This project focuses on how the Na/K pump is regulated by RNA editing. A central premise to our approach is that naturally occurring codon changes, caused by RNA editing, can lead us to functionally important regions of the Na/K pump. They might also be used as tools to compensate for rapid-onset dystonia parkinsonism, genetic disease associated with the human Na/Ka3 subunit. The squid nervous system will be used as a model because RNA editing is extensive in cephalopods and because the editing sites that we have identified cause a gain of function, a rare phenomenon for a mutation. Preliminary data show that Na/K pump mRNAs can be edited at three codons, two in the phosphorylation domain (P), and the other at the top of the 7th transmembrane span (M7). These changes affect critical components of the pumping cycle. For example, the edits in the P domain increase the apparent affinity for ATP and the edit in M7 regulates how Na is released to the outside. Using an electrophysiological approach, the proposal's first two aims will characterize the mechanism by which these edits exert their function. The goal of the last aim is to see whether squid RNA edits can compensate for depressed turnover rates in human Na/Ka3 caused by mutations associated with rapid-onset dystonia parkinsonism. In addition, not only will we study if they can compensate, we will also try to develop a method to introduce these edits into human pumps at the level of mRNA. Preliminary data show that the human editing enzyme ADAR2 is capable of editing squid mRNAs because it recognizes the appropriate secondary structure. We hypothesize that we can trick human ADAR2 into editing human pumps by mimicking the squid secondary structure with an antisense RNA oligo. From the standpoint of public health, this work is significant on several fronts. The Na/K pump creates the ion gradient that is required for excitability and the majority of solute transport across cell membranes. With the first crystal structure recently published, this is an opportune time to learn more about how the Na/K pump operates. Clinically, the Na/K pump is important because it is the receptor of digoxin, a widely prescribed cardiac steroid used to control many cardiac arrhythmias. Further, two neural disorders have been directly correlated with mutations to the Na/K pump: familial hemiplegic migraine, which is linked to the Na/Ka2 subunit, and rapid-onset dystonia parkinsonism, which is linked to the Na/Ka3 subunit. Results from this proposal will be directly relevant to the development of therapeutics for rapid-onset dystonia parkinsonism. The general approach may also prove relevant for a wide variety of genetic disorders.

Public Health Relevance

The Na/K pump plays a vital role in establishing ion gradients across cells. Preliminary data shows that its ability to pump can be regulated by RNA editing. This proposal focuses on understanding how RNA editing regulates the Na/K pump and how it might be used to treat genetic disorders that affect the Na/K pump.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01NS064259-05
Application #
8702244
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Stewart, Randall R
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Puerto Rico Med Sciences
Department
None
Type
Overall Medical
DUNS #
City
San Juan
State
PR
Country
United States
Zip Code
00936
Montiel-Gonzalez, Maria Fernanda; Vallecillo-Viejo, Isabel; Yudowski, Guillermo A et al. (2013) Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing. Proc Natl Acad Sci U S A 110:18285-90
Palavicini, Juan Pablo; Correa-Rojas, Rodrigo A; Rosenthal, Joshua J C (2012) Extra double-stranded RNA binding domain (dsRBD) in a squid RNA editing enzyme confers resistance to high salt environment. J Biol Chem 287:17754-64
Garrett, Sandra; Rosenthal, Joshua J C (2012) RNA editing underlies temperature adaptation in K+ channels from polar octopuses. Science 335:848-51
Castillo, Juan P; De Giorgis, Daniela; Basilio, Daniel et al. (2011) Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid. Proc Natl Acad Sci U S A 108:20556-61
Galarza-Munoz, Gaddiel; Soto-Morales, Sonia I; Holmgren, Miguel et al. (2011) Physiological adaptation of an Antarctic Na+/K+-ATPase to the cold. J Exp Biol 214:2164-74
Colina, Claudia; Palavicini, Juan Pablo; Srikumar, Deepa et al. (2010) Regulation of Na+/K+ ATPase transport velocity by RNA editing. PLoS Biol 8:e1000540