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Comparative Study
. 2012 Feb 17;335(6070):848-51.
doi: 10.1126/science.1212795. Epub 2012 Jan 5.

RNA Editing Underlies Temperature Adaptation in K+ Channels From Polar Octopuses

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Free PMC article
Comparative Study

RNA Editing Underlies Temperature Adaptation in K+ Channels From Polar Octopuses

Sandra Garrett et al. Science. .
Free PMC article

Abstract

To operate in the extreme cold, ion channels from psychrophiles must have evolved structural changes to compensate for their thermal environment. A reasonable assumption would be that the underlying adaptations lie within the encoding genes. Here, we show that delayed rectifier K(+) channel genes from an Antarctic and a tropical octopus encode channels that differ at only four positions and display very similar behavior when expressed in Xenopus oocytes. However, the transcribed messenger RNAs are extensively edited, creating functional diversity. One editing site, which recodes an isoleucine to a valine in the channel's pore, greatly accelerates gating kinetics by destabilizing the open state. This site is extensively edited in both Antarctic and Arctic species, but mostly unedited in tropical species. Thus adenosine-to-inosine RNA editing can respond to the physical environment.

Figures

Fig 1
Fig 1
RNA editing, but not gene level differences, change channel function. A, Current traces for Antarctic and tropical Kv1 genomic channels in response to a voltage step from -80 mV to +60 mV. Traces have been scaled in order to show the near identity in opening and closing kinetics. B, Representative current traces focusing on closing kinetics for genomic Antarctic and I321V edited channels. Currents were activated by a stimulus to +50 mV, but only their decay following a return to -80 mV is shown. C, Channel closing kinetics over a range of repolarization voltages following an activating step to +50 mV. Error bars = s.e.m; n = 16 for genomic and 9 for I321V. ◆ = genomic Antarctic and ■ = I321V edited. All data was recorded from voltage clamped Xenopus oocytes injected with cRNA for the appropriate construct and the temerparature was maintained at 15°C.
Fig 2
Fig 2
mRNAs encoding octopus Kv1 channels are extensively edited. A, Editing percentages for the 12 non-silent sites found in the Antarctic and tropical octopus Kv1 channels calculated by sequencing 50 individual cDNA clones for each channel. B, Octopus editing sites occur in different functional domains. Homologous positions to those altered by RNA editing in octopus Kv1 channels, shown in red, have been mapped on the Kv1.2 crystal structure(23). One full subunit of the tetramer is shown (blue); for the pore region, all four subunits are shown, each in a different color.
Fig 3
Fig 3
The editing site I321V speeds channel closing kinetics by destabilizing the open state. Current traces from cell attached patches containing single Antarctic genomic (A) and I321V (C) Kv1 channels. Channels opened in response to a voltage step from -80 mV to +60 mV. I321V channels close more frequently. Overlapping red lines (insets) show idealizations of traces as a series of open and closed events. B and D, Duration distributions of open events for genomic Antarctic and I321V channels. The average duration of open events in the genomic channel (3.42 ms) was about twice that of I321V channels (1.72 ms). E and F, Simple 5 state models for genomic Antarctic and I321V channel gating and simulations generated from the models. Doubling the backwards rate constant for the final transition recapitulates both the macroscopic difference in closing kinetics and the single channel behavior (G and H) between genomic and I321V channels. Rate constants were assumed to depend exponentially on voltage: k = k0e(zFV/RT) where F is the Faraday’s constant, V is voltage, R is the universal gas constant, and T is the temperature. For the above models: k1f = 700, z = 0.3; k1b = 50, z = 1.6; k2f = 1200, z = 1.8; k2b = 25, z = 1.1; k3f = 600, z = 0.8; k3b = 150, z = 1.5; k4f = 3000, z = 0.02; k4b variable, z = 0.2. A stochastic simulation of 1000 channels pulsed from -80mV to +60mV and back to -80mV showed faster closing with the I321V model. The maximum open probability was similar: 0.59 vs. 0.55 for genomic and I321V, respectively. Closing rates (single exponential fit, overlapping red line) were similar to those obtained from room temperature experimental data: 625 and 1250 (s-1) simulated kinetics vs. 610 and 1423 (s-1) experimental kinetics for genomic and I321V, respectively. For the single channel simulations, the average open duration using the I321V model was half that for the genomic model.
Fig 4
Fig 4
The extent of editing at I321V correlates with the water temperature where octopus species were captured. A, Collection sites for eight octopus species and the water temperatures and habitats at the time of capture. B, representative poison primer extension assay showing the amount of I321V editing among the eight species. D, I321V editing percentages for the eight species, based on poison primer extension assays, versus water temperature at capture site. Error bars = SEM; n = 4.

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