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. 2020 Apr 1;9:e53308. doi: 10.7554/eLife.53308

Figure 4. hsElkin1 and mmElkin1-dependent currents exhibit distinct mechano-sensitivity.

(A) Example traces of M. musculus Elkin1-dependent MA currents in HEK-293T P1KO cells (grey: mmElkin1-iso1; blue mmElkin1-iso1 F271L N292G). (B) Stimulus-response plots of HEK-293T P1KO cells expressing: mmElkin1-iso1 (open triangles, n = 8 cells), mmElkin1-iso1 F271L N292G (blue triangles, n = 9 cells), hsElkin1-iso3 L210F (orange squares, n = 8 cells), hsElkin1-iso3 G231N, (green squares, n = 8 cells). Data points presented as mean ± s.e.m. Cartoons of stimuli adapted from Rocio Servin-Vences et al. (2017). See Figure 4—figure supplement 1 for sequence alignment of hsElkin1 and mmElkin1, Figure 4—figure supplement 2 for surface biotinylation analysis of Elkin1 variants and Figure 4—source data 1 for details on current kinetics.

Figure 4—source data 1. Source data for details of current kinetics.

Figure 4.

Figure 4—figure supplement 1. Sequence alignment of human and mouse Elkin1 protein and the effect of N-terminal deletions on hsElkin1 function.

Figure 4—figure supplement 1.

(A) Sequence alignment of the human (hs) Elkin1-iso1 and -iso3 against the mouse (mm) Elkin1 protein. The predicted transmembrane regions are marked with green, conservative residue variations in teal and non-conservative residue variations in yellow. The two residues that have been studied here are marked with a magenta star. Arrows indicate the start site for cloned truncation proteins. (B) Stimulus-response curve analysis of hsElkin1-iso3 versus hsElkin1 truncation mutants. Note that function is only lost once the first predicted TM domain is disrupted.
Figure 4—figure supplement 2. Cell-surface biotinlyation of Elkin1-GFP fusion proteins.

Figure 4—figure supplement 2.

GFP-tagged Elkin1 variants were overexpressed in HEK-293T P1KO cells. Cells were biotinylated and the cell-surface fraction isolated by pulldown of biotinylated proteins using Neutravidin beads. Input samples and the surface fraction were separated using SDS-PAGE, transferred to a PVDF membrane and Elkin-1-GFP fusion proteins were detected using an anti-GFP antibody. Human (hs) Elkin1 isoforms 1 and 3 and truncations ∆0–169, ∆0–207 were all detected in the plasma membrane. The Elkin1-iso1 mouse (mm) variant, and the mutated hs variants (G292N and L271F) that did not respond efficiently to deflection stimuli were still present in the plasma membrane fraction, as was the mouse variant F271L, N292G. As such, the differences in mechanically activated currents that depend on these isoforms cannot be explained by a change in localisation of the protein. In contrast, the lack of activity of the human ∆0–269 variant can be attributed to a lack of stability of the protein, as this variant is no longer detected in the input samples, or the plasma membrane.