Ca2+ permeability and Na+ conductance in cellular toxicity caused by hyperactive DEG/ENaC channels

Abstract

Hyperactivated DEG/ENaC channels cause neuronal death mediated by intracellular Ca²⁺ overload. Mammalian ASIC1a channels and MEC-4(d) neurotoxic channels in Caenorhabditis elegans both conduct Na⁺ and Ca²⁺, raising the possibility that direct Ca²⁺ influx through these channels contributes to intracellular Ca²⁺ overload. However, we showed that the homologous C. elegans DEG/ENaC channel UNC-8(d) is not Ca²⁺ permeable, yet it is neurotoxic, suggesting that Na⁺ influx is sufficient to induce cell death. Interestingly, UNC-8(d) shows small currents due to extracellular Ca²⁺ block in the Xenopus oocyte expression system. Thus, MEC-4(d) and UNC-8(d) differ both in current amplitude and Ca²⁺ permeability. Given that these two channels show a striking difference in toxicity, we wondered how Na⁺ conductance vs. Ca²⁺ permeability contributes to cell death. To address this question, we built an UNC-8/MEC-4 chimeric channel that retains the calcium permeability of MEC-4 and characterized its properties in Xenopus oocytes. Our data support the hypothesis that for Ca²⁺-permeable DEG/ENaC channels, both Ca²⁺ permeability and Na⁺ conductance contribute to toxicity. However, for Ca²⁺-impermeable DEG/ENaCs (e.g., UNC-8), our evidence shows that constitutive Na⁺ conductance is sufficient to induce toxicity, and that this effect is enhanced as current amplitude increases. Our work further refines the contribution of different channel properties to cellular toxicity induced by hyperactive DEG/ENaC channels.

Keywords: DEG/ENaC channels; calcium permeability; neurotoxicity.

Fig. 1.

An UNC-8/MEC-4 chimera with Ca²⁺ permeability and strong Ca²⁺ block. A: schematic representation of DEG/ENaC subunit topology for UNC-8 (d), MEC-4 (d), and UNC-8(d)/MEC-4(TM2) chimera. Black dots mark the relative position of the hyperactivating mutation in each channel subunit. B: protein sequence alignment of the second transmembrane (TM2) domains of MEC-4 and UNC-8. Black bold denotes the mutated amino acids shown in Table 1. GxS is the proposed “selectivity filter” of DEG/ENaC channels, and the black box shows the TM2a region swapped in UNC-8(d)/MEC-4(TM2a) chimera. C: representative currents from a noninjected oocyte perfused with a solution containing 73 mM Ca²⁺. Currents were elicited by voltage steps from −160 to +50 mV in 20-mV increments; 0 mV was the holding potential. No Ca²⁺-activated Cl⁻ currents are elicited in noninjected oocytes. D: an oocyte expressing the UNC-8(d)/MEC-4 (TM2) chimera was perfused with the same 73 mM calcium solution. In this oocyte the Ca²⁺-activated Cl⁻ current is activated. E: quantification of Ca²⁺-activated Cl⁻ currents measured at −160 mV at the time point of ∼150 ms (at the peak current) in noninjected oocytes and oocytes injected with the UNC-8(d)/MEC-4(TM2) chimera. Data are expressed as means ± SE, n = 4 and 5, respectively. ****P ≤ 0.0001 by Student's t-test, (P = 2.394, E-6). F: representative current traces recorded from an oocyte expressing the UNC-8(d)/MEC-4(TM2) chimera perfused with the physiologic NaCl solution. Currents were elicited by voltage steps from −160 to +100 mV in 20-mV increments starting from a holding potential of −30 mV. The same oocyte shown in F was perfused with a divalent cation-free NaCl solution containing 1 mM EGTA (denoted div.free + 1 EGTA). G: average current amplitude recorded in oocytes expressing the UNC-8(d)/MEC-4(TM2) chimera measured at −100 mV at the 300-ms time point in physiologic NaCl solution (denoted physiologic) and in divalent cation-free NaCl solution (denoted div.free). Data are expressed as means ± SE, n = 18. ****P ≤ 0.0001 by Student's t-test (P = 9.200, E-14). Oocytes were incubated with amiloride 12 h after injection until the recordings were made. H: calcium dose-response for oocytes expressing the UNC-8(d)/MEC-4(TM2) chimera. Currents were recorded at −100 mV at the 300-ms time point in oocytes perfused with increasing concentrations of extracellular calcium (see materials and methods). Currents in the presence of calcium were normalized for the current recorded in same oocyte perfused with divalent cation-free plus EGTA NaCl solution. Data are expressed as means ± SE. Data points were fitted with a sigmoidal curve for a K_i of 9.5 μM, n = 9. I: magnesium dose-response of oocytes expressing the UNC-8(d)/MEC-4(TM2) chimera. Currents were recorded at −100 mV at the 300-ms time point in oocytes perfused with increasing concentrations of extracellular magnesium (see materials and methods). Currents in the presence of magnesium were normalized for the current recorded in same oocyte perfused with divalent cation-free NaCl solution. Data are expressed as means ± SE. Data points were fitted with a sigmoidal curve for a K_i of 536.3 μM, n = 11.

