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Regulation of Na+/K+ ATPase transport velocity by RNA editing.

Colina C, Palavicini JP, Srikumar D, Holmgren M, Rosenthal JJ - PLoS Biol. (2010)

Bottom Line: Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis.In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing.Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurobiology, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico.

ABSTRACT
Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis. In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+) release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

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The I877V edit shifts the voltage dependence of the Na+/K+ pump's turnover rate.Na+/K+ pump currents were studied using the cut-open oocyte voltage-clamp with Xenopus oocytes expressing the unedited and I877V versions of SqNaKα1. (A) Chart recording at a slow sampling rate (1 kHz) of the entire experiment used to measure Na/K pump currents at different voltages. Current-voltage patterns (40 ms steps from −198 mV to 42 mV in 10 mV increments) were recorded twice in a 5 mM K+ external solution and then repeated in the same solution with 100 uM ouabain. The stability of the oocyte was monitored by subtracting I-V patterns recorded under the same condition (1–2 and 3–4). (B) Na+/K+ pump currents were measured by isolating the ouabain sensitive component (2–3) and recording the steady-state value (arrow; average of last 5 ms). The dark trace was recorded at 42 mV and the light trace at −198 mV. Presteady-state transients result from Na+-Na+ exchange. (C) Current measurements normalized to maximum values for SqNaKα1 (cyan circles; n = 6) and SqNa/Kα1 I877V (red circles; n = 8). Error bars (s.e.m.) are only shown when they are larger than the symbols.
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pbio-1000540-g003: The I877V edit shifts the voltage dependence of the Na+/K+ pump's turnover rate.Na+/K+ pump currents were studied using the cut-open oocyte voltage-clamp with Xenopus oocytes expressing the unedited and I877V versions of SqNaKα1. (A) Chart recording at a slow sampling rate (1 kHz) of the entire experiment used to measure Na/K pump currents at different voltages. Current-voltage patterns (40 ms steps from −198 mV to 42 mV in 10 mV increments) were recorded twice in a 5 mM K+ external solution and then repeated in the same solution with 100 uM ouabain. The stability of the oocyte was monitored by subtracting I-V patterns recorded under the same condition (1–2 and 3–4). (B) Na+/K+ pump currents were measured by isolating the ouabain sensitive component (2–3) and recording the steady-state value (arrow; average of last 5 ms). The dark trace was recorded at 42 mV and the light trace at −198 mV. Presteady-state transients result from Na+-Na+ exchange. (C) Current measurements normalized to maximum values for SqNaKα1 (cyan circles; n = 6) and SqNa/Kα1 I877V (red circles; n = 8). Error bars (s.e.m.) are only shown when they are larger than the symbols.

Mentions: The Na+/K+ 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, Figure 3A 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). Figure 3B 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. Figure 3C 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 [10]. From this we estimate that I877V would significantly increase Ip at the resting potential, which is ∼−60 mV in the squid axon [27]. Under physiological conditions the pump's voltage dependence comes mostly from the transitions underlying extracellular Na+ release [5],[6],[8],[10],[28]–[30]. Therefore, these results suggest that the I877V edit targets this process.


Regulation of Na+/K+ ATPase transport velocity by RNA editing.

Colina C, Palavicini JP, Srikumar D, Holmgren M, Rosenthal JJ - PLoS Biol. (2010)

The I877V edit shifts the voltage dependence of the Na+/K+ pump's turnover rate.Na+/K+ pump currents were studied using the cut-open oocyte voltage-clamp with Xenopus oocytes expressing the unedited and I877V versions of SqNaKα1. (A) Chart recording at a slow sampling rate (1 kHz) of the entire experiment used to measure Na/K pump currents at different voltages. Current-voltage patterns (40 ms steps from −198 mV to 42 mV in 10 mV increments) were recorded twice in a 5 mM K+ external solution and then repeated in the same solution with 100 uM ouabain. The stability of the oocyte was monitored by subtracting I-V patterns recorded under the same condition (1–2 and 3–4). (B) Na+/K+ pump currents were measured by isolating the ouabain sensitive component (2–3) and recording the steady-state value (arrow; average of last 5 ms). The dark trace was recorded at 42 mV and the light trace at −198 mV. Presteady-state transients result from Na+-Na+ exchange. (C) Current measurements normalized to maximum values for SqNaKα1 (cyan circles; n = 6) and SqNa/Kα1 I877V (red circles; n = 8). Error bars (s.e.m.) are only shown when they are larger than the symbols.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990702&req=5

pbio-1000540-g003: The I877V edit shifts the voltage dependence of the Na+/K+ pump's turnover rate.Na+/K+ pump currents were studied using the cut-open oocyte voltage-clamp with Xenopus oocytes expressing the unedited and I877V versions of SqNaKα1. (A) Chart recording at a slow sampling rate (1 kHz) of the entire experiment used to measure Na/K pump currents at different voltages. Current-voltage patterns (40 ms steps from −198 mV to 42 mV in 10 mV increments) were recorded twice in a 5 mM K+ external solution and then repeated in the same solution with 100 uM ouabain. The stability of the oocyte was monitored by subtracting I-V patterns recorded under the same condition (1–2 and 3–4). (B) Na+/K+ pump currents were measured by isolating the ouabain sensitive component (2–3) and recording the steady-state value (arrow; average of last 5 ms). The dark trace was recorded at 42 mV and the light trace at −198 mV. Presteady-state transients result from Na+-Na+ exchange. (C) Current measurements normalized to maximum values for SqNaKα1 (cyan circles; n = 6) and SqNa/Kα1 I877V (red circles; n = 8). Error bars (s.e.m.) are only shown when they are larger than the symbols.
Mentions: The Na+/K+ 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, Figure 3A 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). Figure 3B 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. Figure 3C 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 [10]. From this we estimate that I877V would significantly increase Ip at the resting potential, which is ∼−60 mV in the squid axon [27]. Under physiological conditions the pump's voltage dependence comes mostly from the transitions underlying extracellular Na+ release [5],[6],[8],[10],[28]–[30]. Therefore, these results suggest that the I877V edit targets this process.

Bottom Line: Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis.In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing.Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurobiology, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico.

ABSTRACT
Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis. In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+) release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

Show MeSH
Related in: MedlinePlus