Intramolecular Interactions of the Regulatory Region with the Catalytic Core in the Plasma Membrane Calcium Pump

Isoform 2b of the plasma membrane calcium pump differs from the ubiquitous isoform 4b in the following: (a) higher basal activity in the absence of calmodulin; (b) higher affinity for calmodulin; and (c) higher affinity for Ca2+ in the presence of calmodulin [Elwess, Filoteo, Enyedi and Penniston (1997) J. Biol. Chem. 272, 17981–17986]. To investigate which parts of the molecule determine these kinetic differences, we made four chimaeric constructs in which portions of isoform 2b were grafted into isoform 4b: chimaera I contains only the C-terminal regulatory region of isoform 2b; chimaera II contains the N-terminal moiety of isoform 2b, including both cytoplasmic loops; chimaera III contains the sequence of isoform 2b starting from the N-terminus to after the end of the first (small) cytoplasmic loop; and chimaera IV contains only the second (large) cytoplasmic loop. Surprisingly, chimaera I showed low basal activity in the absence of calmodulin and low affinity for calmodulin, unlike isoform 2b. In contrast, the chimaera containing both loops showed high basal activity, and Ca2+ activation curves (both in the absence and in the presence of calmodulin) similar to those of isoform 2b. The rates of activation by calmodulin and of inactivation by calmodulin removal were measured, and the apparent Kd for calmodulin was calculated from the ratio between these rate constants. The order of affinity was: 2b = II>4b = IV>III = I. From these results it is clear that the construct that most closely resembles isoform 2b is chimaera II. This shows that, in order to obtain an enzyme with properties similar to those of isoform 2b, both cytoplasmic loops are needed.

Download Full-text

Modulation of the Human Erythroid Plasma Membrane Calcium Pump (PMCA4b) Expression by Polymorphic Genetic Variants

Membranes ◽

10.3390/membranes11080586 ◽

2021 ◽

Vol 11 (8) ◽

pp. 586

Author(s):

Orsolya Mózner ◽

Boglárka Zámbó ◽

Balázs Sarkadi

Keyword(s):

Plasma Membrane ◽

Regulatory Region ◽

Mechanistic Explanation ◽

Calcium Pump ◽

Luciferase Reporter ◽

Erythroid Cells ◽

Plasma Membrane Calcium Pump ◽

Gene Coding ◽

Reporter Assays ◽

A Minor

In the human ATP2B4 gene, coding for the plasma membrane calcium pump PMCA4b, a minor haplotype results in the decreased expression of this membrane protein in erythroid cells. The presence of this haplotype and the consequently reduced PMCA4b expression have been suggested to affect red blood cell hydration and malaria susceptibility. By using dual-luciferase reporter assays, we have localized the erythroid-specific regulatory region within the haplotype of the ATP2B4 gene, containing predicted GATA1 binding sites that are affected by SNPs in the minor haplotype. Our results show that, in human erythroid cells, the regulation of ATP2B4 gene expression is significantly affected by GATA1 expression, and we document the role of specific SNPs involved in predicted GATA1 binding. Our findings provide a mechanistic explanation at the molecular level for the reduced erythroid-specific PMCA4b expression in carriers of ATP2B4 gene polymorphic variants.

Download Full-text

Vitamin D, the Intestinal Plasma Membrane Calcium Pump Gene Expression and the Calbindins

Vitamin D ◽

10.1515/9783110882513-124 ◽

1994 ◽

pp. 376-384

Keyword(s):

Gene Expression ◽

Vitamin D ◽

Plasma Membrane ◽

Calcium Pump ◽

Plasma Membrane Calcium Pump

Download Full-text

Positive charge modifications alter the ability of XIP to inhibit the plasma membrane calcium pump

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.3.c736 ◽

1996 ◽

Vol 271 (3) ◽

pp. C736-C741 ◽

Cited By ~ 22

Author(s):

W. Xu ◽

C. Gatto ◽

M. A. Milanick

Keyword(s):

Plasma Membrane ◽

Positive Charge ◽

Calcium Pump ◽

Inhibitory Peptide ◽

Ca Pump ◽

Contact Points ◽

Plasma Membrane Calcium Pump ◽

Peptide Analogues ◽

Positively Charged

Exchange inhibitory peptide (XIP; RRLLFYKYVYKRYRAGKQRG) is the shortest peptide that inhibits the plasma membrane Ca pump at high Ca (A. Enyedi, T. Vorherr, P. James, D. J. McCormick, A. G. Filoteo, E. Carafoli, and J. T. Penniston, J. Biol. Chem. 264: 12313-12321, 1989). Sulfosuccinimidyl acetate (SNA)-modified XIP does not inhibit the Ca pump; SNA neutralizes the positive charge on Lys at positions 7, 11, and 17. Peptide 2CK-XIP (RRLLFYRYVYRCYCAGRQKG) inhibits the pump, but the iodoacetamido-modified peptide does not inhibit. Three peptide analogues, in which 7, 11, and 17 were Ala, Cys, or Lys, inhibited about as well as XIP. SNA modification of these analogues (each with 1 Lys) did not inhibit. SNA modification of 2CK-XIP results in a peptide that does not inhibit; thus position 19 is important. Our results suggest that it is critical that position 19 be positively charged, that positions 7, 11, and 17 are important contact points between XIP and the Ca pump (with at least one positively charged), and that, whereas it is not essential that residues 12 and 14 be positive, they cannot be negative.

Download Full-text