Na + -K + -ATPase (NKA) establishes the transmembrane [Na + ] gradient in cells. In heart, phospholemman (PLM) inhibits NKA activity by reducing its apparent Na + affinity, an effect that is relieved by PLM phosphorylation. The NKA crystal structure suggests regions of PLM-NKA interaction, but the sites important for functional effects in live cells are not known. We tested wild type (WT) and CFP-NKA-α1 point mutants (alanine substitution at F956, E960, L964, and F967) for fluorescence resonance energy transfer (FRET) with WT-PLM-YFP in HEK293 cells. NKA-PLM FRET was unaltered with F956A or F967A, reduced with L964A, and nearly abolished with E960A. Mutating the PLM site (F28A) identified by structural analysis to interact with E960-NKA also nearly abolished NKA-PLM FRET. In contrast, NKA-PLM coimmunoprecipitation was only slightly reduced by E960A-NKA or F28A-PLM mutants, consistent with an additional interaction site. FRET titrations indicate that the additional site has higher affinity than that between E960-NKA and F28-PLM. To test whether the FRET-preventing mutations also prevent PLM functional effects, we measured NKA-mediated Na + -transport in intact cells. For WT-NKA, PLM reduced apparent Na + -affinity of NKA and PLM phosphorylation reversed the effect. In contrast, for E960A-NKA the apparent Na + -affinity was unaltered by either PLM or forskolin-induced PLM phosphorylation. We conclude that E960 on NKA and F28 on PLM are critical for PLM effects on both NKA function and NKA-PLM FRET, but also there is at least one additional site that is critical for tethering PLM to NKA.is critical for electrical excitability and coupled transport. In heart, [Na + ] i closely regulates intracellular Ca 2+ , contraction, and rhythmicity via Na + /Ca 2+ exchange (1, 2). Small changes in [Na + ] i can have major effects on both [Ca 2+ ] i and intracellular pH (via Na + /H + exchange) (2). Therefore, [Na + ] i regulation is very important for understanding basic ion homeostatic mechanisms.There are several Na + entry pathways, whereas the Na + /K + pump (NKA) is the main Na + extrusion pathway (2). NKA is a ubiquitous transmembrane protein that establishes and maintains [Na + ] and [K + ] gradients across the plasma membrane. These gradients ensure osmotic balance, resting membrane potential, and cellular excitability. NKA uses energy derived from hydrolysis of ATP to extrude three Na + ions in exchange for two K + ions.Phospholemman (PLM), a 72-amino acid sarcolemmal protein, is a member of the FXYD protein family, which derives its name from the conserved Phe-X-Tyr-Asp motif in the proximal extracellular domain. FXYDs are tissue-specific NKA regulators that bind to and modulate NKA function by affecting the apparent affinity for internal Na + or external K + (3-5). The [Na + ] i for halfmaximal NKA activation (K 0.5 ) in the heart varies with internal and external ionic conditions and is typically 8-22 mM. This is near the resting [Na + ] i in most cells (6). PLM (FXYD1) is highly expressed in heart, brain, and skele...

Currently, the role of water and the membrane assembly in tuning the function of seven-helical transmembrane (7TM) proteins is not well-understood. Here, we focus on the light activation and functional properties of a prototypical example, the Proteorhodopsin (PR) proton pump from marine bacteria, observing how the protein and surrounding hydration water rearrange upon activation. This is made possible by the application of the powerful residue-specific magnetic resonance methods of electron paramagnetic resonance (EPR), which measures protein segment mobility, and Overhauser dynamic nuclear polarization (ODNP), as recently developed for probing local water diffusivity within 10 Å of a nitroxide spin-label. We investigate further how these dynamics are affected by the surrounding environment, encompassing both protein-protein and protein-lipid interactions. With these techniques together with optical absorption spectroscopy, we find that water dynamics (both ps scale translational motion and ns scale ''bound'' water) at the membrane protein surface is dramatically affected by the lipid bilayer or surfactant micelle environment. Furthermore, hydration is correlated to functional changes such that water could modulate the timescale of conformational motion. Specifically, the slowdown of translational water motion at the membrane surface, coupled to a lack of bound waters, may facilitate proton uptake by PR. The association of PR with other PR molecules within the membrane, or oligomerization, has similar functional consequences in addition to effects on the protonation properties of key residues for ion transport (pKa of D97). The implication of our study is that PR-PR association alters the hydrogen bond network within the channel, possibly mediated by an altered interaction with the surfactant and surface hydration water upon oligomerization. This result, combined with the homology of PR with sensory receptors, elicits the intriguing possibility that PR has a functional flexibility mediated by oligomerization.

of Ca 2þ , dissociation of CaM from C28 occurs by a pathway in which Trp 1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp 1093, followed by very slow release of Phe 1110, removal of C28 and return of CaM to its conformation in the free state. The intermediate of dissociation with exposed Trp 1093 has a long lifetime (minutes), and is an activated form of PMCA. This mechanism may explain some unique biological properties of PMCA4b.