Figure 6. RRV cytotoxicity is driven by the influx of both Na⁺ and Ca²⁺.

(a) Schematic of the planar lipid bilayer setup showing the sequence of cis buffer perfusions. (b) The APOL1 channel is readily permeable to Na⁺, but not choline⁺. In symmetrical KCl solutions E_rev was determined as + 1 mV. Then, after cis perfusion with equimolar NaCl buffer (pH 7.2, horizontal bar) there was only a slight change in E_rev (E_rev = −2 mV; 4 mV scale). In contrast, E_rev increased to + 60 mV after exchanging the cis solution for chamber buffer containing equimolar choline chloride. Substituting into the Goldman-Hodgkin-Katz equation (assuming zero permeability to chloride) gives K:Na and K:choline permeability ratios of 1.0:1.1 and 1.0:0.1 respectively. There are two breaks in the record (indicated by //), during which the perfuser was recharged with the appropriate solution. (c) The cytotoxicity of the RRVs in RUSH transfected HEK293 cells is significantly reduced by lowering extracellular Na⁺ from 150 mM to 85 mM. The rescue from cytotoxicity was indistinguishable between replacement with either K⁺ or choline⁺ (n = 9). (d) RRV cytotoxicity was reduced by lowering extracellular Ca²⁺ from 1.8 mM to 0.45 or 0.1125 mM (n = 12). (e) Reduction of both extracellular Ca²⁺ and Na⁺ (replaced by choline⁺) has an additive effect in lowering RRV cytotoxicity, as seen by further rescue from cell death with 0.45 mM Ca²⁺ combined with 85 mM Na⁺ (n = 13). (c–e) Cell death was assayed 12 hr post-biotin treatment with the Promega MultiTox fluorescent assay. Two-way ANOVAs with multiple comparisons were performed. (f) RRV mediated cytotoxicity is driven by the concurrent influx of both Ca²⁺ and Na⁺. CHO cells were co-transfected with either RUSH-G0 or G2, GCaMP6f, and the membrane voltage sensor FliCR. G2 cells exhibiting the established phenotype of Ca²⁺ influx followed by cell swelling were analyzed for changes in membrane voltage (used as a surrogate for the influx of Na⁺) (n = 21). G0 cells treated with biotin were analyzed for comparison (n = 7). Blue boxes and arrows indicate when the sustained increase in Ca²⁺ initiates. The representative cells from this figure can be viewed in Figure 6—video 1.

Figure 6—figure supplement 1. Replacement of Na⁺ with K⁺ significantly reduces cell viability.

RRV cytotoxicity is exacerbated with increased extracellular Ca²⁺. (a) Replacement of extracellular NaCl with KCl for 12 hr significantly reduces cell viability in RUSH-G0 transfected HEK293 cells (no biotin was added) (n = 6). (b) Further replacement of NaCl with choline Cl for 12 hr also reduces cell viability, but only below 86.33 mM Na⁺ (n = 3). (c) Reduction of extracellular CaCl₂ lowers the cytotoxicity of RUSH-G1. (a–c) Cell viability and death were measured 12 hr post-biotin treatment using the Promega MultiTox fluorescent assay. (d–e) Increasing the levels of CaCl₂ in the media exacerbates RRV cytotoxicity in both FT293 (d), n = 10) and RUSH-transfected HEK 293 cells (e), n = 3). Cytotoxicity was assayed 24 hr post induction/biotin treatment via release of lactate dehydrogenase. (a, c–e) Analysis was performed with two-way ANOVAs using multiple comparisons. In (a and c), cells incubated with the indicated salt reductions/replacements were compared with the controls in each treatment group. (b) A one-way ANOVA multiple comparisons test was performed.

Figure 6—figure supplement 2. Validation of the FliCR sensor.

Release of RUSH-G0 from the ER does not elicit a sustained increase in cytoplasmic Ca²⁺ or membrane depolarization. (a) Fluorescent trace of a representative CHO cell transfected with the plasma membrane voltage sensor FliCR. Cells were depolarized by adding 50 mM KCl to the media while imaging. (b) RUSH-G0 does not lead to a sustained increase in GCaMP6f or FliCR fluorescence after biotin treatment (n = 7). Some cells elicit temporary increases in cytoplasmic Ca²⁺ and membrane voltage, which may be due to signaling events or passage through the cell cycle.

Figure 6—figure supplement 3. The RUSH-G2-mediated Ca²⁺ influx occurs concurrently with a modest depolarization of the cell (Na⁺ influx), followed by complete depolarization prior to cell death.

The G2-mediated Ca²⁺-influx occurs alongside a Na⁺ influx (as measured via membrane voltage) (n = 21). Cytoplasmic Ca²⁺ continues to increase for several hours while the membrane potential either steadily increases alongside it, or becomes erratic and then undergoes depolarization prior to cell death.

Figure 6—video 1. The G2-mediated Ca²⁺ influx occurs concurrently with the influx of Na⁺.

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CHO cells were co-transfected with RUSH-G2, GCaMP6f, and FliCR for 24 hr prior to imaging. On the day of the experiment cells were treated with 80 µM biotin and imaged every 5 min from 0.5 to 12 hr post-biotin (the cells represented here, from Figure 5, underwent lysis by 5 hr). Cells that displayed the established phenotype of Ca²⁺ influx followed by cell swelling were selected. Scale bar = 20 µm.