Ion Interactions with the Gramicidin A Transmembrane Channel: Cesium-133 and Calcium-43 NMR Studies

This chapter illustrates the capacity to determine meaningful rate constants by means of NMR relaxation studies of spin 7/2 cesium- 133 and allow these approaches to be considered for spin 7/2 calcium-43. Background NMR data are presented for calcium-43, e.g., CaCI₂concentration dependence and temperature dependence of the longitudinal relaxation time, TI, in D,O, in the presence of phosphatidylcholine lipid and in the presence of phosphatidylcholine lipid plus gramicidin A channels and the transverse relaxation time, T₂for 100 mM and 1 M CaCI₂, in the presence and absence of channels. These data allow determination of the off-rate constant for calcium ions leaving the channel binding site to be of the order of 5 x 10⁷/sec. With the binding constant demonstrated here to be about l/M for the site at the mouth of the gramicidin A channel and with the experimental off-rate constant, it becomes apparent that the lack of a calcium ion current is due to a high central barrier arising from the large repulsive image force which occurs when a divalent charge is separated from the lipid dielectric constant by no more than a single layer of polypeptide backbone.