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. 2008 Dec 1;586(23):5651-63.
doi: 10.1113/jphysiol.2008.161927. Epub 2008 Oct 9.

Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiation

Dina Simkin  1 , Eric J Cavanaugh, Donghee Kim
Affiliations

Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiation

Dina Simkin et al. J Physiol. .

Abstract

TREK-2 expressed in mammalian cells exhibits small ( approximately 52 pS) and large ( approximately 220 pS) unitary conductance levels. Here we tested the role of the N-terminus (69 amino acids long) in the control of the unitary conductance, and role of the alternative translation initiation as a mechanism that produces isoforms of TREK-2 that show different conductance levels. Deletion of the first half (Delta1-36) of the N-terminus had no effect. However, deletion of most of the N-terminus (Delta1-66) resulted in the appearance of only the large-conductance channel ( approximately 220 pS). In support of the critical function of the distal half of the N-terminus, the deletion mutants Delta1-44 and Delta1-54 produced approximately 90 pS and 188 pS channels, respectively. In Western blot analysis, TREK-2 antibody detected two immunoreactive bands at approximately 54 kDa and approximately 60 kDa from cells expressing wild-type TREK-2 that has three potential translation initiation sites (designated M(1)M(2)M(3)) within the N-terminus. Mutation of the second and third initiation sites from Met to Leu (M(1)L(2)L(3)) produced only the approximately 60 kDa isoform and the small-conductance channel ( approximately 52 pS). Mutants designed to produce translation from the second (M(2)L(3)) or third (M(3)) initiation site produced the approximately 54 kDa isoform, and the large conductance channel ( approximately 185-224 pS). M(1)L(2)L(3), M(2)L(3) and M(3) were relatively selectively permeable to K(+), as judged by the 51-55 mV shifts in reversal potential following a 10-fold change in [K(+)](o). P(Na)/P(K) values were also similar for M(1)L(2)L(3) ( approximately 0.02), M(2)L(3) ( approximately 0.02) and M(3) ( approximately 0.03). Arachidonic acid, proton and membrane stretch activated, whereas dibutyryl-cAMP inhibited all three isoforms of TREK-2, indicating that deletion of the N-terminus does not abolish modulation. These results show that the small and large conductance TREK-2 channels are produced as a result of alternative translation initiation, producing isoforms with long and short N-termini, and that the distal half of the N-terminus controls the unitary conductance.

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Figures

Figure 1
Figure 1. Different conductance levels of TREK-2 expressed in HeLa cells
A, current recordings from cell-attached patches. Pipette potential was held at +40 (cell membrane potential of ∼−40 mV) or −40 mV. S and L indicate small and large conductance levels indicated by dotted lines. B, current recordings from cell-attached patches containing only the small (TREK-2-S) or large (TREK-2-L) conductance channel. C, plot of the mean current amplitude at +40 and −40 mV (n = 5; mean ±s.e.m.). Asterisk indicates a significant difference (P < 0.05). D, relative channel activity of TREK-2-S and TREK-2-L measured from cell-attached patches (n = 10; mean ±s.e.m.). Asterisk indicates a significant difference from other bar (P < 0.05).
Figure 2
Figure 2. Deletion of the N-terminus on the conductance level
The amino acid sequence of the N-terminus (residues 1–69) is shown at the top. The region containing the residues 48–56 indicated by the line has a predicted α-helical structure, and the regions on either side of the α-helix are predicted random coil, based on secondary structure prediction programs (DPM, HNNC, Predator, nnPredict and PHDsec). Current tracings show single channel recordings from wild-type TREK-2 and deletion mutants, as indicated. For all deletions, the beginning amino acid was changed to methionine. Membrane potential was +40 mV. Small and large conductance levels are indicated by dotted lines (S and L).
Figure 3
Figure 3. Western blot analysis and single channel recordings of TREK-2 and its mutants
A, cells expressing TREK-2, TREK-1, TRAAK or TREK-2 tagged with HA epitope were prepared for Western blot analysis. Two immunoreactive bands were detected using the TREK-2 antibody, whereas only one band was detected with the HA antibody. B, TREK-2, various mutants designed to elicit translation of initiation at specific sites were expressed in HeLa cells, and immunoreactive proteins were analysed with TREK-2 antibody. In cells expressing c-myc-tagged TREK-2, antibody to c-myc was used. C, single channel currents from cell-attached patches from HeLa cells expressing TREK-2 mutants, and the corresponding amplitude histograms (+40 mV) are shown.
Figure 4
Figure 4. Permeability properties of TREK-2 isoforms to K+ and Na+
A, large outside-out patches were formed from cells expressing TREK-2 mutants each designed to produce one type of isoform. Pipette solution contained 140 mm K+, and the bath solution contained different ratio of K+ and Na+ as indicated. Arachidonic acid (5 μm) was added to the pipette to activate the channels. Macroscopic currents were determined at various membrane potentials and plotted as shown for M1L2L3, M2L3 and M3. B, shifts in reversal potential (Erev) are plotted as a function of external [K+], and fitted to a linear line. Dotted line indicates the calculated values from the Nernst equation. C, large outside-out patches with pipette that has 140 mm K+. Bath solution contains 140 mm Na+. D, the bar graph shows the reversal potentials for M1L2L3, M2L3 and M3 from experiments in C (n = 4; mean ±s.e.m.). No significant difference was present (P > 0.05).
Figure 5
Figure 5. Responses of TREK-2 isoforms to three activators
A, inside-out patches expressing M1L2L3 were formed, and three activators (pH 5.8), suction (−80 mmHg) or arachidonic acid (20 μm) was applied briefly. Membrane potential is held at −40 mV. B, same experiments as A except that patches express M3. C, channel activities before and after activation are plotted for M1L2L3, M2L3 and M3. Each bar is the mean ±s.e.m. of 4–5 determinations. All three activators produced significant increases in activity from the corresponding basal level (P < 0.05).
Figure 6
Figure 6. Intermediate conductance levels of TREK-2 and its deletion mutants (M2L3 and M3)
Current recordings are from cell-attached patches at a holding membrane potential of ∼+40 mV. A, channel openings of wild-type TREK-2 show small, intermediate and large conductance levels indicated by dotted lines. B, channel openings of M2L3 show large and intermediate levels. Amplitude histogram obtained from the second tracing shows the ∼120 pS subconductance level. C, channel openings of M3 show mainly the large conductance level. Amplitude histogram shows the ∼150 pS subconductance level.
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