Figure 5. S1PR3 modulates KCNQ2/3 channels to regulate AM excitability.

All experiments were performed in S1pr3^mCherry/+ or ^-/- DRG neurons. (A) (Left) Example traces of a single mCherry +neuron in whole cell current clamp before and after S1P application. (Right) % change in rheobase after S1P application for S1pr3^mCherry/+ (left, n = 7) and KO (right, n = 12) neurons (p_WT,KO = 0.012, 0.287; two-tailed paired t-tests). (B) % ∆ in input resistance after S1P or vehicle application (p=0.017; two-tailed paired t-test; n = 4 cells per group). (C) The S1P-sensitive current is carried by potassium. The current-voltage relationship was determined by subtraction of the post-S1P current from the pre-S1P current and reverses at −60.125 mV; n = 6 cells. Data were fitted with a Boltzmann equation. Pre- and post-S1P currents were measured at the indicated voltage (−100 mV to +80 mV, 20 mV increments) following a +100 mV step (100 ms). Current was quantified using the peak absolute value of the slowly-deactivating current 0–10 ms after stepping to indicated voltage. Unless indicated otherwise, all error bars represent mean ± SEM. (D) (Graphic, top) Averaged current traces of a single mCherry+ neuron in whole cell voltage clamp recording comparing tail currents (∆_{I tail}) pre- and post-S1P using indicated voltage step protocol. (graphic, bottom) Averaged current traces of a single mCherry+ neuron in whole cell voltage clamp recording with XE991 treatment. Holding phase (−40 mV, 150 ms) was truncated in traces. (Left graph) % ∆ in outward tail current (average ±SD after indicated treatments (1 µM S1P, 3 µM XE 991, or both) for S1pr3^mCherry/+ medium-diameter neurons; (p=0.58; one-way ANOVA; n = 6, 8, 14 cells) using protocol depicted at right. (Right graph) % ∆ in inward tail current after indicated treatments (LINO = 100 µM linopirdine) for S1pr3^mCherry/+ medium-diameter neurons; (p=0.47; two-tailed paired t-test; n = 12 cells).

Figure 5—figure supplement 1. S1P selectively modulates potassium tail currents to increase DRG neuron excitability.

Related to Figure 5. (A) (Left) Example trace of a single mCherry+ neuron in S1P before and after current injection. (Right) Resting membrane potential (RMP) in millivolts before and after addition of S1P (p=0.23; two-tailed paired t-test; n = 6 cells). (B) Rheobase pre- and post-S1P application in DRG neurons; p_WT = 0.011; p_KO = 0.28 (two-tailed paired t-test). Same data are represented in Figure 5A. (C) Sodium I-V relationship for a representative S1pr3^mCherry/+ medium-diameter neuron pre- and 5 min post- 1 µM S1P using voltage step from −100 to +80 mV (150 ms steps, −80 mV holding). (D) Steady-state I-V relationship for same neuron. (E) (Left) % ∆ in peak sodium current (Na⁺) after S1P or 1% DMSO vehicle application for medium-diameter mCherry+ neurons; p=0.39 (two-tailed paired t-test; n = 7 cells per group). (Right) % ∆ in peak steady-state current (S.S. K⁺) after S1P or 1% DMSO vehicle application for medium-diameter mCherry+ neurons; p=0.948 (two-tailed paired t-test; n = 7 cells per group). (F) % ∆ in inward tail current (∆_{I tail}) after S1P or 1% DMSO vehicle application for S1pr3^mCherry/+ and KO medium-diameter neurons using a pre-pulse stimulation of +80 mV followed by a step to −80 mV, where (∆_{I tail}) was calculated by subtracting the steady-state current from the absolute peak of the slowly-deactivating current at −80 mV (p=0.014; one-way ANOVA; n = 10, 13, 10 cells). Tukey Kramer post hoc p-values indicated on graph. (G) Dose-response relationship between % ∆ in tail current and S1P concentration for 1 nM, 50 nM, 100 nM, and 1 µM S1P (n = 7 cells). EC₅₀ (48.8 nM), marked by thin dotted lines, was estimated from sigmoidal fit (thick dotted line).