Transmembrane Helix 11 Is a Genuine Regulator of the Endoplasmic Reticulum Ca2+ Pump and Acts as a Functional Parallel of β-Subunit on α-Na+,K+-ATPase

Abstract

Background:The ubiquitous sarco/endoplasmic reticulum Ca 2ϩ ATPase SERCA2b has a C-terminal extension that increases the Ca 2ϩ affinity. It consists of a transmembrane helix (TM11) and a luminal extension. Results: Both parts control Ca 2ϩ affinity independently. Conclusion: TM11 is an independent and highly conserved functional region of SERCA2b. Significance: This study shows that TM11 acts as a genuine regulator of the Ca 2ϩ pump.

Regulation of the Sarcoplasmic Reticulum Calcium Pump by Divergent Phospholamban Isoforms in Zebrafish

“…Compared with human PLN, the zfMID chimera was a super-inhibitor of SERCA (K Ca values of 0.88 and 1.1 M calcium, respectively), while maintaining the characteristic increase in maximal activity (Table 1). Finally, the Cys 46 to Tyr mutation had a small effect on the ability of human PLN to alter the apparent calcium affinity (K Ca values of 0.89 and 0.88 M, respectively) and maximal activity of SERCA (V max values of 6.6 and 6.1 mol mg Ϫ1 min Ϫ1 , respectively). However, the Cys 46 to Tyr substitution resulted in a monomeric variant of human PLN by SDS-PAGE (data not shown), despite the fact that both human and zebrafish PLN can form pentamers.…”

“…Finally, the Cys 46 to Tyr mutation had a small effect on the ability of human PLN to alter the apparent calcium affinity (K Ca values of 0.89 and 0.88 M, respectively) and maximal activity of SERCA (V max values of 6.6 and 6.1 mol mg Ϫ1 min Ϫ1 , respectively). However, the Cys 46 to Tyr substitution resulted in a monomeric variant of human PLN by SDS-PAGE (data not shown), despite the fact that both human and zebrafish PLN can form pentamers. 53 -PheHis-Gly-Met 57 ), which is lacking in all other known PLN sequences.…”

“…We first focused on the cytoplasmic domain chimeras (zfHEAD and zfMID) and a single residue change in the trans-membrane domain (Cys 46 to Tyr; Fig. 4 and Table 1).…”

“…6). In co-reconstituted proteoliposomes with SERCA, these two chimeras resulted in only minor changes in the apparent calcium affinity and maximal 46 to Tyr substitution; zfPLN(Ϫ5), zfPLN lacking last five residues; zfPLN(Ϫ6), zfPLN lacking last six residues; hPLN(ϩ5), hPLN chimera containing the last five residues of zfPLN; hPLN(ϩ6), hPLN chimera containing the last six residues of zfPLN; zfPLN-SLN tail , zfPLN chimera containing the last five residues of SLN; F54A, Phe 54 to Ala mutant of zfPLN; H55A, His 55 to Ala mutant of zfPLN; F54A,H55A, double mutant. For statistical analysis, between subjects one-way analysis of variance followed by the Holm-Sidak test was used for pairwise comparisons against SERCA in the absence and presence of wild-type human PLN.…”

“…Mutagenesis of the Zebrafish Luminal Domain-Aromatic residues play a key role in regulating SERCA from the luminal side of the SR membrane, as observed for the luminal domain of SLN (4,8,44) and the C-terminal tail of the ubiquitous SERCA2b splice variant (45,46). For this reason, we focused on the role of Phe 54 and His 55 in the luminal tail of zfPLN.…”