifically, cholesterol enrichment of rabbit atrial cardiomyocytes led to enhanced channel activity while cholesterol depletion suppressed I(K,ACh). Moreover, a high-cholesterol diet resulted in up to 3-fold increase in I(K,ACh) in rodents. In accordance, elevated currents were observed in Xenopus oocytes expressing the Kir3.1/Kir3.4 heteromer that underlies I(K,ACh). Furthermore, our data suggest that cholesterol affects I(K,ACh) via a mechanism which is independent of both PI(4,5)P(2) and Gßγ. Interestingly, the effect of cholesterol on I(K,ACh) is opposite to its effect on I(K1) in atrial myocytes. The latter are suppressed by cholesterol enrichment and by high-cholesterol diet, and facilitated following cholesterol depletion. These findings establish that cholesterol plays a critical role in modulating I(K,ACh) in atrial cardiomyocytes via a mechanism independent of the channel's major modulators.


Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/biosíntesis , Hiperhomocisteinemia/metabolismo , Miocitos Cardíacos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Regulación hacia Arriba , Animales , Colesterol/metabolismo , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Hiperhomocisteinemia/inducido químicamente , Hiperhomocisteinemia/genética , Miocitos Cardíacos/patología , Fosfatidilinositol 4,5-Difosfato/genética , Conejos , Xenopus laevis
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