Figure 1.
The stimulus-response relationship between medium osmolarity and NHE1 Na+ transport activity in atRBCs.
Unidirectional Na+ influx rates (22Na+ initial rates) were measured in hyperosmotic media at a fixed media [Na+] = 100 mM, following complete activation of NHE1 by pre-incubation in thermodynamically nulled media of matched osmolarity. The data are fit to a sigmoidal curve by non-linear regression (Hill slope = 4.1), yielding a maximal Na+ influx rate of 13.2 mmoles Na+ kg−1 dcs minute. (data are n≥5 each; ± SE).
Figure 2.
Na+ transport kinetics of NHE1 in hyperosmotic medium.
Michaelis-Menten Na+ transport kinetics were determined for NHE1 in atRBCs following complete activation in hyperosmotic (1.6×IR) media, with (closed triangles) or without (open triangles) 500 nM CLA (data are compiled from n≥3 independent experiments; means ± SE). The data are fit by regression to a simple hyperbola, and the corresponding kinetic constants are reported in Table 1. The Na+ transport kinetics (activity) are virtually identical in the two conditions.
Table 1.
Na+ transport kinetic constants for NHE1 in Amphiuma RBCs maximally stimulated to steady-state in hyperosmotic (1.6×IR) media with or without 500 nM CLA treatment.
Figure 3.
Na+ transport kinetics of NHE1 in mildly hyperosmotic medium.
The Michaelis-Menten Na+ transport kinetics were determined for NHE1 in atRBCs following complete activation in mildly hyperosmotic (1.2×IR) medium alone (closed triangles), hyperosmotic (1.2×IR) medium with 500 nM CLA (open triangles), or CLA treatment alone in IR (squares). Data for resting atRBCs in isosmotic medium are shown for comparison (small squares, dashed curve). The data are fit by regression to a simple hyperbola, and the corresponding kinetic constants are reported in Table 2. The Na+ transport affinity is not significantly different across the three conditions. However, the maximal transport rate Jmax is significantly increased in hyperosmotic media together with CLA treatment, relative to either treatment alone (p<0.05). (data are compiled from n≥3 independent experiments; means ± SE).
Table 2.
Na+ transport kinetic constants for NHE1 in Amphiuma RBCs stimulated to steady-state in isosmotic solution with 500 nM CLA, or mildly hyperosmotic (1.2×IR) solution ± CLA.
Figure 4.
In situ [32P]-orthophosphate labeling of NHE1 during cell suspension in hyperosmotic medium.
(upper) 32P incorporation into immunoprecipitated NHE1 from atRBCs. autoradiograph images corresponding to immunoprecipitated NHE1 from membrane fractions of [32P]-orthophosphate labeled atRBCs suspended in isosmotic medium, or hyperosmotic media (n1.6×IR) with or without CLA treatment. Below each autoradiograph band is the corresponding NHE1 Western blot band detected on the same PVDF membrane. (lower) A quantitative comparison of data from the autoradiograph bands described in panel A (normalized to NHE1 Western blots from the same PVDF membrane). Relative phosphorylation of immunoprecipitated NHE1 is significantly increased by suspension of cells in hyperosmotic (n1.6×IR) solution with or without CLA treatment relative to the isosmotic control (*p<0.05 , **p<0.01; n = 6±SEM). No significant difference is detected between the two treatment conditions (hyperosmotic ± CLA; n.s.).
Figure 5.
Ion Trap MS/MS spectrum of a fragmented peptide containing phosphorylated serine 711 of atNHE1.
NHE1 was immunoprecipitated from atRBC membranes, SDS-PAGE purified and in-gel digested with trypsin. The resulting peptides were extracted an analyzed by LC-MS/MS. MS/MS spectra were generated by collision-induced dissociation of individual peptides, which preferentially fragments peptides at peptide bonds to generate N-terminal (b ions) and C-terminal (y ions) fragments with characteristic charge/mass (m/z) ratios identifying the amino acid composition of the collection of fragments. In this representative spectrum, the (2+) y ions are labeled in red and positioned above the b ions labeled in blue. Phosphorylation of serine 711 is shown as a gain of 80-kDa corresponding to H3PO4. MS/MS spectra identifying other sites of NHE1 phosphorylation are on file in the Tranche database at ProteomeCommons.org (see Methods).
Table 3.
NHE1 phosphorylation sites detected in situ in atRBCs by LC-MS/MS.
Figure 6.
Comparison of C-terminal amino acid consensus sequence and phosphorylation sites in human NHE1 versus Amphiuma NHE1.
Human NHE1, Amphiuma NHE1 and consensus sequences are shown for regions of the cytosolic C-terminus that contain phosphorylation sites identified by LC-MS/MS. A. The distal C-terminus. Shown in bold with asterisks are the locations of 4 conserved phosphorylated serine residues within this region: (Amphiuma) S701, S711, S783 and S794. T727, a site unique to Amphiuma NHE1, is also shown in bold. Residues with similarity are noted with + in the consensus sequence. B. The proximal C-terminus. Three conserved phosphorylation sites within this region are shown in bold with asterisks: (Amphiuma) S607, S610 and S613. C. Comparison of sequence information and phosphorylation sites of the volume-sensitive calmodulin (CaM) binding region in human versus Amphiuma NHE1. The helical domains necessary for CaM binding shown (α1 and α2) are based on structural studies by Köster et al [42], indicating CaM binding domains that are necessary for cell shrinkage-induced transport activity [41]. This region is 97% identical between human and Amphiuma NHE1. Shown in bold with an asterisk is the location of (Amphiuma) threonine 693, a conserved phosphorylation site within this region. The location of human S648 is also noted (#). Positive and negatively charged residues within the CaM binding regions are denoted below with + or −.