Thermal destabilization of transmembrane proteins by local anaesthetics

Local anaesthetics, in addition to anaesthesia, induce the synthesis of hea t shock proteins (HSPs), sensitize cells to hyperthermia, and increase the aggregation of nuclear proteins during heat shock. Anaesthetics are membran e active agents, and anaesthesia appears to be due to altered ion channel a ctivity; however, the direct effect of heat shock is protein denaturation. These observations suggest that local anaesthetics may sensitize cells to h yperthermia by interacting with and destabilizing membrane proteins such th at protein denaturation is increased. Tt is shown, using differential scann ing calorimetry (DSC), that the local anaesthetics procaine, lidocaine, tet racaine and dibucaine destabilize the transmembrane domains of the Ca2+-ATP ase of sarcoplasmic reticulum and the band III anion transporter of red blo od cells. The transmembrane domain of the Ca2+-ATPase has a transition temp erature (T-m) of denaturation of 61 degrees C which is decreased, for examp le, to 53 degrees C by 15mM lidocaine. The degree of destabilization (Delta T-m) by each anaesthetic is proportional to the lipid to water partition c oefficient, and the increased sensitization by anaesthetics with larger par tition coefficients and at higher pH suggests that the uncharged forms of t he anaesthetics are responsible for destabilization. A Hill analysis of Del ta T-m for the Ca2+ -ATPase as a function of the concentration of anaesthet ic in water gives dissociation constants (Kd) On the order of 10(-4) M, if binding occurs directly from the aqueous phase. This demonstrates moderate affinity binding. However, dissociation constants of 1-3 M are obtained, if binding occurs through the lipid phase, which demonstrates low affinity bi nding. Thus, the interaction of local anaesthetics with the Ca2+ ATPase may be moderately specific or non-specific depending on the mechanism of inter action. The observation that local anaesthetics also destabilize the transm embrane domain of the band III protein of erythrocytes suggests that destab ilization of transmembrane proteins is a general property of anaesthetics, which is at least in part a mechanism of sensitization to hyperthermia.