¹⁴N NMR of Lipid Bilayers: Effects of Ions and Anesthetics

The interaction of divalent and trivalent metal cations, ferricyanide, a lipophilic ion (tetraphenylborate), and a local anesthetic (tetracaine) with the phosphocholine head group of egg lecithin was investigated by using wide-line 14N and 31P NMR. Measurements of the N quadrupolar splittings in the presence of a variety of perturbing agents demonstrated that the N NMR technique can be used to directly monitor ion or anesthetic binding. The 14N quadrupolar splitting (Δvq) is a measuee of the order parameter of the Cβ-N bond segment, and changes in ΔvQ as large as 3.5 kHz were observed. Moreover, a comparison of the changes in the quadrupolar splittings induced by the binding of ions or anesthetics provided a sensitive method of discriminating between these perturbing agents in their ability to alter the orientational order of the Cβ-N bond segment of the phosphocholine moiety. Without exception, addition of metal ions or anesthetics always resulted in a decrease of the N ΔvQ. This reduction reflects a change in the average orientation or degree of motional averaging at the Cβ-N bond segment position. In the case of metal ion binding, the strength of the interaction increased with the charge of the metal ion in the order Ca₂₊ < Ln₃₊, in agreement with a previous 2H NMR study [Akutsu, H., & Seelig, J. (1981) Biochemistry 20, 7366-7373]. However, distinct differences were also noted between ions of the same charge, and in the case of the trivalent lanthanide ions, the 14N ΔvQ decreased in the sequence La3+ > Pr3+ > Eu3+ > Lu3+, following the order of the lanthanide contraction. The 14N and 31P line shapes in the presence of lanthanide ions showed that it is possible to clearly distinguish between the effects of paramagnetic (Pr3+, Eu3+, Dy3+, Tm3+) and diamagnetic (La3+, Lu3+) ions. Unusual, distinctive “asymmetric' N NMR line shapes were observed in the presence of the paramagnetic lanthanides, apparently due to incomplete averaging of the magnetic dipolar interaction between the bound lanthanide ion and the nitrogen nucleus. Addition of the lipophilic anion tetraphenylborate led to a slight reduction in the 14N ΔvQ, together with a dramatic decrease in the absolute value of the 31P chemical shielding anisotropy (Δσ). By contrast, the reduction of the I4N ΔvQ due to the presence of tetracaine was accompanied by a substantial increase in the 31P |Δsigma;|. In general, the measurements of the N ΔvQ as well as the 14N spin-lattice (T1) relaxation times support the notion that there is a rapid exchange of ions from the bulk medium to the ligand binding sites and among the various binding sites, in agreement with H NMR studies. The above results suggest that 14N NMR can provide a useful complement to 3IP and 2H NMR techniques in studies of the influence of various perturbing agents on the orientational order and dynamics of the phosphocholine head groups in membranes.