A CALORIMETRIC INVESTIGATION OF THE ANION TRANSPORT PROTEIN OF THE HUMAN ERYTHROCYTE MEMBRANE AND THE EFFECT OF ANESTHETIC CHARGE ON ANESTHETIC-PHOSPHOLIPID INTERACTIONS
Abstract
Recent calorimetric studies on the human erythrocyte membrane have revealed five distinct endotherms which presumably correspond to the heat disruption of separate structures in the membrane. We have identified one of these endotherms, termed the C transition, as a conformational transition of the membrane-spanning domain of the erythrocyte anion transport protein, band 3. This identification is based on numerous observations. The most compelling evidence for this identification is (i) the fluorescent properties of probe molecules specific for the membrane-spanning domain of band 3 change dramatically during the C transition, (ii) the removal of proteins other than the membrane-spanning domain was found to have no significant effect on the C transition, and (iii) reconstituted membranes containing the purified membrane-spanning domain render a C transition with normal calorimetric properties. The identification of the C transition enabled us to examine the effect of the local anesthetic and inhibitor of anion transport, lidocaine, on the structural stability of the anion channel. Lidocaine was found to destabilize the structure of the anion channel (lower the temperature of the C transition) over the same concentration range where it inhibits anion transport. Further, only the neutral form of lidocaine was found to be effective as an inhibitor of anion transport and as a destabilizer of band 3. Based on these results, a mechanism of local anesthesia was proposed which attributes an anesthetic's potency to its ability to penetrate the bilayer, destabilize and, thus, distort the structure of the sodium channel. In a separate study, we examined the effect that anesthetic charge has on the ability of an anesthetic molecule to perturb the thermotropic phase behavior of anionic and zwitterionic phospholipids. Neutral lidocaine was found to modify the phase behavior of both anionic and zwitterionic phospholipids. Cationic lidocaine, however, was found to modify only anionic phospholipids. Cationic lidocaine is generally regarded as the more potent form of lidocaine. Thus, these results suggest that if anesthetics exert their effect by perturbing a phospholipid structure in the nerve membrane, then this structure consists primarily of anionic phospholipid.
Degree
Ph.D.
Subject Area
Biochemistry
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