Plasma Membrane Ca2+ Pump PMCA4z Is More Active Than Splicing Variant PMCA4x.

Gerardo R Corradi,Lucia Rochi,Luciana R Mazzitelli,Guido D Petrovich,Felicitas De Tezanos Pinto,Hugo P Adamo

doi:10.3389/fncel.2021.668371

Abstract

The plasma membrane Ca2+ pumps (PMCA) are P-ATPases that control Ca2+ signaling and homeostasis by transporting Ca2+ out of the eukaryotic cell. Humans have four genes that code for PMCA isoforms (PMCA1-4). A large diversity of PMCA isoforms is generated by alternative mRNA splicing at sites A and C. The different PMCA isoforms are expressed in a cell-type and developmental-specific manner and exhibit differential sensitivity to a great number of regulatory mechanisms. PMCA4 has two A splice variants, the forms “x” and “z”. While PMCA4x is ubiquitously expressed and relatively well-studied, PMCA4z is less characterized and its expression is restricted to some tissues such as the brain and heart muscle. PMCA4z lacks a stretch of 12 amino acids in the so-called A-M3 linker, a conformation-sensitive region of the molecule connecting the actuator domain (A) with the third transmembrane segment (M3). We expressed in yeast PMCA4 variants “x” and “z”, maintaining constant the most frequent splice variant “b” at the C-terminal end, and obtained purified preparations of both proteins. In the basal autoinhibited state, PMCA4zb showed a higher ATPase activity and a higher apparent Ca2+ affinity than PMCA4xb. Both isoforms were stimulated by calmodulin but PMCA4zb was more strongly activated by acidic lipids than PMCA4xb. The results indicate that a PMCA4 intrinsically more active and more responsive to acidic lipids is produced by the variant “z” of the splicing site A.

Highlights

The Ca2+ transporting P-ATPases known as Ca2+ pumps are key elements for the control of intracellular Ca2+ homeostasis (Chen et al, 2020)
K616 cells cannot proliferate in media containing low concentrations of Ca2+ (10 mM EGTA), but this phenotype can be rescued by the expression of a high-affinity Ca2+ transport system
We have previously shown that the expression of PMCA4xb does not allow the growth of K616 cells in Ca2+ depleted media and successful complementation requires the expression of hyperactive forms of the protein, in which the autoinhibitory mechanism has been disrupted Activated versions of PMCA4xb can be produced either by introducing mutations in the A domain or by the removal of the C-terminal autoinhibitory segment, as in the case of the mutant CT120 lacking the C-terminal 120 amino acid residues (Enyedi et al, 1993; Bredeston and Adamo, 2004; Mazzitelli and Adamo, 2014)

Summary

Introduction

The Ca2+ transporting P-ATPases known as Ca2+ pumps are key elements for the control of intracellular Ca2+ homeostasis (Chen et al, 2020). The relative contribution of the PMCA to these processes depends on the cell type and the activity of other Ca2+ transporters. The SERCA pump and the plasma membrane Na+/Ca2+ exchanger (NCX) are mostly responsible for removing Ca2+ from the cytosol, while the function of PMCA seems more suited for the control of Ca2+ dependent microdomain processes (Strehler, 2015). Consistent with this idea, in excitable cells, PMCA is a PMCA4z Splicing Variant part of protein networks that by including voltage-gated Ca2+ channels, couple influx, and clearance of Ca2+ (Müller et al, 2010). The loss of PMCA function has been associated with several human diseases, including neurologic pathologies, such as familial spastic paraplegia (Mohamed et al, 2011, 2016; Li et al, 2014)

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Conclusion