Structural mechanisms of PIP activation and SEA0400 inhibition in human cardiac sodium-calcium exchanger NCX1.

Na/Ca exchangers (NCXs) transport Ca across the plasma membrane in exchange for Na and play a vital role in maintaining cellular Ca homeostasis. Our previous structural study of human cardiac NCX1 (HsNCX1) reveals the overall architecture of the eukaryotic exchanger and the formation of the inactivation assembly by the intracellular regulatory domain that underlies the cytosolic Na-dependent inactivation and Ca activation of NCX1. Here we present the cryo-EM structures of HsNCX1 in complex with a physiological activator phosphatidylinositol 4,5-bisphosphate (PIP), or pharmacological inhibitor SEA0400 that enhances the inactivation of the exchanger.

Dual network structure of the AV node.

Biological systems, particularly the brain, are frequently analyzed as networks, conveying mechanistic insights into their function and pathophysiology. This is the first study of a functional network of cardiac tissue. We use calcium imaging to obtain two functional networks in a subsidiary but essential pacemaker of the heart, the atrioventricular node (AVN).

Cardiac function is regulated by the sodium-dependent inhibition of the sodium-calcium exchanger NCX1.

The Na-Ca exchanger (NCX1) is the dominant Ca extrusion mechanism in cardiac myocytes. NCX1 activity is inhibited by intracellular Na via a process known as Na-dependent inactivation. A central question is whether this inactivation plays a physiological role in heart function.

Structural mechanisms of the human cardiac sodium-calcium exchanger NCX1.

Na/Ca exchangers (NCX) transport Ca in or out of cells in exchange for Na. They are ubiquitously expressed and play an essential role in maintaining cytosolic Ca homeostasis. Although extensively studied, little is known about the global structural arrangement of eukaryotic NCXs and the structural mechanisms underlying their regulation by various cellular cues including cytosolic Na and Ca.

An epilepsy-associated K1.2 charge-transfer-center mutation impairs K1.2 and K1.4 trafficking.

We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability.

The Cardiac Na -Ca Exchanger: From Structure to Function.

Ca homeostasis is essential for cell function and survival. As such, the cytosolic Ca concentration is tightly controlled by a wide number of specialized Ca handling proteins. One among them is the Na -Ca exchanger (NCX), a ubiquitous plasma membrane transporter that exploits the electrochemical gradient of Na to drive Ca out of the cell, against its concentration gradient.

Suppression of ventricular arrhythmias by targeting late L-type Ca2+ current.

Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of ICa,L, compromising inotropy.

Acute Genetic Ablation of Cardiac Sodium/Calcium Exchange in Adult Mice: Implications for Cardiomyocyte Calcium Regulation, Cardioprotection, and Arrhythmia.

Background Sodium-calcium (Ca) exchanger isoform 1 (NCX1) is the dominant Ca efflux mechanism in cardiomyocytes and is critical to maintaining Ca homeostasis during excitation-contraction coupling. NCX1 activity has been implicated in the pathogenesis of cardiovascular diseases, but a lack of specific NCX1 blockers complicates experimental interpretation. Our aim was to develop a tamoxifen-inducible NCX1 knockout (KO) mouse to investigate compensatory adaptations of acute ablation of NCX1 on excitation-contraction coupling and intracellular Ca regulation, and to examine whether acute KO of NCX1 confers resistance to triggered arrhythmia and ischemia/reperfusion injury.

BK Channels Regulate LPS-induced CCL-2 Release from Human Pulmonary Endothelial Cells.

We recently established a role for the stretch-activated two-pore-domain K (K2P) channel TREK-1 (K2P2.1) in inflammatory cytokine secretion using models of hyperoxia-, mechanical stretch-, and TNF-α-induced acute lung injury. We have now discovered the expression of large conductance, Ca-activated K (BK) channels in human pulmonary microvascular endothelial cells and primary human alveolar epithelial cells using semiquantitative real-time PCR, IP and Western blot, and investigated their role in inflammatory cytokine secretion using an LPS-induced acute lung injury model.

Modulation of the cardiac Na-Ca exchanger by cytoplasmic protons: Molecular mechanisms and physiological implications.

A precise temporal and spatial control of intracellular Ca concentration is essential for a coordinated contraction of the heart. Following contraction, cardiac cells need to rapidly remove intracellular Ca to allow for relaxation. This task is performed by two transporters: the plasma membrane Na-Ca exchanger (NCX) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA).

Na/Ca exchange in the atrium: Role in sinoatrial node pacemaking and excitation-contraction coupling.

Na/Ca exchange is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. Although our knowledge of exchanger function (NCX1 in the heart) was originally established using biochemical and electrophysiological tools such as cardiac sarcolemmal vesicles and the giant patch technique [1-4], many advances in our understanding of the physiological/pathophysiological roles of NCX1 in the heart have been obtained using a suite of genetically modified mice. Early mouse studies focused on modification of expression levels of NCX1 in the ventricles, with transgenic overexpressors, global NCX1 knockout (KO) mice (which were embryonic lethal if homozygous), and finally ventricular-specific NCX1 KO [5-12].

Molecular determinants of pH regulation in the cardiac Na-Ca exchanger.

The cardiac Na-Ca exchanger (NCX) plays a critical role in the heart by extruding Ca after each contraction and thus regulates cardiac contractility. The activity of NCX is strongly inhibited by cytosolic protons, which suggests that intracellular acidification will have important effects on heart contractility. However, the mechanisms underlying this inhibition remain elusive.

Na+/Ca2+ exchange and Na+/K+-ATPase in the heart.

