RNA editing of ion channels and receptors has important consequences to their function. RNA editing is a co-transcriptional modification of pre-mRNA performed by an enzyme that converts adenosine into inosine (A to I). Inosine is interpreted as guanosine by the translational machinery. Thereby, RNA editing provides a substantial expansion of the genetic pool of an organism. As might be intuitively expected, A to I conversions often target critical positions of the encoded protein, where changes in function are essential for the physiology of a cell. Over the past six years, we have become increasingly interested in understanding how nature functionally tunes membrane proteins by RNA editing. RNA editing allows multiple protein products from a single gene. This increase in genomic capability does not appear to be random, rather it targets regions of a protein that are functionally important. The classical example is in the GluRB subunit of glutamate-gated ion channels, important for fast excitatory synaptic transmission in the central nervous system. Editing underlies the conversion of glutamine to arginine in the channelfs pore (Sommer et al., 1991), which renders the receptor impermeable to calcium ions (Kohler et al., 1993). Although editing seems to be common among membrane proteins (Hoopengardner et al., 2003), the functional consequences of editing had been explored in only a few examples (Kohler et al., 1993;Burns et al., 1997;Patton et al., 1997;Wang et al., 2000;Berg et al., 2001;Rosenthal and Bezanilla, 2002;Bhalla et al., 2004). We have joined efforts with the laboratory of Josh Rosenthal to approach the subject of RNA editing of membrane proteins in a comprehensive manner. In excitable cells, precisely synchronized ionic currents generate the electric potentials that are the currency of communication. For the system to operate, the timing of turning the currents off is as important as the timing of turning them on. Accordingly, ion channels have developed intricate systems to switch off that override the signals to stay on, a process known as inactivation. In rodent and human KV1.1 channels, RNA editing recodes a highly conserved isoleucine to a valine (I400) (Hoopengardner et al., 2003). This conversion is regulated in different regions of the nervous system. Structural (Long et al., 2005) and functional (Liu et al., 1997) data show that I400 is in the lining of the permeation pathway, in a region known as the channelfs intracellular cavity. I400V selectively targets the process of fast inactivation (Bhalla et al., 2004), allowing edited channels to recover from inactivation about 20 times faster than their unedited counterparts. For a neuron, this change in function would greatly influence action potential shape, signal propagation and the firing pattern (Connor and Stevens, 1971;Aldrich et al., 1979;Debanne et al., 1997;Hoffman et al., 1997;Giese et al., 1998;Johnston et al., 1999). In this study we investigated the precise mechanism by which I400V alters fast inactivation. The robust functional consequence of editing on inactivation is conserved in phylogenetically diverse KV channels, and mimicking I400V in Shaker KV channels (I470V) produces an increase in the speed of recovery from inactivation comparable to that seen in the human isoform (Bhalla et al., 2004). Using an intracellular cysteine-less construct of shaker, we studied the mechanistic details underlying the I400V phenotype by changing the chemistry at this site by mutating it to a cysteine, and attaching different chemical moieties. Our results are consistent with the simple idea that nature uses RNA editing to increase the unbinding kinetics of the inactivation particle. It accomplishes this by selectively reducing the hydrophobic interaction between the tip of the N-terminus and its receptor, the edited codon within the intracellular cavity. Further, our experiments sugggest that to obstruct permeation the inactivation particle must penetrate deeply into the intracellular cavity in an extended conformation. Transporters are essential for ion channel function because they provide and maintain the ionic gradient that allows ions to diffuse through channels. Recently, by comparing genomic and cDNAs sequences, new targets of RNA editing have been identified, among them, proteins involved in ion homeostasis (Stapleton et al., 2006). Interestingly, there is no report in the literature of how RNA editing might alter the function of any transporter. We are taking advantage of the apparent high levels of editing in squid (Patton et al., 1997;Rosenthal and Bezanilla, 2002) to examine RNA editing in transporters, initially focusing our attention on the Na/K pump, a transporter that I have studied over the past 15 years. After cloning the full-length cDNA and genomic DNA for the squid's Na/K-ATPase, we have found at least four potential RNA editing sites. We showed that RNA editing may regulate ion homeostasis by making specific changes within Na/K pump mRNAs. In particular, the editing event I877V, an amino acid located withiing the seventh transmembrane segment affects the Na/K pumpfs intrinsic voltage dependence. The principal effect of I877V is to shift the Ip-V curve 25 mV to more negative potentials. To better understand the mechanism by which I877V shifts the Na/K pumpfs Ip-V relationship, we studied the process of external Na binding/release and occlusion/deocclussion in isolation by removing all K and maintaining the intracellular ATP concentration at high levels. It is know that these state determine the voltage dependence of the Na/K pump. We found that I877V shifts the occupancy of the states of the transport cycle associated with the release of Na, so that the pump's I-V function is displaced negatively. These results imply that the Na/K pumps voltage dependence allows for the turnover rate to be adjusted.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$534,269
Indirect Cost
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