Historically, the Na+
ATPase of the squid giant axon has been one of the most actively studied native pumps. In a previous report we identified the mRNA sequences for the underlying α (EF467998) and β (EF467996) subunits 
. Because other squid transcripts are regulated by RNA editing 
, we examined whether the Na+
ATPase mRNAs were as well. Sequences of 50 individual cDNA clones for the squid NaKα1 subunit, isolated from the giant axon system, showed adenosine-or-guanine variation at specific sites, a hallmark of RNA editing. To explore whether this variation was indeed due to RNA editing, we cloned the gene that encodes squid NaKα1 mRNAs (). The squid NaKα1 gene, which spans over 20 KB, is highly fragmented, containing 19 exons. At four positions, the gene sequence contains an A whereas some or all of the cDNA sequences contain a G (e.g., ). Three of the sites lie at the junction with a nearby intron, as is commonly the case with other RNA editing sites () 
. Two sites lie within the same codon. Because both were guanosine in all cDNAs sequenced, the lysine at this position was always converted to glycine. To further support the idea that the A→G conversions are caused by RNA editing, we tested whether a squid editing enzyme (SqADAR2.1A (FJ478450.1); 
) could edit these codons in vitro (Figure S1A
). Using the genomic form of the full-length, mature squid NaKα1 mRNA as a substrate, recombinant SqADAR2.1A could edit all four codons. It is notable that all the information required for editing resides within the exons and that intron sequence was not required, as is commonly the case for other editing sites. Similarly, the structure that drives editing of human Kv1.1 channel mRNAs is entirely exonic 
. Interestingly, human ADAR2 (BC065545.1) can also edit codons K666G and I877V, but not R663G. Predicted folds for NaKα1 mRNA using MFOLD software show an obvious hairpin surrounding the I877V codon (Figure S1B
). Using this approach, similar structures are not apparent around codons R663G and K666G. In any case, the combination of our cloning data and the in vitro editing assays verify that the A/G variation observed in Na+
ATPase mRNAs is due to RNA editing.
mRNAs for the squid Na+/K+ pump are edited.
The editing sites R663G and K666G are located within the phosphorylation domain which accepts ATP's γ phosphate during the transport cycle, while the I877V edit lies at the extracellular end of the seventh transmembrane segment. All editing sites recode a highly conserved amino acid (R663G, K666G, and I877V; ). In fact, a survey of over 200 NaKα1 cDNA sequences from both vertebrates and invertebrates shows the unedited codon at these positions to be almost invariant. There are a few exceptions, however. The ovine and bovine NaKα1 sequences (emb CAA26582.1 and gb AAI23865.1), and a NaK sequence from planaria (dbj BAA32798.1), have an arginine at codon 666, conceivably being produced by editing at the codon's second position (AAR→AGR). At position 877, an electric eel NaK cDNA is the only sequence with a valine. As with squid, this could have been caused by RNA editing. Overall, however, our bioinformatics search uncovered little evidence of editing in distantly related organisms. Because of this we tested whether the squid sites are edited in another cephalopod. Using squid specific primers, NaKα1 cDNA and genomic DNA was amplified from Octopus bimaculata collected from Catalina Island, CA. Based on 50 individual cDNA clones, the R663G edit was edited, but at a much lower rate than in Loligo (12% versus 96%). No editing was apparent in codons K666 or I877. These data suggest that editing sites are evolving rapidly within cephalopods.
In the giant axon, R663G and K666G are edited almost to completion while I887V is scarcely edited. Why undergo a complex process such as editing when a simple mutation to the gene would produce much the same result? One possibility is that these sites are used for regulating pump function. If this is the case, we would expect the extent of editing at these sites to differ between neuronal tissues. To test this idea, we collected tissue from 10 different regions of the nervous system, both central and peripheral. Using a poison-primer extension assay 
, we estimated the editing efficiency in each sample (). The extent of variation differed dramatically between sites (). R663G varied only from ~65%–85%. Editing at codon 666 was more complicated. Because it can be incompletely edited at the first two positions (AA
G), a mixed population of pumps with either arginine, glycine, or lysine (unedited) can result. In some tissues, as in the giant axon, K666G predominates, while in others K666R or K666 is the dominant species. Although K666E is theoretically possible (GAG), this edit was never observed. The I877V edit is also highly tissue- specific. Barely present in the giant axon system and other peripheral regions, it occurs close to 50% of the time in parts of the central nervous system such as the Inferior Frontal Lobe neurons. These results strongly suggest that RNA editing could be used to regulate Na+
RNA editing efficiency is highly regulated between different areas of the nervous system.
For any cell maintaining ion homeostasis, the most important aspect of Na+
pump function is the velocity of ion transport. Accordingly, we were interested in determining whether any of the RNA editing events regulates the Na+
pump's turnover rate. Because the Na+
pump is electrogenic and its stoichiometry does not change with voltage 
, the pump current (Ip
) is an accurate reflection of the turnover rate at any voltage. Under physiological conditions, the voltage dependence of Ip
is approximately sigmoid, reaching a maximum at positive potentials and approaching zero at very negative potentials. We first measured the maximum turnover rate for the unedited pump and all single edited versions (Figure S2
). To estimate this parameter we expressed these constructs in Xenopus
oocytes and measured Ip
at positive voltages, where it reaches its maximum, while estimating the pump density in the same oocytes. The maximum turnover rate for the unedited pump is 27.0±4.7 cycles·s−1
(at 22°C). None of the rates determined for the edited versions differed significantly from the unedited construct when compared at the same temperature. Thus, at positive voltages, editing has little effect.
