Supplementary Material

Title: FHF2 isoforms differentially regulate Nav1.6 mediated resurgent sodium currents in dorsal root ganglion neurons.

Author Block:

1.  Cindy Barbosa1,2:

2.  Yucheng Xiao 1,2:

3.  Andrew J. Johnson1,2:

4.  Wenrui Xie3:

5.  Judith A. Strong3:

6.  Jun-Ming Zhang3:

7.  *Theodore R. Cummins1,2,4:

1 Dept. of Pharmacology and Toxicology, Indiana Univ., Indianapolis, IN; 2 Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN; 3 Dept. of Anesthesiology, Univ. of Cincinnati, Cincinnati, OH; 4 Dept. of Biology, Indiana University – Purdue University Indianapolis.

*Corresponding Author:

Theodore R. Cummins

Department of Biology

723 W. Michigan St., SL306

Indianapolis, IN 46202-2266

Phone: 317-278-9343

Supplementary Figure S1: Effect of LID and sham LID surgeries on mechanical and cold sensitivity. Two baseline measurements were made and the average value is plotted on POD0. On POD 0 the L5 DRG was inflamed or the corresponding sham surgery performed. This resulted in a marked decrease in the static mechanical threshold (von Frey test; a), increase in dynamic allodynia (withdrawal responses to stroking the paw with a fine cotton wisp; b) and increase in cold allodynia (withdrawal responses to a drop of acetone; c) in the ipsilateral hindpaw that were significantly larger in the LID group compared to the sham. *, p<0.05; **, p<0.01; ***, p<0.001, significant difference between the two groups at the indicated time point, 2-way repeated measures ANOVA with Bonferroni posttest. N = 4 rats per group.

Supplementary Figure S2: F2A(β4) but not FHF2B induced fast resurgent currents mediated by Nav1.5 in HEK cells. HEK293 cells stably expressing Nav1.5 were transfected with tagged-F2A(β4) or tagged-FHF2B. Non-transfected cells were used as a control. Resurgent currents were elicited with a standard resurgent current voltage protocol, in which cell membrane was initially depolarized to +30mV for 20ms, and then repolarized to voltages ranging from +15mV to -85 for 100ms in 5mV increments. A, Representative traces of Nav1.5 resurgent currents are shown for control (black), FHF2B (purple) and F2A(β4) (green). Trace obtained at -40 mv is highlighted for each group. B, Resurgent current amplitude was normalized to peak transient current. F2A(β4) (green triangles) generated a biphasic current voltage curve typically observed for resurgent currents, whereas, control (blue circles) or FHF2B (purple diamonds) did not. C, Effects of FHF2B and F2A(β4) on Nav1.5’s voltage dependence of activation. The V1/2 values are -47.5 ± 0.9mV (control), -47.6 ± 0.3mV (FHF2B) and -45.5 ± 0.4mV (F2A(β4)), respectively. D, Effects of FHF2B and F2A(β4) on voltage dependence of inactivation. The V1/2 values are -92.4 ± 1.2mV (control), -87.5 ± 0.5 mV (FHF2B) and -85.3 ± 1.0mV (F2A(β4)), respectively. The holding potential was -120 mV. Data are mean ± SEM and n=5 for each group