Sodium-calcium exchanger

IntroductionIntroduction

spinqualitylabelsall models Structure of NCX_Mj exchanger from Methanocaldococcus jannaschii complex with Cd++ (PDB code 3v5s) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Methanococcus janaschii sodium calcium exchanger (NCX_Mj) is a member of the NCX (sodium/calcium exchanger) family of proteins, expressed in the archaebacteria Methanococcus jannaschii. NCX_Mj is a membrane protein capable of transporting calcium ions across the plasma membrane (PM) by utilizing the electrochemical gradient of sodium ions. In physiological conditions, it's function is most probably related to calcium homeostasis in the cell. NCX_Mj is to date, the only member of the NCX protein family which it's structure had been resolved, and only in the outward facing (OF) conformation.

Calcium Cation Antiporter Superfamily:Calcium Cation Antiporter Superfamily:

The calcium cation antiporter (CaCA) superfamily is a family of membrane bound secondary transporters, which couple the translocation of different ions along with the extrusion of calcium ion across the PM. The CaCA superfamily is a vast and diverse family with many members in both prokaryotic and eukaryotic organisms. Family members are defined by a core structure of at least ten TM helices and the presence of two highly conserved α-repeat regions (named α-1 and α-2 repeats). These repeats are found in two clusters of inversely oriented hydrophobic domains separated by an intracellular loop of varying length. These highly conserved domains, include 12 residues which coordinate cation binding, probably taking part in both ion selectivity and transport mechanism. The CaCA superfamily contains five subfamilies.

CaCA subfamilies:

• K+ independent Na+/Ca2+ exchangers (NCX)
• K+ dependent Na+/Ca2+ exchangers (NCKX)
• Cation/Ca2+ exchangers (CCX)
• H+/Cation exchangers (CAX)
• YRBG like exchangers

A notable family member is the Mg²⁺/H⁺ exchanger (MHX) which can be found as an alternative for NCX in land plants. To date it is classified under the NCX family.

Sodium Calcium Exchanger FamilySodium Calcium Exchanger Family

The sodium calcium exchanger (NCX) family is a family of electrogenic membrane bound secondary transporters, capable of calcium transport across the cell membrane and organelles to regulate cytosolic Ca²⁺ level, with a stoichiometry of 3 Na⁺ and 1 Ca²⁺ ions. NCX members are encoded in the SLC8 gene. Major work was done on the gene's structure evolution, phylogenetics and sequence homology, but they will not be covered in this article. Invertebrates carry only one SLC8 gene that does not go through alternative splicing while vertebrates often carry several and offer alternative splice variants. For example: Three mammalian SLC8 genes were discovered: SLC8A1, SLC8A2 and SLC8A3 encoding NCX1, NCX2 and NCX3 gene products respectively. The first and last are capable of alternative splicing. An additional gene, SLC8A4 is present at teleost, reptilian and amphibian species. The newest members of the family are NCLX proteins encoded in the SLC8B1 gene. These exchangers are mitochondrial Na⁺/Ca²⁺ exchangers with the unique ability to transport either Na⁺ or Li⁺ in exchange for Ca²⁺ ions.

Calcium, due to its coordination chemistry, is an important and versatile second messenger. It regulates several signal transduction pathways in the cell including excitation-contraction coupling, release of neurotransmitters, apoptosis and proliferation and many other important cellular functions. Resting cytosolic free calcium is therefore tightly maintained. Derangement in the expression and/or regulation of NCX proteins in humans can lead to several pathologic conditions including heart failure, cardiac arrhythmias, ischemia and reperfusion injury and hypertension. NCX apparently also has roles in stroke and cerebral ischemia as well as in insulin secretion.

Structure:Structure:

NCX_mj represents our current understanding of NCX protein's structure and is composed of several structural elements most probably present in other members of the NCX family as well as other members of the CaCA super family. These element will be discussed below.

General Structure:General Structure:

NCX_mj is a monomer, 302 residues long, composed of 10 transmembrane (TM) helices. Both protein's termini are located at the extracellular side. NCX_mj's basic structure is of two inverted, nearly identical functional halves (TM helices 1 - 5 and 6 - 10), embedded in the PM, connected by a short linker (13 residues). These two portions make the ion binding and transporting mechanism. This structural motif is common for secondary transporter proteins and is most probably the result of an earlier gene duplication and fusion events. TM helices 2 - 5 and 7 - 10 are inserted perpendicularly into the PM and are densely packed against each other creating a tight protein core flanked by conserved residues (including the α-1 and α-2 repeats) from TM helices 2, 3, 7 and 8. TM segments 1 and 6 are longer, situated further away from the protein core and are at a 45° to the plane of the PM.

