Sodium ion internalized within phospholipid membranes

Article abstract of DOI:10.1021/ja065702o

Seven phospholipids, modified with ester groups in their hydrophobic chains, were synthesized and examined for their ability to promote sodium ion flux across vesicular membranes. It was found by ²³Na NMR that only the phospholipids having short chain segments beyond their terminal ester groups catalyze sodium ion transfer by up to 2 orders of magnitude relative to a conventional phospholipid, POPC. The rates increase with the concentration of the ester-phospholipid admixed with POPC in the bilayer. More surprisingly, the rates increase with the time allowed for the vesicles to age. This was attributed to ester-phospholipid migrating in the bilayers to form domains that solubilize the sodium ion within the hydrocarbon interior of the membrane. Such membrane domains explain why shift reagent-modified NMR spectra display three ²³Na signals representing sodium outside the vesicles, sodium within the vesicular water pools, and sodium within the membranes themselves. Copyright

Full text of DOI:10.1021/ja065702o

Published on Web 10/06/2006
Sodium Ion Internalized within Phospholipid Membranes
Fredric M. Menger,* Ashley L. Galloway, Mary E. Chlebowski, and Shaoxing Wu
Department of Chemistry, Emory UniVersity, Atlanta, Georgia 30322
Received August 21, 2006; E-mail: menger@emory.edu
Ion passage through phospholipid membranes is a venerable1-6
and honored7,8 area of bio-organic/biophysical chemistry. Briefly,
ions can pass through phospholipid bilayers via channel compounds
(e.g., gramicidin A, amphotericin B, and blepharismin 19) or via
ion carriers (e.g., valinomycin and monensin). Transport occurs
either along diffusion gradients (“passive”) or against diffusion
gradients (“active”), the latter requiring energy input. In previous
work, mobile ions were present on both sides of the membrane,
but they have never been detected, that we know of, within the
membrane itself. This is not surprising because the interior of a
phospholipid membrane is comprised of hydrocarbon chainsshardly
a propitious environment for ionic species. Indeed, it is this very
environment that provides the barrier to ion flux. In the present
communication, we provide 23Na NMR evidence that Na+ can
locate itself inside the actual phospholipid bilayer. To achieve such
behavior, we used a vesicular membrane composed of a conven-
tional phospholipid (1-palmitoyl-2-oleoyl phosphatidylcholine or
POPC) admixed with a lesser amount of a “designer” phospholipid
that possesses ester groups along its normally all-hydrocarbon
chains. We will demonstrate that the ester groups create membrane
“pockets” in which sodium ions reside.
Figure 1. Structures of seven ester-modified phospholipids.
Seven ester-modified phospholipids were synthesized (Figure 1)
according to multistep routes given in the Supporting Information.
Suffice it to mention here that each lipid was twice chromatographed
to give, in low overall yields, pure compounds that had the expected
1H and 13C NMR’s and high-resolution mass spectra (ESI). Among
the seven lipids in Figure 1, the total methylene count per
hydrocarbon chain ranges from 8 to 17. Terminal segments (an
important variable as it turns out) vary from 2 to 10 carbons per
chain. Five of our phospholipids each possess two ester groups per
chain, whereas two phospholipids have chains with only a single
ester group.
Figure 2. 23Na NMR spectra of vesicular A and B (30 mol %) admixed
with POPC (0.15 M sodium chloride in the presence of Tm[DOTP]5-).
The spectrum of B is identical to the spectrum of the pure POPC system.
the 23Na spectra relative to those from pure POPC vesicles (Figure
2). Both 23Na signals of B, E, and F systems have normal shifts
and line widths even at the high 30 mol % concentration and after
2 days of aging in the presence of the shift reagent. Since the three
“normal-acting” ester-lipids are the only ones with long terminal
segments (i.e., 10 hydrocarbon carbons), we can conclude that short
terminal segments are critical for promoting rapid Na+ interchange.
Momentarily we will explain why.
Vesicles (60-75 nm in diameter as determined by DLS) were
prepared by hydrating a thin film of a POPC/ester-lipid mixture in
0.15 M NaCl/D2O and then extruding the resulting suspension (30
mM lipid) 19 times through a 100 nm polycarbonate filter. Sodium
flux was monitored using a procedure pioneered by F. G. Riddell
in the mid-1980s:10 To a vesicle suspension (0.75 mL) was added
10 mg of thulium shift reagent, Tm[DOPTP]5- 11
Thereupon, the
.
intense 23Na NMR signal of sodium external to the vesicles was
shifted downfield by about 30 ppm, whereas the weak signal of
Na+ inside the vesicle cavities remained unaltered. The signal of
internal Na+ is weak owing to the small “capture volume” of the
dilute vesicle system. We used an INOVA 600 MHz NMR
operating at 158 MHz (21-22 °C, 6000 scans).
Four of the seven ester-lipids in Figure 1 (A, C, D, G), admixed
with POPC at 30 mol %, lack the inner sodium peak shown by a
pure POPC system (Figure 2). Inside/outside Na+ exchange across
the vesicle bilayer must be so fast that the inner Na+ signal melds
into the dominant outer Na+ signal. Defects are apparently created
by the four ester-lipids, allowing fast Na+ exchange on the NMR
time scale. In contrast, 30 mol % of B, E, and F has no effect on
A detailed examination of ester-lipid G is particularly illuminat-
