An animal toxin-antidote system kills cells by creating a novel cation channel
Fig 5
PEEL-1 and PMPL-1 create a monovalent cation channel.
Current–voltage plots of whole-cell patch-clamp electrophysiology on (A) transfected cells, (B) Control cell line, and (C) Experimental cell line. High intracellular potassium (140 mM K+/8.6 mM Na+) and high extracellular sodium (145 mM Na+/4 mM K+) solutions are used. Currents elicited by a family of 0.5 s voltage steps from a −30 mV holding potential, from −100 mV to 60 mV, in 10 mV increments. Currents normalized to cell capacitance (pF). Negative and positive currents indicate cation flow into or out of the cell, respectively. (A) Plots from HEK293 cells acutely transfected with peel-1::eGFP or pmpl-1::mCherry. (B) Plots from Control cell line expressing constitutive eGFP and tetracycline-inducible pmpl-1::mCherry. (C) Plots from Experimental cell line expressing constitutive peel-1::eGFP and tetracycline-inducible pmpl-1::mCherry. Control and Experimental cell lines are shown without tetracycline or with tetracycline at the indicated time after addition of tetracycline. All plots shown as mean with SEM. (D) Western blot for PMPL-1::mCherry in Control and Experimental cell lines without tetracycline (−tet, left) and 18 hrs after addition of tetracycline (+tet, right). Leaky expression of PMPL-1 is seen in both cell lines in the absence of tetracycline. Less background PMPL-1 expression is seen in Experimental cells than in Control cells, likely because of selection against higher background PMPL-1 expression when in combination with PEEL-1 but not eGFP. GAPDH loading control shown. Original blots available in S1 Raw images. (E) Permeability of indicated ions was assayed in Experimental cells without tetracycline. Test ionic solutions substituted previous bath solution (145 mM Na+/4 mM K+) with 140 mM pure cations (external) or 20 mM anions (internal), except Ca2+ (20 mM external, 1 mM internal Ca2+ with 2.5 mM EGTA). All plots show mean with SEM. Statistical tests compare all results to NMDG (treated as control) in one-way ANOVA with Dunnett’s multiple comparisons test (*p < 0.05; ***p < 0.001). (F) Schematic of planar lipid bilayer experiment. Liposomes containing purified PEEL-1 or PMPL-1 are added to the cis well to deliver proteins to the lipid bilayer. (G) Conductance traces of one experiment (left) and a histogram of the trace (right, 2 pS bin width, normalized based on probability density) at an applied voltage of +180 mV. SDS–PAGE gels of purified proteins are shown in S15A Fig and more example conductance traces and controls are shown in S16 Fig. Underlying data are available in S1 Data.