pump's turnover rate at negative voltages, where it is partially inhibited, is a more relevant measurement because the pump predominantly operates over these potentials. Next we investigated whether editing affects the voltage dependence of the pump's transport velocity. To illustrate our approach, shows a current record of the entire experiment recorded on a slow time scale. The oocyte is held under voltage clamp at 0 mV, where the Ip
is maximal. The rapid vertical deflections are the current changes in response to 40 ms voltage pulses from the holding potential to various potentials between −198 mV and +42 mV (in 10 mV increments). After each step the voltage was returned to the holding potential. The voltage protocol was repeated in each experimental condition to verify the stability of the preparation. After the application of 100 µM ouabain, the current trace visibly becomes smaller due to inhibition of Ip
. To isolate Ip
, current traces after ouabain application (3) were subtracted from those before (2). shows an example of these traces at the extreme voltages (−198 mV in gray, +42 mV in black). Steady-state Ip
(arrow) was determined at all voltages by averaging the final 5 ms of each trace, after the transients had settled. Similar measurements were performed for the unedited pump, and all single edited versions. I877V differed substantially from the unedited version. shows the normalized voltage dependence of the pump velocity for I877V and the unedited pump. The principal effect of I877V is to shift the Ip
-V curve ~25 mV to more negative potentials, thereby relieving voltage dependent inhibition. Because there is ~2-fold less extracellular Na+
in oocyte strength solutions than in those used for squid, both curves would be shifted approximately 60 mV to the right, as we have previously shown 
. From this we estimate that I877V would significantly increase Ip
at the resting potential, which is ~−60 mV in the squid axon 
. Under physiological conditions the pump's voltage dependence comes mostly from the transitions underlying extracellular Na+
. Therefore, these results suggest that the I877V edit targets this process.
The I877V edit shifts the voltage dependence of the Na+/K+ pump's turnover rate.
To better understand the mechanism by which I877V shifts the Na+
-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 (). As before, the membrane was stepped to a wide range of potentials and stability was assessed by repeating voltage protocols in each condition (). Under these ionic conditions ouabain sensitive currents contain only transient components, reflecting the redistribution of external Na+
between occluded and deocluded states (see ). Examples of these currents for the unedited pump at three potentials are given in . Analysis of these traces shows that there are three kinetic components, as in the squid axon where each is thought to reflect the sequential release of one of the three Na+ 
. We first focused on the slowest component (τ~12 ms at 0 mV) because it tracks the rate-limiting transition for Na+
release and is therefore responsible for determining the Ip
-V relationship's shape. Its voltage-dependence was estimated by integrating the slow component of the off transients and the results are plotted in . As with the steady-state pump currents (), the I877V edit shifts the charge distribution 32 mV towards more negative potentials, indicating that the voltage dependence of the distribution between (Na3
)E1-P and P-E2
)Na states has been targeted. Is this due to a change in rates associated with this transition? The rate constants between these two states can be estimated by fitting the kinetics of the slow component to a simple model, derived from a Hill equation, that has been used to describe this transition in pumps from a variety of preparations 
, including the squid clone expressed in Xenopus
oocytes (). Conceptually, the model reduces Na+
release to two basic steps: a slow voltage independent conformational change between the occluded and deoccluded states, and a rapid redistribution of ions across a narrow pore that spans part of the membrane's electrical field, which is the step that renders the process voltage dependent 
. In this model, the relaxation rates reach asymptotes at extreme voltages. At positive potentials, the relaxations approach the forward rate, while at negative potentials they approach the sum of the forward and backward rates (see legend for ). The steepness of the curve is largely determined by the electrical depth of the access channel, a value that is unchanged by the I877V edit. Data in show that the forward rates for both constructs reach a similar asymptote at positive voltages and fits to the model indicate that the backwards rates do so as well. The small changes that I877V does cause to these rate constants are not sufficient to account for the shift in the voltage dependence of Ip
. Of greater significance, the model predicts that I877V considerably reduces the apparent affinity for extracellular Na+
, a change that could be caused by very different physical factors. Because the pump's cation binding sites are thought to be far from position 877, it is unlikely that this edit directly reduces the pump's affinity for Na+
. In addition, the amino acid change caused by the edit is conservative, making it unlikely that it changes the electrostatics along the ion permeation pathway 
, another mechanism that could plausibly affect the apparent affinity 
. An alternative that is more consistent with the amino acid change is that I877V shifts the state occupancy from deeply occluded states towards those that favor release.
The I877V edit shifts the voltage dependence of Na+ release.
The I877V edit shifts the relative proportion of each charge moving step.
Which transitions does I877V affect? Just as the transition from (Na3)E1-P↔P-E2(Na2)·Na can be tracked by the slow component of the relaxations, the transitions between P-E2(Na2)·Na↔P-E2(Na)Na and P-E2(Na)·Na↔P-E2·Na can be tracked by the medium (τ~1.5 ms at 0 mV) and fast components (τ<200 µs at 0 mV), respectively (). In order to estimate the transition rates between these states we would have to accurately measure the kinetics of each component. In our experimental set-up this is not possible because the time constant of the fast component is comparable to that of the clamp. However, by focusing on the proportion of charge carried by each component in the off transients, we can get a snapshot of the state occupancy during the conditioning pulse (). A visual inspection of off transients following a prepulse to −58 mV shows a clear difference caused by I877V (): the fast component is more pronounced and the slow component is reduced. A more rigorous quantification over a broad range of voltages shows that the reduction of the slow component in I877V comes at the expense of the fast component, a trend that is particularly apparent at positive voltages (). The medium component, on the other hand, carries about the same proportion of charge in both pumps at all voltages. From these data we conclude that I877V selectively targets the release of the last Na+ (fast component), thereby shifting the entire equilibrium towards release.