Phospholemman Transmembrane Structure Reveals Potential Interactions with Na+/K+-ATPase

Abstract

Phospholemman (PLM) is a 72-residue bitopic cardiac transmembrane protein, which acts as a modulator of the Na ؉ /K ؉ -ATPase and the Na ؉ /Ca 2؉ exchanger and possibly forms taurine channels in nonheart tissue. This work presents a high resolution structural model obtained from a combination of site-specific infrared spectroscopy and experimentally constrained high throughput molecular dynamics (MD) simulations. Altogether, 37 experimental constraints, including nine long range orientational constraints, have been used during MD simulations in an explicit lipid bilayer/water system. The resulting tetrameric ␣-helical bundle has an average helix tilt of 7.3°a nd a crossing angle close to 0°. It does not reveal a hydrophilic pore, but instead strong interactions between various residues occlude any pore. The helix-helix packing is unusual, with Gly Human phospholemman (PLM)2 is a member of a family of single-span transmembrane proteins characterized by the invariant extracellular motif FXYD (1) and is also known as FXYD1. It is found in liver, skeletal muscle, and most abundantly in the cardiac sarcolemma (2). Over recent years, conclusive experimental evidence has been presented that PLM acts as a tissue-specific modulator of the Na ϩ /K ϩ -ATPase similar to the other members of the FXYD family, each of which is prevalent in a different tissue, summarized in several recent reviews (3, 4). The Na ϩ /K ϩ -ATPase consists of a catalytic ␣-subunit with 10 transmembrane segments and a ␤-subunit involved in membrane insertion and as a modulator of transport properties (5). In particular, cross-linking and modeling studies revealed the direct interaction between the Na ϩ /K ϩ -ATPase ␣-subunit and members of the FXYD family (␥ and CHIF), which involves the binding of a single transmembrane domain into a groove formed by M2, M6, and M9 transmembrane helices of the Na ϩ /K ϩ -ATPase (6, 7). PLM appears to be a major control point in the function of heart cells, although its specific physiological role is unclear, complicated by the possibility that its regulatory effect on the Na ϩ /K ϩ -ATPase may depend on the phosphorylation state of PLM (8) as well as other less well established functional roles (e.g. interaction with the Na/Ca 2ϩ exchanger (9, 10) or independent channel formation). PLM knock-out mice expressed a complex response, including increased cardiac mass, larger cardiomyocytes, and ejection fractions in the absence of hypertension, whereas the overall Na ϩ /K ϩ -ATPase activity was reduced by 50% (11). There is growing evidence that PLM regulates the activity of Na/Ca 2ϩ exchanger 1 (NCX1) from heterologous expression studies of PLM and NCX1 in HEK239 cells (10) and overexpression of PLM in rat cardiomyocytes (12, 13) using different approaches to measure NCX1 activity. It appears that phosphorylation of PLM abolishes its inhibitory effect on the Na ϩ /K ϩ -ATPase (14, 15), whereas the phosphorylated form of PLM inhibits NCX1 (9, 12).Electrical measurements of PLM in artificial lipid bilayers and oocytes sh...

Phosphomimetic Mutations Enhance Oligomerization of Phospholemman and Modulate Its Interaction with the Na/K-ATPase

“…The data are compatible with a mixture of monomers and tetramers. That the major oligomeric species is a tetramer was previously suggested by in vitro experiments of another group (15,16,26) and is consistent with our own observation (in FRET experiments) that the oligomer size is three or more protomers (18). We found that the concentration of tetramers increased with average fluorescence intensity (Fig.…”

“…For the PLM oligomer, the average donor-acceptor separation distance was calculated from FRET max using a computational model of intra-oligomeric FRET (25) as described previously (20,23). This model assumes a ring-shaped arrangement of subunits, which we regard as the most likely geometry (26). For values of R Ն R 0 , FRET efficiency is strongly dependent on donor-acceptor separation distance and only weakly dependent on the number of subunits in the complex (25).…”

Swelling-Induced Taurine Transport: Relationship with Chloride Channels, Anion-Exchangers and Other Swelling-Activated Transport Pathways

“…The structure of the protein would, at first sight, appear to favour the latter suggestion. However, a recent report suggests that phospholemman tetramers have the potential to form a hydrophilic pore which could accommodate taurine [118]. It can be predicted, on the basis of evidence gathered from functional investigations, that molecular studies could eventually reveal the existence of more than one system for osmosensitive taurine transport.…”

Phospholemman, a major regulator of skeletal muscle Na+/K+-ATPase, is not mutated in probands with hypokalemic periodic paralysis

“…FXYD1 is highly expressed in skeletal muscle and has been demonstrated to have an important influence on sodium pump function (28)(29)(30). Studying the relationship between FXYD1 and HypoPP may yield an improved understanding of the mechanism of the disease.…”