Alcohol Modulation of Human BK Channels Evidence for β-Subunit Dependent Plasticity in Functional Ethanol Tolerance: A Dissertation

Abstract

Alcoholism is responsible for more than 6% of deaths internationally per annum. The development of acute tolerance to ethanol (EtOH) is a critical component of alcoholism. Previous studies identified large conductance calcium-activated potassium (BK) channels as potential EtOH targets in a variety of species and cells. In order to elucidate mechanisms underlying tolerance development, I used inside-out patch clamp techniques to measure EtOH induced changes in channel activity (measured as open probability) of hSlo, hSlo+β1, and hSlo+β4 channels exogenously expressed in HEK 293 cells. I show that the human BK channels have subunit dependent responses to acute application of EtOH, and the magnitude of potentiation was dependent on the concentration of ethanol used and the type of β-subunit expressed. In addition the subunit dependent effects on the channels were a function of cytosolic calcium concentration. Furthermore, to determine if BK channels in ripped-off patches can become tolerant to EtOH, I monitored changes in channel activity in response to a second application of the drug, 10-minutes after washing-out the first exposure. I found that channels were less responsive to the second exposure, indicative of tolerance. I examined long-term consequences of EtOH exposure by repeating these experiments on cells cultured in 25 mM EtOH in the culture medium for 24-hours. Under these conditions, all three channel types show chronic tolerance has developed as revealed by the response to acute EtOH applications. Subunit-dependent differences to the development of acute tolerance were apparent, however. In response to a second application to EtOH, hSlo+β4 channels were now inhibited. Overall, these results indicate that BK channels respond to and develop tolerance to EtOH in the absence of cellular context, suggesting the possibility that alcohol tolerance within organisms may be in part mediated by changes imparted by EtOH on BK channels directly.