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Figure 1. The extracellular portion of Shakerâs paddle motif is required for SMase D sensitivity. (A) Hydrolysis scheme of sphingomyelin by SMase D. (B) Amino acid sequence of Shakerâs paddle motif. Residues from I325 through V367 were replaced with a glycine triplet in the âdeletionâ mutant (mut) in E, F, and H. (CâF) Wild-type currents in 100 mM of extracellular K+ ([K+]ext; C and D) and âdeletionâ mut currents in 20 mM [K]ext (E and F) elicited by stepping membrane voltage of Xenopus oocytes (XO) from the â80-mV holding voltage to between â80 and 20 mV in 10-mV increments at 3-s intervals before (âpreâ; C and E) and after (âpostâ; D and F) SMase D treatment, where for comparison, the current traces at â40 mV are colored maroon. Dashed lines indicate zero current levels. (G and H) G-V curves before (open squares) and after (closed circles) SMase D treatment for wild-type (G) and âdeletionâ mut (H), where all data are presented as means ± SEM (n = 5). The curves are fits of Boltzmann functions, yielding the midpoint (V1/2) of â29.0 ± 0.6 mV and the apparent valence (Z) of 3.8 ± 0.3 (open squares) or V1/2 = â42.9 ± 0.9 mV and Z = 3.4 ± 0.3 (closed circles) for G, and V1/2 = â34.6 ± 0.5 mV and Z = 2.3 ± 0.1 (open squares) or V1/2 = â32.6 ± 0.2 mV and Z = 2.7 ± 0.1 (closed circles) for H.
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Figure 2. SMase D stimulates KV1.3 channels expressed in Xenopus oocytes and CHO cells. (A) G-V curves before (open squares) and after (closed circles) treatment with SMase D for KV1.3 expressed in Xenopus oocytes, where all data are presented as means ± SEM (n = 6). The curves are fits to single Boltzmann functions, yielding V1/2 = â24.2 ± 0.1 mV and Z = 5.0 ± 0.1 (open squares), and V1/2 = â46.2 ± 0.5 mV and Z = 5.0 ± 0.4 (closed circles). (B) KV1.3 currents recorded from a CHO cell in the presence of 5 mM [K+]ext elicited by repeatedly stepping the voltage every 15 s from the â100-mV holding potential to the â40-mV test potential; the addition of SMase D caused the current to increase gradually (top). Time course of the KV1.3 current increase after the addition of SMase D (bottom). (C and D) KV1.3 currents of a CHO cell (K+]ext = 20 mM) elicited by stepping membrane voltage from the â100-mV holding voltage to between â80 and 10 mV in 10-mV increments at 15-s intervals before (C) and after (D) SMase D treatment, where for comparison, the current traces at â30 mV are colored maroon. Dashed lines in BâD indicate zero current levels. (E) G-V curves of KV1.3 before (open squares) and after (closed circles) 3-min SMase D treatment, where all data are presented as means ± SEM (n = 5). The curves are fits to single Boltzmann functions, yielding V1/2 = â27.2 ± 0.4 mV and Z = 4.1 ± 0.2 (open squares), and V1/2 = â42.9 ± 0.4 mV and Z = 4.6 ± 0.3 (closed circles). (F) Voltage dependence of activation (circles) and deactivation (squares) time constants (ÏA and ÏD) of KV1.3 before (open symbols) and after (closed symbols) 3-min SMase D treatment, where all data are presented as means ± SEM (n = 5).
