Mario E. Lloret-Torres¹, Stephanie Palacios², Katherine Cordero Padilla^1,3, Jordan, G.A. Yudowski^1,3, Steven N. Treistman³ andCristina Velázquez-Marrero^1,3

¹University of Puerto Rico Medical Sciences Campus, San Juan P.R.; ²University of Puerto Rico, Biology Department, Rio Piedras Campus, San Juan, P.R.; and ³Institute of Neurobiology, University of Puerto Rico, San Juan, P.R.

Introduction: The large conductance voltage- and calcium-dependent (BK) channel is known to play an essential role in alcohol tolerance. As BK is needed for post-action potential hyperpolarization changes in BK activity and expression can strongly impact firing frequency. We have shown that internalization of the ZERO, alcohol-sensitive, BK channel isoform in heterologous systems and hippocampal neurons occurs via the Wnt/b-catenin signaling pathway in response to EtOH. This internalization is isoform-specific as the STREX exon-containing variant (STREX) does not show acute sensitivity to alcohol. Wnt/b-catenin signaling also causes declustering ZERO from lipid rafts. Whether this declustering plays a role in BK internalization remains unstudied. The fate of this internalized protein is also unknown.

Methods: To further understand the molecular processes governing the development of ethanol tolerance, we used TIRF imaging to examine changes in STREX internalization and lipid raft declustering after exposure to ethanol in a HEK293 heterologous expression system. We also used Western Blots to assess whether ethanol exposure affects overall protein expression.

Results: Our results show that STREX does not internalize, or redistribute away from lipid rafts, nor is its overall expression affected by ethanol exposure. Furthermore, key aspects of Wnt/b-catenin signaling triggered by EtOH exposure do not occur when STREX is expressed in HEK293 cells, including b-catenin accumulation or TCF/LEF transcriptional activation.

Conclusion: STREX-containing BK channels are resistant to the effect of Wnt signaling suggesting Wnt/beta catenin signaling may be essential for molecular alcohol tolerance.

Aknowledgements: This work was supported by NIH R01AA027808 (CVM) and NIMHD S21MD001830 (CVM).

Keywords: BK channel, STREX, HEK 293, tolerance, ethanol,