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na(+)/Ca(2+) exchange (NCX) and Na(+)/K(+)-ATPase (NKA).

Na/Ca exchange and contraction of the heart.

Sodium-calcium exchange (NCX) is the major calcium (Ca) efflux mechanism of ventricular cardiomyocytes. Consequently the exchanger plays a critical role in the regulation of cellular Ca content and hence contractility. Reductions in Ca efflux by the exchanger, such as those produced by elevated intracellular sodium (Na) in response to cardiac glycosides, raise sarcoplasmic reticulum (SR) Ca stores.

The cardiac Na+-Ca2+ exchanger has two cytoplasmic ion permeation pathways.

The Na(+)-Ca(2+) exchanger (NCX) is a ubiquitously expressed plasma membrane protein. It plays a fundamental role in Ca(2+) homeostasis by moving Ca(2+) out of the cell using the electrochemical gradient of Na(+) as the driving force. Recent structural studies of a homologous archaebacterial exchanger, NCX_Mj, revealed its outward configuration with two potential ion permeation pathways exposed to the extracellular environment.

NCX1: mechanism of transport.

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) plays a critical role in the maintenance of Ca(2+) homeostasis in a variety of tissues. NCX accomplishes this task by either lowering or increasing the intracellular Ca(2+) concentration, a process which depends on electrochemical gradients. During each cycle, three Na(+) are transported in the opposite direction to one Ca(2+), resulting in an electrogenic transport that can be measured as an ionic current.

20 years from NCX purification and cloning: milestones.

The Na(+)/Ca(2+) exchanger protein was first isolated from cardiac sarcolemma in 1988 and cloned in 1990. This allowed study of Na(+)/Ca(2+) exchange at the molecular level to begin. I will review the story leading to the cloning of NCX and the research that resulted from this event.

Ca2+-dependent structural rearrangements within Na+-Ca2+ exchanger dimers.

Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane.

Interactions between Ca2+ binding domains of the Na+-Ca2+ exchanger and secondary regulation.

The Na(+)-Ca(2+) exchanger (NCX) is a plasma membrane protein particularly abundant in cardiomyocytes where it plays a prominent role in Ca(2+) extrusion. In addition to being transported, cytoplasmic Ca(2+) and Na(+) regulate NCX activity by activating and inhibiting ion transport, respectively. There are two Ca(2+) binding domains within the exchanger, CBD1 and CBD2, which have been crystallized and detailed structural information obtained.

Roles of two Ca2+-binding domains in regulation of the cardiac Na+-Ca2+ exchanger.

We expressed full-length Na(+)-Ca(2+) exchangers (NCXs) with mutations in two Ca(2+)-binding domains (CBD1 and CBD2) to determine the roles of the CBDs in Ca(2+)-dependent regulation of NCX. CBD1 has four Ca(2+)-binding sites, and mutation of residues Asp(421) and Glu(451), which primarily coordinate Ca(2+) at sites 1 and 2, had little effect on regulation of NCX by Ca(2+). In contrast, mutations at residues Glu(385), Asp(446), Asp(447), and Asp(500), which coordinate Ca(2+) at sites 3 and 4 of CBD1, resulted in a drastic decrease in the apparent affinity of peak exchange current for regulatory Ca(2+).

Structure and functional analysis of a Ca2+ sensor mutant of the Na+/Ca2+ exchanger.

The mammalian Na(+)/Ca(2+) exchanger, NCX1.1, serves as the main mechanism for Ca(2+) efflux across the sarcolemma following cardiac contraction. In addition to transporting Ca(2+), NCX1.

How does regulatory Ca2+ regulate the Na+-Ca2+ exchanger?

Spatial and temporal regulation of intracellular Ca2+ concentrations is a fundamental requirement for life. The mammalian cardiac Na+-Ca2+ exchanger serves as the main mechanism for Ca2+ efflux after heart contraction. Exchange activity is highly regulated by intracellular Ca2+, which binds two regulatory domains (CBD1 and CBD2) and triggers the full activity of the exchanger.

Conformational changes of a Ca2+-binding domain of the Na+/Ca2+ exchanger monitored by FRET in transgenic zebrafish heart.

The Na(+)/Ca(2+) exchanger is the major Ca(2+) extrusion mechanism in cardiac myocytes. The activity of the cardiac Na(+)/Ca(2+) exchanger is dynamically regulated by intracellular Ca(2+). Previous studies indicate that Ca(2+) binding to a high-affinity Ca(2+)-binding domain (CBD1) in the large intracellular loop is involved in regulation.

The RCK2 domain of the human BKCa channel is a calcium sensor.

Large conductance voltage and Ca(2+)-dependent K(+) channels (BK(Ca)) are activated by both membrane depolarization and intracellular Ca(2+). Recent studies on bacterial channels have proposed that a Ca(2+)-induced conformational change within specialized regulators of K(+) conductance (RCK) domains is responsible for channel gating. Each pore-forming alpha subunit of the homotetrameric BK(Ca) channel is expected to contain two intracellular RCK domains.

Dynamic modulation of intracellular glucose imaged in single cells using a FRET-based glucose nanosensor.

To study intracellular glucose homeostasis, the glucose nanosensor FLIPglu-600 microM, which undergoes changes in fluorescence resonance energy transfer (FRET) upon interaction with glucose, was expressed in four mammalian cell lines: COS-7, CHO, HEK293, and C2C12. Upon addition of extracellular glucose, the intracellular FRET ratio decreased rapidly as intracellular glucose increased. The kinetics were fast (tau=5 to 15 s) in COS and C2C12 cells and slow (tau=20 to 40 s) in HEK and CHO cells.