ing. As mentioned, the baseline near 0 ppm at 30 mol % G is
featureless, but at lower G levels (5-27.5 mol %), an internal Na+
signal (Figure 3a) is visible. More interesting, a new broad peak
develops at 22.5 mol % G but disappears at 25 mol %. The mystery
of this third 23Na signal was compounded by the discovery that
the signal is time-dependent. Thus, all spectra in Figure 3a were
taken 30 min after vesicle preparation. If, however, we waited 0.5-
13 h for the vesicles to age prior to taking an NMR, a 20 mol %
G preparation gave spectra shown in Figure 3b. It is seen that within
3 h the single peak splits into two peaks. Although both peaks
broaden with time, the “new” peak on the left shifts further
downfield by 0.6 ppm over several hours.
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C O M M U N I C A T I O N S
Figure 4. A cross-section of an ester-lipid domain containing a sodium
ion and its associated water. The exact location of the ion within the bilayer
may vary with time.
pockets where, presumably, Na+ is coordinated with both water
and the ester carbonyl groups.14 If the domains serve as a conduit
for Na+ flux, then the NMR signal of the inner Na+ will decrease
as the domains form.
When the terminal chain segments are long (as in ester-lipids
B, E, and F), the lipids “ideally” intermix with the POPC, domain
formation does not occur, and catalyzed flux is not observed. Lipids
with short terminal segments (A, C, D, and G) are, however, able
to migrate and form domains that solubilize Na+ within the
hydrocarbon interior.
Figure 3. (A) Details of the inner sodium peak region for various mol %
of G in POPC. Tm[DOTP]5- ) 10 mg/0.75 mL. (B) 23Na NMR of 20 mol
% of G in POPC studied over time. Tm[DOTP]5- ) 3.3 mg/0.75 mL.
We estimate from the line width of the higher field signal,
representing internal Na+, that Na+ flux after 10.5 h is roughly
200 times faster than that occurring with pure POPC vesicles. The
same time-dependent double-peak formation was observed using
We cannot help wondering about unchartered chemistry that is
likely concealed within inner sanctums of live membranes.
7-
Dy(PPPi)2 as the shift reagent, except that the two peaks now
appeared downfield from the intense external Na+ signal.
What is the origin of this extra 23Na signal located slightly
downfield from the internal Na+ signal in Figure 3b? Since one
NMR signal with pure POPC vesicles comes from Na+ in the bulk
water and the other NMR signal comes from Na+ in the vesicular
water pools, the third signal in Figure 3b must derive from Na+
residing in or on the membrane phase. Evidence suggests that this
“membrane Na+” lies within the bilayer as opposed to binding at
the vesicle surface: (a) The existence of a membrane Na+ signal
indicates that the ion is not rapidly exchanging on the NMR time
scale (in the millisecond regime). It is difficult to imagine Na+
ions residing at the vesicle surface, or within a surface pocket, that
exchange only slowly with excess Na+ in the water. (b) The
chemical shift of the membrane Na+ (being only slightly downfield
from that of internal Na+) indicates that membrane Na+ is not in
close contact with shift reagent.12 Membrane-buried Na+, in contrast
to surface-bound Na+, would have this property (assuming, of
course, that shift reagent, with its penta-anionic charge, cannot enter
the membrane). The broader line width of membrane Na+, relative
to internal Na+ (Figure 3b), may result from loss of motional
freedom within the membrane pocket.
Acknowledgment. This work was funded by the National
Institutes of Health. A.L.G. was supported by a Woodruff Fellow-
ship, and M.E.C. by an ARCS Scholar Award. We appreciate
helpful discussions with Dr. Dan Lundberg.
Supporting Information Available: Synthetic routes to the new
lipids and their spectral characterization. This material is available free
of charge via the Internet at http://pubs.acs.org.
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It remains to explain why the appearance of membrane-buried
Na+ should be time-dependent. A likely rationale is that ester-lipid
G molecules are initially randomly distributed among the excess
POPC molecules in the vesicle bilayer. This is followed by an intra-
leaflet migration in which ester-lipid G slowly gathers into a
domain,13 thereby creating a membrane “defect” containing Na+
and water (Figure 4). A need to create opposing domains in the
two membrane leaflets, possibly requiring a slow gathering of
domain fragments, may impede the process. Simple calculations
show that there is roughly one membrane Na+ per lipid-ester in
(10) Ridell, F. G.; Hayer, M. K. Biochim. Biophys. Acta 1985, 817, 313.
(11) Tm[DOPTP]5- ) thulium(III)(1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetramethylenephosphonate). Macrocyclics M-155.
(12) Dipolar shifts decrease with the cube of the distance.
(13) Binder, W. H.; Barragan, V.; Menger, F. M. Angew. Chem., Int. Ed. 2003,
42, 5802.
(14) We have also considered the possibility that the double high-field peaks
(Figure 3b) arise from a population of empty vesicles plus a population
of vesicles containing a small number of shift reagent molecules. This
seems unlikely intuitively and because the NMR signal had collapsed after
a waiting period in the absence of shift reagent (and shift reagent only
then added for monitoring purposes).
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