Recombinant tandem of pore-domains in a Weakly Inward rectifying K+ channel 2 (TWIK2) forms active lysosomal channels

Abstract

Recombinant TWIK2 channels produce weak basal background K⁺ currents. Current amplitudes depend on the animal species the channels have been isolated from and on the heterologous system used for their re-expression. Here we show that this variability is due to a unique cellular trafficking. We identified three different sequence signals responsible for the preferential expression of TWIK2 in the Lamp1-positive lysosomal compartment. Sequential inactivation of tyrosine-based (Y₃₀₈ASIP) and di-leucine-like (E₂₆₆LILL and D₂₈₂EDDQVDIL) trafficking motifs progressively abolishes the targeting of TWIK2 to lysosomes, and promotes its functional relocation at the plasma membrane. In addition, TWIK2 contains two N-glycosylation sites (N₇₉AS and N₈₅AS) on its luminal side, and glycosylation is necessary for expression in lysosomes. As shown by electrophysiology and electron microscopy, TWIK2 produces functional background K⁺ currents in the endolysosomes, and its expression affects the number and mean size of the lysosomes. These results show that TWIK2 is expressed in lysosomes, further expanding the registry of ion channels expressed in these organelles.

Figure 1

Comparison of human, rat and mouse TWIK2 channels in heterologous expression systems. TWIK2 currents were recorded in Xenopus oocytes 24 h after cRNA injection. (A) Current amplitude at +50 mV of human, rat and mouse TWIK2 compared to human K_2P channels TREK1 and TASK3. TWIK2 current is small (<1 µA) but significantly different from non-injected cells (non injected n = 56, human TWIK2 n = 23, p < 0.001, rat TWIK2 n = 47, p < 0.001, mouse TWIK2 n = 33, p < 0.001). Each value is the mean ± sem, statistic evaluation was done using Mann-Whitney test. (B) I-V relationships deduced from recordings with voltage pulses ranging from −120 mV to +60 mV in 10 mV steps from a holding potential of −80 mV (non injected n = 56, human n = 23, rat n = 47, mouse TWIK2 n = 33). The “inset” represents the evoked current shape of mouse TWIK2, 20 mV steps. (C) Human and rodent TWIK2 co-localization in transfected MDCK cells. Human TWIK2 was stained with rabbit polyclonal TWIK2 antibody and rat and mouse TWIK2HA with mouse monoclonal HA antibody. Overlap of green and red fluorescence appears in yellow. (C) TASK3 (in green) and TREK1 (in green) in transfected MDCK cells.

Figure 2

Localization of TWIK2 in lysosomal membranes. Representative images of MDCK cells expressing (A) GFP-coupled compartment markers and rat TWIK2HA, and (B) Lamp1-GFP together with mouse, rat or human TWIK2HA. TWIK2 was stained with HA antibody (red) and nuclei were visualized with Hoechst33342 (blue). Scale bar: 10 µm. (C) Image of a living MDCK cell expressing rat TWIK2-GFP and incubated with lysotracker (red), staining the lysosomal lumen. Scale bar: 10 µm. (D) Electronic microscopy images of a cell expressing rat TWIK2 (lower panel) or of a control cell (upper panel). Scale bar: 2 µm. Lysosomes are dense bodies (plain arrow).

Figure 3

TWIK2 currents in endolysosomes. Endolysosomal currents were recorded from HEK293T cells expressing rat TWIK2. (A) Currents recorded using a high K⁺-containing bath solution (145 mM K⁺, pH 7.2). Step protocols from −100 to +100 mV with a 20 mV step were applied from a holding potential of 0 mV. (B) Currents recorded using a high Na⁺-containing bath solution (145 mM Na⁺, pH 7.2). Same step protocol as in (A). (C) I-V relationships constructed from the currents recorded in (A) and (B). (D) Averaged current densities at 60 mV. (E) Potassium I-V curves (defined as $I_{\mathrm{cyto}\mathrm{high}{\mathrm{K}}^{+}}-I_{\mathrm{cyto}\mathrm{high}{\mathrm{Na}}^{+}}$). (F) Representative continuous recordings using ramp protocols (−100 mV to +100 mV in 500 ms, every 5 s, from a holding potential of 0 mV). (G) Recordings at time points (black and red arrows) in (F) were used for the I-V relationships. (H) Current recorded at cytosolic pH 7.4 and pH 4.6. Outward current denotes flow of positive charge into the lumen (pipette) from the cytosol (bath). Numbers of patched endolysosomes are shown in parentheses. Data are represented as mean ± s.e.m. Same step protocol as in (A). (I) I-V relationships constructed from the currents recorded in (H).

Figure 4

Identification of trafficking motifs in the C-ter of TWIK2. (A) Sequence of human, rat and mouse TWIK2 C-ter. The trafficking motifs are shown in bold. Overview of the set of generated mutants, in which underlined amino acids were replaced by an alanine residue. (B) Current amplitudes at +50 mV of the corresponding rat TWIK2 mutants recorded in Xenopus oocytes (number of recorded oocytes) expressed as mean current amplitude ± sem. Mutant rat TWIK2HA channels were transfected into MDCK cells and stained with HA antibody. % cell surface expression corresponds to the percentage of transfected cells showing a clear expression of TWIK2 at the plasma membrane. (C) Representative images of MDCK cells expressing the different TWIK2 mutations shown in (B) and TREK1. Scale bar: 10 µm.

Figure 5

Human TWIK2 shows the same properties as rat TWIK2 when expressed in heterologous expression systems. Human wild type and mutated TWIK2 channels were expressed as indicated in (A) Xenopus oocytes (current amplitudes at +50 mV, (number of recorded oocytes)), (B) MDCK cells for immunodetection with anti-HA antibody, (C) HEK cells for electrophysiological recordings (n = 15), and (D) for immunodetection with anti-HA antibody.

Figure 6

Cytoplasmic TWIK2 C-ter drives channel targeting to lysosomal membranes. (A) Co-transfection of rat TWIK2 with TASK3HA containing an extracellular HA-tag or TASK3HA-TW2Ct and staining with anti-HA (green) and anti-TWIK2 (red) antibody. (B) MDCK cell transfection with TASK3HA, TASK3HA-TW2Ct or TASK3HA-TW2Ct mutated at Y308A and IL289/290AA. (C) MDCK cell transfection with TASK3HA or TASK3HA-TW2Ct fused to GFP and stained without permeabilisation. (D) Co-expression of mouse TWIK1 or rat TWIK2 with Dynamin-GFP or Dynamin-K44A-GFP. Scale bar: 10 µm.

Figure 7

Glycosylation of rat TWIK2 in the M1-P1 extracellular/lumenal loop. (A) TWIK2 membrane topology and localization of the N-glycosylation sites. (B) TWIK2 deglycosylation. Protein lysates from COS cells expressing TWIK2 were incubated with PNGaseF and/or EndoH. The blot images have been cropped, only bands corresponding to glycosylated and deglycosylated TWIK2 isoforms are shown. (C) Mutation of the glycosylation sites (N to L) has the same effect as incubation with PNGaseF and EndoH. The blot images have been cropped, only bands corresponding to glycosylated and deglycosylated TWIK2 isoforms are shown. (D) Immunolabelling in MDCK transfected cells. TWIK2 N₇₉L and TWIK2 N₈₅L have a lysosomal distribution whereas TWIK2 N₇₉LN₈₅L is detected in the endoplasmic reticulum. Scale bar: 10 µm.