Large conductance, Ca2+-activated K+ channels (MaxiK, BK) are key regulators of a plethora of cell functions including vascular tone, neuronal function, and immunity. As such, knowing their natural maturation steps from synthesis to the establishment of interactions with proteins that guide them to their functional sites is key to understand the basis of their function. Thus, the long-term goal of this research is to identify the regulatory mechanisms of MaxiK channel transcription, co(post)-translational modifications, and traffic that determine its availability and function at the right time and place. We have learned about the molecular composition of MaxiK channels in different systems, their role in animal physiology by means of silencing subunit genes, and have started to grasp information on the mechanisms of their cellular traffic and on their potential networks. We will now test the general hypothesis that, MaxiK's pore-forming a subunit (Slo) gene and protein have intrinsic sequences that rule their expression, vectorial traffic, and protein-lipid interactions localizing them in strategic cell compartments according to physiological needs. Our preliminary data indicate that: i. mSlo promoter region responds to estrogen (E2) and contains potential hormone-response sequences that may rule channel expression by E2; ii. basolateral MaxiK targeting may be driven by a Slo splice variant insert; and iii. Slo can be myristoylated. We will use a multidisciplinary approach, in particular, avidin-Slo constructs for visualization of single-molecule movements with quantum dots and high resolution confocal microscopy.
Specific Aims are to: 1) map the transcription start site(s) and functional E2-regulatory sequences in Slo promoter(s), and define the genomic mechanism(s) of E2-mediated regulation of Slo transcription; 2) investigate the role of Slo splice variant(s) in determining differential trafficking and targeting; and 3) investigate the mechanism and site of MaxiK myristoylation and its functional consequences. These studies should provide new information on the mechanisms that regulate MaxiK channel gene and protein expression and targeting, and identify new therapeutic pathways to alleviate cardio- or cerebro-vascular diseases. ? ?
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