Michela Ottolia, PhD - US grants

? DESCRIPTION (provided by applicant): In heart cells, a major route for Ca2+ to move across the plasma membrane is through the highly specialized protein called the Na+-Ca2+ exchanger (NCX). This transporter moves Ca2+ against its gradient using the electrochemical gradient of Na+ as driving force. By maintaining Ca2+ homeostasis, NCX regulates cardiac function and alterations in its activity or expression are arrhythmogenic. Accordingly, NCX inhibitors have been suggested as therapy for arrhythmia, providing strong motivation for learning more about the structure and regulation of NCX. This is needed as, in spite of the recently resolved crystal structure of an archaebacterial homolog, salient features of this important transporter are still unknown. As a consequence, the regions of the protein involved in ion transport are not well defined and the molecular mechanisms of ion translocation are unknown. Also unclear is the physiological role of Ca2+ regulation since it is difficult to distinguish the role of Ca2+ as an activator and as transport substrate in intact cells. Another feature of NCX that has been largely overlooked is the functional role of its dimeric state and whether this property alters excitation contraction coupling is still uncharted. The objective of this proposal is to improve our understanding of NCX structure and regulation. To accomplish this we have combined cutting edges tools such as mutagenesis, electrophysiology and optical techniques, including a newly developed approach to measure conformational changes of plasma membrane proteins by FRET. Three specific aims are proposed. Aim1 will map the residues of the protein involved with ion translocation by utilizing cysteine scanning mutagenesis and electrophysiology. We will also identify and characterize the local rearrangement of NCX transmembrane segments (TMS) occurring during ion translocation using Voltage Clamp Fluorometry. This is a sophisticated and very powerful technique which permits to resolve in real time membrane protein motion (detected as changes in fluorescence) and the associated ionic flux, under voltage clamp. Aim2 will focus on elucidating if in vivo cytoplasmic Ca2+ modulates NCX activity preferentially via its allosteric regulation or transport site. These studies will resolve a major controversy in the fiel. Finally, Aim3 will define for the first time if NCX dimerizes in heart cells and determine whether NCX oligomerization is necessary for transport or regulation. This research proposal will better define the role of NCX in physiological and pathophysiological conditions and help in the rational design of drugs targeting NCX, which have promising therapeutic effects. Their development has been markedly limited by the lack of structural and mechanistic information on NCX. The studies outlined herein will help in this endeavor and greatly advance our understanding of NCX.

The purpose of this MPI proposal between the Goldhaber and Ottolia laboratories is to improve our mechanistic understanding of how sodium-calcium exchange (NCX), the dominant calcium (Ca) efflux mechanism in cardiac cells, functions to regulate cellular Ca, which in turn controls contractility and pacemaker activity. Our labs have been studying the exchanger for more than two decades, despite a number of challenges: 1) there is no proven specific blocker of NCX that can be administered extracellularly; 2) NCX current is sometimes difficult to interpret because the transporter is influenced not only by voltage, but also by temperature, pH and the intracellular and extracellular concentrations of Na and Ca; 3) intracellular Ca is not only transported by the exchanger, but also serves a regulatory function that can influence transport activity. In addition to our longstanding expertise isolating and studying NCX, for this proposal we have generated new highly innovative NCX mouse models: a tamoxifen-inducible cardiac knockout of NCX, which allows us to investigate how Ca regulation adapts over time to the acute removal of NCX in the adult mouse; a unique pH- insensitive NCX knockin mouse to investigate the physiological impact of NCX pH regulation; and the first ventricular-specific plasma membrane Ca pump 1 (PMCA1) KO mouse to determine the relative contribution of NCX and PMCA to Ca homeostasis and EC coupling. Our three specific aims are to study: 1. Atrial-Specific NCX KO?Effects on Nodal Rhythm and Atrial EC Coupling; 2. Acute and Chronic Adaptations of Ventricular EC Coupling and Ca regulation to Genetically Altered Levels of NCX; 3. NCX pH dependence ? implications for EC coupling and arrhythmia. These aims will test the hypotheses that NCX is an essential component of atrioventricular node (AVN) conduction and impulse generation, that acute ablation of NCX in adult mice activates a Ca regulatory and EC coupling adaptation program that is distinct from chronic adaptation, that PMCA1 is a critical alternative to NCX as a Ca efflux mechanism, and that pH sensitivity of NCX is critical for maintaining Ca regulation under conditions of low pH. Our approach is to use our existing and new innovative mouse models, along with state-of-the-art single cell and tissue electrophysiology combined with high speed subcellular Ca imaging techniques, high-quality proteomics and next generation RNA sequencing, to determine how NCX contributes to cardiac function through regulation of Ca. When completed, these studies will improve our mechanistic understanding of the role of NCX and related Ca handling proteins in cellular Ca regulation, EC coupling, and cardiac pacemaker (SAN and AVN) function. Such information is critical to develop effective and safe approaches to improving contractility and cellular pacemaker function in cardiac diseases such as heart failure with reduced ejection fraction, and high degree heart block from AVN disease.

PROJECT SUMMARY/ABSTRACT Na+ and Ca2+ ion homeostasis are essential for heart excitability and contractility. At the cellular level the plasma membrane protein Na+-Ca2+ exchanger (NCX) plays a vital role in regulating the ionic homeostasis of both Na+ and Ca2+. It does so by extruding one Ca2+ out of the cell in exchange for three extracellular Na+ ions. In addition to being transported, both these ions allosterically regulate the activity of NCX. Intracellular Ca2+ increases NCX activity while cytoplasmic Na+ inactivates NCX via a process known as Na+-dependent inactivation. Despite the potential physiological and pathophysiological relevance of this regulation, whether the Na+-dependent inactivation occurs in vivo is unknown and its impact has yet to be determined. Since this is such an exquisite controlling system, but heretofore uninvestigated, the investigators hypothesize that small changes in cellular Na+ concentrations may have significant effects on Ca2+ homeostasis by directly affecting NCX activity and thereby affect excitability and contractility of the heart. Therefore, the goal of this application is to investigate the physiological impact of NCX Na+ modulation and determine how it ultimately shapes heart contractility. These studies have been hampered by the difficulties of studying this process in intact myocytes under controlled conditions. However, with the development of genomic modification via CRISPR technology, this experimental paradigm, heretofore out of reach, can now be addressed. Using CRISPR, the investigators have inserted a single site mutation (K229Q) in the native cardiac NCX gene of mice, which will exclusively abolish Na+- dependent inactivation. By combining electrophysiology and calcium imaging techniques, the collected novel preliminary data demonstrating that the inhibition of NCX by cytoplasmic Na+ alters the electrical and mechanical properties of both single cells and intact hearts. The work proposed here is organized into two aims. Aim 1 will investigate how the absence of Na+-dependent inactivation alters excitation-contraction coupling in mouse adult ventricular myocytes by comparing, action potentials, Ca2+ transients and ionic currents measured from adult ventricular myocytes isolated from either control (WT) or the genetically altered mice (K229Q). Aim 2 will conduct similar recordings but in intact perfused hearts. Additionally, the cardiac function of live K229Q mice will be assessed using echocardiography. These investigations are groundbreaking as they will detail the potential function of NCX allosteric Na+ regulation in cardiac function. This work may also have pathophysiological applications by defining the regulation of Na+ as a potential target for controlling NCX activity.