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Figure 3. SMase D activates native KV1.3 channels in human T cells. (A) KV1.3 currents of a T cell recorded in the presence of 5 mM [K+]ext elicited by stepping the voltage every 15 s from the â100-mV holding potential to the â40-mV test potential. After the addition of SMase D, the current gradually increased. (B) Time course of KV1.3 current increase after the addition of SMase D. (C and D) KV1.3 currents ([K+]ext = 20 mM) elicited by stepping the membrane voltage from the â100-mV holding potential to between â80 and 10 mV in 10-mV increments at 15-s intervals before (C) and after (D) the addition of SMase D to the bath solution, where the current traces for â30 mV are colored maroon. Dashed lines in A, C, and D indicate zero current levels. (EâH) G-V curves of KV1.3 channels in T cells (E and F) and Jurkat cells (G and H) before (open squares) and after (closed circles) either a 3-min treatment with SMase D (E and G) or a 3-min control period in the absence of SMase D (F and H), where all data are presented as means ± SEM (n = 5). The curves are fits of Boltzmann functions, yielding V1/2 = â26.1 ± 0.4 mV and Z = 3.7 ± 0.2 (open squares), or V1/2 = â42.4 ± 0.6 mV and Z = 4.0 ± 0.3 (closed circles) for E; V1/2 = â27.0 ± 0.4 mV and Z = 3.8 ± 0.2 (open squares) or V1/2 = â33.2 ± 0.5 mV and Z = 4.1 ± 0.3 (closed circles) for F; V1/2 = â28.8 ± 0.4 mV and Z = 4.3 ± 0.2 (open squares) or V1/2 = â38.6 ± 0.3 mV and Z = 4.5 ± 0.2 (closed circles) for G; and V1/2 = â30.6 ± 0.5 mV and Z = 3.5 ± 0.2 (open squares) or V1/2 = â32.1 ± 0.5 mV and Z = 3.6 ± 0.2 (closed circles) for H.
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Figure 4. SMase D shifts the G-V curves of NaV and CaV channels expressed in Xenopus oocytes in the hyperpolarized direction. (A) NaV1.4-IR currents elicited by stepping the voltage every 4 s from the â100-mV holding potential to a â120-mV prepulse, and then to the â50-mV test potential; the dashed line indicates the zero current level. After the addition of recombinant SMase D to the bathing solution, the current gradually increased (top). Time course of NaV1.4-IR current increase after the addition of SMase D (bottom). (BâD) G-V curves before (squares) and after (circles) treatment with SMase D for NaV1.4-IR (B), CaV2.1 (C), and CaV2.2 (D), where all data are presented as means ± SEM (n = 3â9). The curves are fits of Boltzmann functions, yielding the midpoint (V1/2) of â28.2 ± 0.4 mV and the apparent valence (Z) of 5.2 ± 0.1 (open squares), or V1/2 = â48.5 ± 0.2 mV and Z = 5.2 ± 0.2 (closed circles) for B; V1/2 = â1.0 ± 0.1 mV and Z = 6.3 ± 0.2 (open squares), or V1/2 = â8.4 ± 0.2 mV and Z = 5.6 ± 0.3 (closed circles) for C; V1/2 = 1.5 ± 0.1 mV and Z = 6.6 ± 0.2 (open squares), or V1/2 = â8.9 ± 0.2 mV and Z = 5.1 ± 0.2 (closed circles) for D.
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Figure 5. SMase D stimulates NaVα + β1 channels in oocytes and slows their inactivation. (AâD) rNav1.2α + β1 (A and B) or rNav1.4α + β1 (C and D) currents from an oocyte, before (A and C) and after (B and D) SMase D treatment, by stepping the membrane voltage from the â120-mV holding voltage to various test potentials between â90 and 10 mV. (E and F) Steady-state inactivation curves (E) and I-V relations (F) before (open squares) and after (closed circles) SMase D treatment; all data are presented as means ± SEM (n = 3â6). Curves in E are fits to single Boltzmann functions, yielding V1/2 = â52.0 ± 0.2 mV and Z = 4.7 ± 0.2 (open squares), and V1/2 = â56.1 ± 0.8 mV and Z = 3.6 ± 0.2 (closed circles).
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Figure 6. Effects of high Mg2+ and the NaVβ1 subunit on SMase D sensitivity of NaV channels. (A and B) rNaV1.4α + β1âs I-V curves (A) and inactivation time courses at â20 mV (B), collected in the presence of 10 mM of extracellular Mg2+ without (open squares) or with (closed circles) SMase D; all data are presented as means ± SEM (n = 3). (CâG) Currents of hNaV1.4α (C), hNaV1.4α + β1 (D), hNaV1.5α (E), hNaV1.5α + β1 (F), and mutant hNaV1.4α containing hNaV1.5αâs S5âS6 linkers (G), elicited by depolarizing the membrane from â120 to â20 mV before (black) and after (maroon) SMase D treatment. Dashed lines indicate the zero current level.
