Interactions between membrane lipids and integral proteins: Effects of bilayer structure on the reconstituted calcium-activated potassium channel from rat brain.

Abstract

The plasma membrane isolates the interior of cells from the external environment. In addition to acting as an insulating barrier, it permits selective interactions across the cell membrane through the presence of membrane-integral proteins which playa major role in the regulation of the internal environment of the cell. One class of membrane-integral proteins forms channels through which solutes pass in and out of the cell. In this dissertation, the properties of one such channel--the high conductance calcium-activated potassium (BK) channel--are examined subsequent to reconstituting the channel into bilayer membranes whose structure is experimentally altered. The central theme of this dissertation is to ask whether an alteration in membrane lipids, which results in different structural properties, provides information on the regulation of membrane-integral proteins such as ion channels. It is known that the lipid composition of cell membranes changes during development and aging, during adaptation to different temperatures, and in some disease states. A better understanding of lipid-channel interactions is therefore likely to provide key information concerning cellular homeostasis. Natural and induced changes in membrane lipid composition, and hence membrane structure, alter the physico-chemical environment at the membrane--protein interface. I propose in this dissertation that changes in membrane structure are ultimately expressed as physical changes that affect ion channel behavior in predictable ways. To test the hypothesis that the lipid environment modifies channel function, I examined the properties of the BK channel reconstituted from rat brain into lipid bilayers of different compositions. The bilayer was modified with phospholipids of different headgroups (altering charge and size), or with phospholipids which have different fatty acid chains (altering the order parameter). Changing the bilayer surface charge is expected to change the concentration of ions near the channel thereby. Therefore the properties of BK channels is expected to be changed due to the interactions of calcium and potassium ions with the surface charge. Furthermore, the interaction between negatively charged lipid and calcium is known to order the lipid structure and may introduce structure stress in bilayers. This structural stress in bilayers may act on the BK channel and modify its properties. Altering the size of phospholipid headgroups and the order of fatty acid chains are likely to change the packing of lipids in the bilayer. Also, such structural alteration in lipid changes the lateral elastic and curvature stress within the bilayer. Addition of cholesterol to phospholipid bilayers is known to increase the orderliness of the fatty acid chains and increase the modulus of compressibility of the membrane, thus increasing the lipid structural stress. Adding general anesthetics into bilayers has been shown to disorder the lipid structure but also increase the lipid structural stress. These physical changes in bilayers may, in tum, act on the channel protein and altering its properties. The results showed that increasing the negatively charged lipid in the bilayer surface resulted in an increase in channel mean opentime, open probability and conductance of the BK channel. Increasing the lipid structural stress, in general, reduces channel mean opentime and open probability. In the case of cholesterol, the conductance is also reduced in addition to mean opentime and open probability. Taking together, these results suggest that the lipid environment plays a profound role in shaping ion channel properties.