Fig. 2.

UNC-8(d)/MEC-4(TM2) current properties. A: normalized current measured at −100 mV at the 300-ms time point in oocytes expressing UNC-8(d) perfused with the solutions indicated on the x-axis. Data are expressed as means ± SE, n = 6. B: similar as in A normalized current measured at −100 mV at the 300-ms time point in oocytes expressing the UNC-8(d)/MEC-4(TM2) chimera perfused with the solutions indicated on the x-axis. Data are expressed as means ± SE, n = 13. Unlike UNC-8(d) but similarly to MEC-4(d), the UNC-8(d)/MEC-4(TM2) chimera is more permeable to Li⁺ ions. C: currents recorded in oocytes expressing UNC-8(d) (open circles) in the presence of increasing concentrations of amiloride at −100 mV at the 300-ms time point were normalized against currents recorded in divalent cation-free plus EGTA NaCl solution at −100 mV at the 300-ms time point. Data were fitted with a sigmoidal curve for a K_i of 287 μM. Data are expressed as means ± SE, n = 8. D: the same as in C for oocytes expressing the UNC-8(d)/MEC-4(TM2) chimera (open squares). Data were fitted with a sigmoidal curve for a K_i of 94 μM. Data are expressed as means ± SE, n = 8. There is a significant difference between UNC-8(d) and UNC-8(d)/MEC-4(TM2) chimera amiloride sensitivity (P = 0.0068 by Student's t-test). E: representative current traces recorded in oocytes expressing the UNC-8(d)/MEC-4(TM2) chimera, UNC-8(d)/MEC-4(TM2) coexpressed with MEC-2, and UNC-8(d)/MEC-4(TM2) coexpressed with both MEC-2 and MEC-6 perfused with a physiologic saline. F: average current amplitude measured at −100 mV at the 300-ms time point recorded from oocytes expressing the UNC-8(d)/MEC-4(TM2) chimera, UNC-8(d)/MEC-4(TM2) + MEC-2, and UNC-8(d)/MEC-4(TM2) + MEC-2 + MEC-6. Data are expressed as means ± SE, n = 18, 11, and 18 respectively. There was no statistical difference between the groups. G: average current amplitude measured at −100 mV at the 300-ms time point recorded in oocytes expressing MEC-4(d), MEC-4(d) + MEC-2, and MEC-4(d) + MEC-2 + MEC-6. Data are expressed as means ± SE (n = 5, 12, and 10, respectively). *, ****Significant difference compared with MEC-4(d) expressed alone by ANOVA with Bonferroni correction (P < 0.0001 and 0.0002, respectively). H: average current amplitude measured at −100 mV at the 300-ms time point recorded in oocytes expressing UNC-8(d), UNC-8(d) + MEC-2, and UNC-8(d) + MEC-2 + MEC-6. Data are expressed as means ± SE (n = 8, 8, and 9, respectively). Oocytes were incubated in ND96 plus 500 μM amiloride 12 h after injection until recordings were made. Amiloride was washed away prior to recordings.

