Cholesterol and Phospholipid Modulation of BK[subscript Ca] Channel Activity and Ethanol Sensitivity: a dissertation

The large conductance Ca H -activated K+ channel (BKca) regulates neuronal excitability through the effux ofK , in response to membrane depolarization and increases in intracellular Ca . The activity of the BKca channel is increased by acute exposure to ethanol (EtOH), which is thought to underlie , in part, the influence of the drg on peptide hormone release from neurohypophysial nerve terminals (Dopico et al. 1996 , 1998). Moreover, chronic EtOH exposure attenuates acute drg action on hormone release, and reduces the sensitivity ofBKca channels to acute EtOH exposure (Knott aI. 2002). The factors regulating EtOH action on BKca channels are not well understood. Several lines of evidence suggest, however, that the lipid composition of the plasma membrane may influence channel sensitivity to the drug. The plasma membrane is highly complex in its organization (Welti and Glaser, 1994; Brown and London, 1998). There is a growing body of literature indicating that the local lipid composition of the membrane can influence the function of ion channels , including BKca (Chang et al. 1995a, b; Moczydlowski et al. 1985; Park et al. 2003; Tumheim et al. , 1999). Interestingly, chronic exposure to EtOH in animal models results in alterations in the composition of synaptic plasma membranes, including changes in the amount and distrbution of membrane cholesterol (CHS) (Chin et al. 1978; Chin et al. 1979; Wood et al. 1989). The significance of these alterations is unclear. Here, we set out to determine the ability of membrane lipids to modulate BKca channel activity and EtOH sensitivity. To address this , we implement the planar lipid bilayer technique, allowing control of both the protein and lipid components of the membrane. Native BKca channels retain EtOH sensitivity in this reductionist preparation (Chu et al. 1998), and we extend the study here to examine cloned human brain (hslo) BKca channels. We show here that hslo channels maintain their characteristic large conductance voltage and Ca ++ -dependent gating, and sensitivity to 50 ro EtOH in bilayers cast from a 3: 1 mixture of 1-pamiltoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1pamiltoyl-2-oleoyl-phosphatidylserine (POPS). The addition of CHS to the bilayer decreases both the basal activity and EtOH sensitivity of the channels , in a concentrationdependent manner. This lends support to the notion that alterations in plasma membrane CHS levels following chronic EtOH exposure may reflect adaptations to the acute actions of the drug on ion channels. Furthermore, the EtOH sensitivity and CHS modulation of these reconstituted hslo channels are greatly reduced in the absence of negatively charged POPS in the bilayer (pure POPE). Based on these findings , we look to gain mechanistic insight into the lipid headgroup and acyl chain properties that may regulate BKca channel modulation by EtOH and CHS. When POPS is replaced with the uncharged lipid 1palmitoyl-2-oleoyl-phosphatidylcholi (POPC), the hslo response to EtOH and CHS is restored, suggesting that the loss of negative surface charge or PS headgroup structue itself cannot explain the lack of channel modulation by these agents in POPE bilayers. Moreover, increases in the proportion of unsaturated acyl chains in the bilayer cannot significantly influence the hslo response to EtOH. The loss of EtOH sensitivity in pure POPE and CHS-containing bilayers may, therefore , reflect the propensity of POPE and CHS to form nonlamellar (nonbilayer) structures. Regarding the basal activity of the channel , we demonstrate that decreases in negative surface charge, increases in the proportion of unsatuated acyl chains , and increases in the complexity of head group interactions can all influence the steady-state activity of reconstituted hslo channels relative to control POPEIPOPS (3: 1) bilayers. Overall , these data further suggest the ability of the local lipid environment to regulate the basal function and EtOH sensitivity of an ion channel protein. Parts of this dissertation have appeared in separate publications: Treistman, S. , O' Connell , R.J. , and Crowley, J.J. (2002). Arificial Bilayer Techniques in Ion Channel Study. In Methods in Alcohol-Related Neuroscience Research D. Lovinger and Y. Liu, eds. (Boca Raton, Florida: CRC Press) Crowley, J. Treistman, S. , and Dopico , A.M. (2003). Cholesterol antagonizes ethanol potentiation of human BKcA channels in binary phospholipid bilayers. Mol. Pharm. 64(2):364-372.