232

N MR sheds more light on ion transport

The effect of paramagnetic ions on the NMR spectra of ~H and :"P has long prom- ised to provide unique information on transport across membranes. However, until recently the physiological relevance of such studies was severely limited by three factors: rapid transport of ions across the membrane could not be followed; only small liposomes could be investigated; and lanthanides, the paramagnetic ions usually employed, are poor models for biologically important ion transport. Recent develop- ments have now made it possible to gather information on more physiologically rel- evant ion transport systems.

When paramagnetic ions such as Pr a~ and Eu a+ closely approach nuclei which give rise to NMR signals, these signals are

i.

!'i: @

Fig. I. NMR can dBtinguish between modes of transport. The :'*PMR spe('trum o]'small unilamel- lear liposomes. (at Jmt after addition oJa para- magnetic shift reagent u~ the external compartment. (b~.l) After some inward trar~port o f the reagent has occurred fl)r (b) an 'all or nothing'. (c) a 'one ion at a time', or (d) a 'one ion at a time with dis- perved permeabilities' mode of transport.

shifted. For example, Eu :~' or P?" ions out- side small unilamellar liposomes (SULs) shift the :'~P or 'H NMR peaks of the phos- pholipids in the outer layer of the liposomes ~ :'. As the ions cross the mem- brane, the peaks of the internal phos- pholipids can be seen to shift. Thus, by monitoring the peaks of the internal phos- pholipids the transport of these paramag- netic ions can be studied:L

The particular advantage of the NMR approach is that it can distinguish between the following modes of transport (see Fig. 1 ). In an 'all or nothing' process (e.g. when SULs with Pt=' on the outside were sonicateda), the peak of the internal phos- pholipids gradually decreases in intensity, and at another position in the spectrum (close to that of the external phospholipids) another peak arises and increases in inten- sity. By contrast, a 'one (ion)at a time' pro- cess with identical rates of transport for all liposomes is indicated by a shift in the peak of the internal phospholipids towards its final position, without a decrease in inten- sity or broadening (e.g. lysophosphatidyl choline-induced Eu a' transport into SULs*4). In a third mode of transport (e. g. A-23187-mediated transport of P(~ into SULs a t - a 'one (ion) at a time' process, but with individual liposomes having different rates of transport - the internal peak broadens as it shifts towards the external

peak. NMR studies could only follow transport

processes which were slow compared with the time needed to obtain an NMR spectrum with a reasonable signal-to-noise ratio (usually about I mint. This limitation has been largely eliminated by using the line width of NMR signals to measure transport occurring approximately during the 'T2' relaxation of the NMR signal, e.g. in some millis'econds. When Pr :'~ and non- paramagnetic La a' are added to a suspen- sion of inverted micelles in benzene such that the inverted micelles contained neither, either, or both of the ions, two distinct lines are observed 5. With successive additions of ionophore, these two lines broaden, come closer, fuse, and the resulting peak becomes narrower and narrower. Thus, the flow of Pr a~ between individual inverted

77BS -July 1982

micelles was shown to depend linearly on the concentration of the ionophore ~.

Springer's group at Stony Brook has also developed a method for studying the trans- port of non-paramagnetic ions across the membranes of larger liposomes (>50 nm diameter) 6. They followed the transport of sodium by sodium-NMR, using dys- prosium nitrilotriacetate :' complex as an anionic paramagnetic shift reagent.

The transport of Na ~ into large unilamel- lar liposomes (LULs) was measured by having Li ~ and the shift reagent on both sides of the LUL membranes and adding a reagent that destroys the shift reagent, in the Na+-containing outside medium. The Na ~ peak shifted downfield. When gramicidin was added, a peak became viv ible at the original position, increasing in height with time. Since under these condi- tions the shift would depend on the concen- tration of Na' inside the liposome", this is consistant with the idea that gramicidin transports Na" by an 'all or none' mechan- ism: once a gramicidin pore is formed in a liposome, Na' equilibrates across the membrane of that particular liposome.

These three innovations offer the oppor- tunity of gaining unique information about transport mechanisms; living cells are now being studied by similar techniquesL

I thank Jan Berden for discussions and Arthur Simonetti for the cartoon.

R e f e r e n c e s I Michaelson, D. M., Horwitz, A. F. and Klein.

M. P. (1973) Biochemislry 12. 2637-2645 2 Hunt, G. R. A. (1975)FEBS Lett. 58. 194--196 3 Ting, D. Z. . Hagan, P. S.. Chan. S. [., Doll. J. D.

and Springer, C. S., Jr. (1981) Biophys. J. 34, 189-216

4 Lee, Y, and Chan, S. 1. (1977)Bio(hemist O' 16. 13O3-13O9

5 ('hen, S.-T. and Springer. C. S . Jr. (1981)

Biophys. (hem, 14. 375-388 6 Pike, M. M,. Simon, S R., Balschi, J. A. and

Springer. C. S., Jr. (1982) Proc. Natl Acad. Sci. U.S.A. 79, 810-814

7 Balschi, J. A., Cirillo, V. P. and Springer, (" S.

Biophys. J. 38, (in press)

HANS V WESTERHOFF

Lalx)ratorium w×)r Bic, chemie. University ot Amster- dam. Plantage Muidergracht 12. I()lg TV Amster-

dam, The Netherlands.

Elsevier BIomediud Press ](;~2 utT~ no,~7/~2'c~rpi; (~xxl /$Ol/Xl