Fig. 3.

Eliminating Na⁺ ions from the extracellular solution reduces current amplitude. A: examples of currents recorded in an oocyte expressing UNC-8(d) + MEC-2 + MEC-6 perfused with 0.005 mM Ca²⁺ (reduced calcium divalent cation-free NaCl solution) (top left) and 0 Na⁺, 0.005 mM Ca²⁺ (reduced calcium divalent cation free choline solution) in which sodium was replaced with the impermeant ion choline (top right). Middle: same solutions as in top for oocytes expressing the UNC-8(d)/MEC-4(TM2) chimera + MEC-2 + MEC-6. Bottom: same solutions as in top for oocytes expressing MEC-4(d) + MEC-2 + MEC-6. Currents were elicited by voltage steps from −160 to +100 mV in 20-mV increments, starting from a holding potential of −30 mV. B: comparison of average current amplitudes measured at −100 mV at the 300-ms time point in oocytes expressing UNC-8(d) + MEC-2 + MEC-6 (open circles), UNC-8(d)/MEC-4(TM2) + MEC-2 +MEC-6 (open squares), and MEC-4(d) + MEC-2 + MEC-6 (open triangles) in physiologic 0.005 mM Ca²⁺ (reduced calcium NaCl) solution (black column bars) vs. 0 Na⁺, 0.005 mM Ca²⁺ (reduced calcium choline) solution (gray column bars). Data are expressed as means ± SE, n = 22 and 4 for UNC-8(d) + MEC-2 + MEC-6 (open circles), n = 14 and 5 for UNC-8(d)/MEC-4(TM2) + MEC-2 +MEC-6 chimera (open squares), and n = 17 and 7 for MEC-4(d) + MEC-2 + MEC-6 (open triangles), respectively. ****P < 0.0001 by Student's t-test for comparison within each group in 0.005 mM Ca²⁺ (reduced calcium NaCl) vs. 0 Na⁺, 0.005 mM Ca²⁺ (reduced calcium choline). We also compared data in physiologic 0.005 mM Ca²⁺ across groups using one-way ANOVA with Bonferroni correction (P values are indicated in the graph).

Fig. 4.

Eliminating Na⁺ ions rescues cell death in UNC-8(d) but not in Ca²⁺-permeable MEC-4(d)-expressing oocytes. A: representative photographs of an intact oocyte (left) and a dead oocyte (right) after 72 h of incubation in reduced calcium solution. B: black column bars represent cell death of oocytes incubated in 0.005 mM Ca²⁺ (reduced calcium ND96) solution in which the major cation was Na⁺. Gray column bars represent cell death for oocytes incubated in 0 Na⁺, 0.005 mM Ca²⁺ (reduced calcium modified choline ND96) solution. After 48 h of incubation, cell death was quantified as the fraction of dead oocytes over the total number of incubated oocytes. For all conditions cell death of oocytes incubated in respective buffer plus 500 μM amiloride was subtracted before plotting. Data are expressed as means ± SE and are an average of four independent experiments with n = 10 oocytes per group per condition. Data within each group in 0.005 mM Ca²⁺ (reduced calcium NaCl) vs. 0 Na⁺, 0.005 mM Ca²⁺ (reduced calcium choline) were compared using the Student's t-test. ****P < 0.0001, **P = 0.0091, *P = 0.0478. We also compared data in physiologic 0.005 mM Ca²⁺ across groups using one-way ANOVA with Bonferroni correction, P values are indicated in the graph. C: same as in B for oocytes incubated in physiologic (ND96) solution (black column bars) and in 0 Na⁺ (modified ND96 containing choline instead of Na ions) solution (gray column bars). Data are expressed as means ± SE and are an average of four independently performed experiments with n = 10 oocytes per group per condition. No statistical significance was detected between the groups.