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Figure 7. Effect of SMase D on native NaV channels in CHO cells. (AâC) NaV currents from a CHO cell before (A) and after (B) the addition of SMase D, elicited by stepping the membrane voltage from the â100-mV holding voltage to various test potentials between â80 and 40 mV in 10-mV increments. The current traces at â20 mV from A (black) and B (maroon) are superimposed in C. Dashed lines indicate zero current levels. (D) I-V relations before (open squares) and after (closed circles) SMase D treatment; all data are presented as means ± SEM (n = 5). (EâH) Steady-state inactivation curves (E and G) and voltage dependence of inactivation time constants (F and H) from cells expressing (G and H) or not expressing (E and F) hNaVβ1 before (open squares) and after (closed circles) SMase D treatment; all data are presented as means ± SEM (n = 5). Curves are fits to single Boltzmann functions, yielding V1/2 = â26.8 ± 0.4 mV and Z = 4.7 ± 0.3 (open squares), and V1/2 = â36.8 ± 0.3 mV and Z = 4.6 ± 0.2 (closed circles) in E; and V1/2 = â40.9 ± 0.4 mV and Z = 3.5 ± 0.2 (open squares), and V1/2 = â53.9 ± 0.7 mV and Z = 3.6 ± 0.3 (closed circles) in G.
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Figure 8. SMase D stimulates native NaV channels in N2A cells. (AâC) NaV currents from an N2A cell before (A) and after (B) the addition of SMase D, elicited by stepping the membrane voltage from the â100-mV holding voltage to a â120-mV prepulse and then to various test potentials between â80 and 40 mV in 10-mV increments. The current traces at â20 mV from A (black) and B (maroon) are superimposed in C. Dashed lines indicate zero current levels. (DâF) I-V relations (D), steady-state inactivation curves (E), and voltage dependence of inactivation time constants (F) before (open squares) and after (closed circles) SMase D treatment; all data are presented as means ± SEM (n = 5). Curves in E are fits to single Boltzmann functions, yielding V1/2 = â67.2 ± 0.4 mV and Z = 3.1 ± 0.1 (open squares), and V1/2 = â76.0 ± 0.3 mV and Z = 3.0 ± 0.1 (closed circles).
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Figure 9. SMase D shifts the action potential threshold of N2A cells in the hyperpolarized direction. (AâC) Passive voltage responses and action potentials of an N2A cell elicited by several series of 2-ms current pulses between â¼200 and 500 pA in 30-pA increments before (A) and after (B) 3-min SMase D treatment. The action potentials indicated by arrows and the most depolarized passive voltage responses are replotted as black (A) and maroon (B) curves in C. (D) Peak voltages (A, open squares, and B, closed circles) plotted against the instantaneous voltage at the end of the 2-ms current pulse. (EâG) Passive voltage responses and action potentials of an N2A cell elicited by several series of 2-ms current pulses between â¼200 and 500 pA in 30-pA increments before (E) and after (F) 3-min control in the absence of SMase D treatment. The action potentials indicated by arrows and the most depolarized passive voltage responses are replotted as black (E) and maroon (F) curves in G. (H) Peak voltages (E, open squares, and F, closed circles) plotted against the instantaneous voltage at the end of the 2-ms current pulse.
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Figure 10. SMase D causes a negative shift in the action potential threshold and reduces the amount of injected current required to depolarize the membrane potential to the action potential threshold. (A) Averaged action potential threshold voltage (means ± SEM; n = 5) before (white) and after (black) SMase D treatment (P < 0.01) or before (white) and after (black) 3-min control without SMase D treatment. (B) Average injected current required to depolarize the membrane potential to the threshold (means ± SEM; n = 5) before (white) and after (black) SMase D treatment (P < 0.01) or before (white) and after (black) 3-min control without SMase D treatment. The p-values were calculated with a paired-sample, two-tailed t test method.
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