Gating bundle:Gating bundle:

NCX_Mj is assumed to function in a ping pong alternative access transport mechanism as was suggested in several works done. This conclusion, along with experimental work done on solving the structure of a CAX transporter led to the following hypothesis: TMs 1 and 6 act (at least in part) as the gating bundle of NCX_Mj and quite possibly other members of CaCA superfamily. These TMs alternatively transpose, straighten/bend and rotate along their long axes (each helix along its own axis) in response to ligand binding. These changes might induce steric and/or electrical hindrances that alternatively expose / cover the ion binding sites from different sides of the membrane. How ligand binding induces these allosteric conformational changes is yet to be elucidated.

Ion binding sites:Ion binding sites:

Four possible ion binding sites are suspected to lie at the core of the protein, about halfway into the PM. These sites are arranged in a diamond shape and coordinated by residues from helices 2, 3, 7 and 8. Experimental evidence indicate that these residues are highly conserved among counterpart NCX proteins, and related NCKX proteins and that mutation of these residues in mammalian NCX orthologs leads to a loss-of-function.

Binding Sites:Binding Sites:

• S_ext - located close to the extracellular side and implicated in binding Na⁺.
• S_mid - found in the middle of the diamond and suspected to bind one water molecule. One of it's coordinating residues is suspected to be protonated.
• S_int - close to the intracellular side, believed to bind Na⁺.
• S_Ca - also situated at the middle of the diamond. This site exhibited anomalous diffraction patterns in the X-ray structure. These patterns are believed to originate from Ca²⁺ occupancy of this site in an unknown fraction of the proteins in the crystal.

Binding Site	Coordinating Residue
Sext	S77, E54, A206, T209, S210
Smid	E54, N81, E213, D240
Sint	A47, T50, S51, E213, S236
SCa	T50, E54, E213, T209

Linker:Linker:

While fairly unremarkable in the NCX_mj, this short linker (sometimes referred to as the "f-loop") between helix 5 and 6 is an important element of eukaryotic NCX proteins. This intracellular loop is of major importance for the regulation of ion transport activity. In eukaryotic NCX1 proteins this loop is much longer (~500 residues) and contains two folds defined as calcium binding domains (CBDs) connected via a very short linker (12 residues). These domains, (designated CBD1 for the N terminal domain and CBD2 for the C terminal domain) when connected as a tandem, bind calcium (and to a lesser degree, magnesium) ions and up- or downregulate sodium and calcium exchange activity.

Function:Function:

Biochemical and electrophysiological studies have concluded that NCX works through a ping pong mechanism in which one calcium and three sodium ions are sequentially translocated in separate steps instead of simultaneously across the PM. Examination of the binding sites suggested that Na⁺ ions binding have an antagonistic effect on Ca²⁺ binding and vice versa. Under physiological conditions, NCX extrudes calcium from the cell while under altered conditions (e.g. high intracellular sodium concentration or high positive membrane potential), it can work in the reverse mode as well. Experimental work done also found calcium/calcium and sodium/sodium exchanging activity although it is probably not main mode of function for the NCX_Mj.

Regulation:Regulation:

NCX is regulated by cytosolic Na⁺ and Ca²⁺ concentrations, pH, ATP and PIP₂.

3D structures of s0odium/calcium exchanger3D structures of s0odium/calcium exchanger

Updated on 23-January-2019

3v5s – MjNCX + Cd – Methanocaldococcus jannaschii
5hya – MjNCX + Na + Ca
3v5u, 5hxc, 5jdg, 5jdh – MjNCX (mutant) + Na + Ca
5hxe – MjNCX + Na
5hwx, 5hwy – MjNCX (mutant) + Na
5hxh, 5hxr, 5jdf – MjNCX (mutant) + Ca
5hxs, 5jdl – MjNCX (mutant) + Sr
5jdm, 5jdn, 5jdq – MjNCX (mutant) + Na + Sr
6bv7 – dNCX 1 THB domain 306-359 + Na – dog - NMR
2dpk – dNCX 1 Ca-binding domain 1 402-541 + Ca + guanidine
2fws – dNCX 1 Ca-binding domain 1 + Ca - NMR
3gin – dNCX 1 Ca-binding domain 1 (mutant)
2qvk, 2qvm – dNCX 1 Ca-binding domain 2 533-724 + Ca
2kls, 2klt – dNCX 1 Ca-binding domain 2 - NMR
2fws – dNCX 1 Ca-binding domain 2 + Ca - NMR
3us9 – dNCX 1 Ca-binding domain 1+2 (mutant) + Ca