Cytosolic Ca2+ Buffers Are Inherently Ca2+ Signal Modulators

Abstract

For precisely regulating intracellular Ca²⁺ signals in a time- and space-dependent manner, cells make use of various components of the "Ca²⁺ signaling toolkit," including Ca²⁺ entry and Ca²⁺ extrusion systems. A class of cytosolic Ca²⁺-binding proteins termed Ca²⁺ buffers serves as modulators of such, mostly short-lived Ca²⁺ signals. Prototypical Ca²⁺ buffers include parvalbumins (α and β isoforms), calbindin-D9k, calbindin-D28k, and calretinin. Although initially considered to function as pure Ca²⁺ buffers, that is, as intracellular Ca²⁺ signal modulators controlling the shape (amplitude, decay, spread) of Ca²⁺ signals, evidence has accumulated that calbindin-D28k and calretinin have additional Ca²⁺ sensor functions. These other functions are brought about by direct interactions with target proteins, thereby modulating their targets' function/activity. Dysregulation of Ca²⁺ buffer expression is associated with several neurologic/neurodevelopmental disorders including autism spectrum disorder (ASD) and schizophrenia. In some cases, the presence of these proteins is presumed to confer a neuroprotective effect, as evidenced in animal models of Parkinson's or Alzheimer's disease.

Figure 1.

Conserved amino acids in the Ca²⁺-binding loop and structure of selected EF-hand proteins. (A) Consensus sequence of the canonical EF-hand Ca²⁺-binding loop of 12 amino acids. Amino acids X, Y, Z, and –Z provide side-chain oxygen ligands, * provides the backbone carbonyl oxygen, and at –X a water molecule is hydrogen-bonded to a loop residue. Amino acids most often present at a given position are shown below and shaded residues are the most conserved ones (Marsden et al. 1990). At positions X and –Z, Asp (D) and Glu (E) are generally present, respectively. The seven oxygen ligands coordinating the Ca²⁺ ion are located at the seven corners of a pentagonal bipyramid and the Ca²⁺ ion (not shown) is in the center (right). (B) Solution structure of Ca²⁺-bound human α parvalbumin (PV) (PDB: 1RJV). Both, the CD domain (green) and EF domain (yellow/red) bind one Ca²⁺ ion each (green spheres) in canonical Ca²⁺-binding loops of 12 amino acids. The orthogonally oriented helices E and F (gray-shaded) are connected by the Ca²⁺-binding loop. Both Ca²⁺-binding sites in PV are of the Ca²⁺/Mg²⁺ mixed type. The amino-terminal AB domain (blue) is necessary for protein stability. (C) Nuclear magnetic resonance (NMR) solution structure of bovine CB-D9k (PDB: 1B1G). The shown structure takes into account the Ca²⁺ ions and explicit solvent molecules. The amino-terminal domain EF1 is a pseudo (Ψ) EF-hand with a larger loop of 14 amino acids, whereas the second domain (EF2) has a canonical Ca²⁺-binding loop of 12 amino acids. In both loops, the Glu residue at the position –Z with the two carboxyl oxygen atoms (red) serves as a bidentate ligand representing two corners of the pentagonal bipyramid. This residue, most often Glu (rarely Asp), is a critical determinant for the Ca²⁺ affinity of the entire loop; Ca²⁺ ions are shown as green spheres. The two Ca²⁺-binding loops are in close proximity and stabilized via short β-type interactions (gray-shaded area). (D) 3D NMR structure of CB-D28k (PDB: 2G9B). CB-D28k has a relatively compact structure comprising three Ca²⁺-binding units, each unit consisting of a pair of EF-hands. Ca²⁺-binding is restricted to the Ca²⁺-binding loop 1 in the amino-terminal unit (blue), to both loops 3 and 4 in the middle unit (green) and to loop 5 in the carboxy-terminal pair (yellow/red). EF-hands 2 and 6 are nonfunctional with respect to Ca²⁺-binding. The Ca²⁺-binding loops flanked by two almost perpendicular α-helical regions are numbered from 1 to 6. (Images B–D were generated with PDB ProteinWorkshop 1.50; Moreland et al. 2005. From Schwaller 2010; reprinted, with permission, from the author.)

Figure 2.

Comparison of mechanisms hypothesized to lead to PV down-regulation and Pvalb neuron impairment in autism spectrum disorder (ASD) (light blue) and schizophrenia (violet). Both in ASD and schizophrenia genetic (G, orange) and environmental (E, green) factors are likely involved. Identified human ASD risk genes and mouse models based on these risk genes are listed. Full names of the proteins encoded by these ASD- and schizophrenia-associated genes and additional details are found in the literature. G factors, E factors, and/or G x E interactions disturb normal neurodevelopment evidenced as excitation/inhibition (E/I) imbalance, impaired synaptic transmission, as well as hyper-/hypoconnectivity of short- or long-range neuronal circuits (yellow box). This is assumed to result in PV down-regulation (red box). In both disorders, PV down-regulation is proposed as the hub or point-of-convergence. The differences lie in the different kinetics of PV down-regulation. A decrease in PV expression during early neurodevelopment, that is, detectable already at PND25 in mice, as shown for mice deficient for PV, SHANK1 and 3 (Filice et al. 2016), CNTNAP2 (Lauber et al. 2018), and VPA mice (Lauber et al. 2016) is correlated with an ASD-like phenotype previously observed in mice or in the case of VPA in rats (Schneider et al. 2008; Peça et al. 2011; Peñagarikano et al. 2011; Wöhr et al. 2011, 2015). In mouse schizophrenia models, in which a phenotype is most often apparent in adult mice (>3 months old), it is hypothesized that a slower PV down-regulation leads to a compensatory/adaptive increase in mitochondria volume and subsequently to augmented ROS production in Pvalb neurons impairing their function. This Pvalb neuron dysfunction will precipitate at the behavioral level, as a schizophrenia-like phenotype. The precise order of events, that is, PV down-regulation-mediated increased oxidative stress leading to Pvalb neuron dysfunction or oxidative stress-mediated Pvalb neuron impairment leading to PV down-regulation is currently unknown and under investigation (adapted from data in Steullet et al. 2016, 2017b).