Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA107

Poster Communications

A new mode of binding between Calmodulin and the human Na+/H+ exchanger SLC9A1 with implications for the SLC9A1 function

L. M. Sjøgaard1, A. Prestel1, E. Pedraz-Cuesta1, J. G. Olsen1, E. S. Pedersen1, B. B. Kragelund1, S. F. Pedersen1

1. Department of Biology, University of Copenhagen, Copenhagen Ø., Denmark.

The plasma membrane Na+/H+ exchanger NHE1 is a ubiquitously expressed ion transporter with essential physiological roles. It functions as a dimeric hub protein with many binding partners, one of which is the calcium-binding protein calmodulin (CaM) (1). CaM is an important regulator of NHE1 activity (2) and is shown to bind two consecutive sites in the NHE1 C-terminal tail (1,3). The aim of the present work was to delineate the structural and functional roles of CaM for NHE1. For in vitro analyses, we created two NHE1 peptides: CaM Site1 (residues 622-657) and Site1+2 (622-692). Using circular dichroism (CD) spectroscopy to monitor thermal unfolding, we showed that the N-lobe of Ca2+-saturated CaM was stabilized by both peptides (Tm, CaM N-lobe alone = 44±2°C, Tm,Site1 = 77±1°C, Tm,Site1+2 = 82±2°C) (±SEM, n=1). Isothermal titration calorimetry (ITC) showed a CaM:peptide stoichiometry in solution of 1:2 (n=2) for the Site1 peptide. HSQC NMR spectra of 15N-labeled CaM titrated with unlabeled Site1 peptide demonstrated that the peptide initially bound the CaM C-lobe then the CaM N-lobe (n=1). Notably, addition of the Site1+2 peptide showed NMR peak saturation at a 1:2 stoichiometry of CaM:peptide similarly to Site1 spectra peaks. Importantly, this demonstrates that CaM can also bind two molecules of NHE1 in vitro. Two phosphorylation sites, S648 and T653, are located in the sequence covered by the Site1 peptide and have been assigned NHE1 regulatory roles. Exploiting the lack of endogenous NHE1 in the cell line PS120 (4), we generated six cell lines expressing WT NHE1 or mutant variants; 1K3R1D3E (K641D, R643E, R645E, R647E) and 1Q2R3E (Q640E, R636E, R632E) to disrupt CaM binding, and S648A, S648D and T653A to inhibit or mimic phosphorylation at these sites. BCECF-AM was used to measure intracellular pH in cells bathed in Ringer solution pH 7.4, followed by acidification using the NH4Cl prepulse technique. Untransfected cells were used as control. Preliminary results showed that WT NHE1 cells had the fastest rate of recovery (28.4±0.1*10-3 pH/s) followed by the S648D (13.8±0.1*10-3 pH/s), the S648A (12.3±0.2*10-3 pH/s) and lastly the 1K3R1D3E (6.8±0.1*10-3 pH/s) (±SEM, n=4). This suggested that CaM-binding is necessary for NHE1 activity and that substitution of S648 to Ala or Asp is important too, but less so. Ongoing work investigates the kinases involved. In conclusion, we characterize a new mode of binding between CaM and NHE1 with a stoichiometry of 1:2 and potential implications for Ca2+ mediated regulation of NHE1 dimerization. Ongoing systematic work on the functional importance of CaM binding and phosphorylations around CaM binding site 1 in NHE1 will expand the current understanding of the physiological relevance of Ca2+/CaM signaling for NHE1.

Where applicable, experiments conform with Society ethical requirements