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. 2016 Feb 19;5:e10960. doi: 10.7554/eLife.10960

Figure 3. Effect of hβ2 on hNav1.2 toxin pharmacology.

(a) Co-expression of hNav1.2 with hβ2 decreases the degree of inhibition by 100 nM ProTx-II. Left trace shows ProTx-II strongly inhibiting WT hNav1.2 whereas right trace displays attenuated inhibition in the presence of hβ2. Black trace is control condition without toxin, red is in the presence of ProTx-II. Traces depict a 50 ms depolarization to -15 mV from -90 mV. Scale bar is 10 ms on horizontal axis and given nA vertically. (b) Normalized conductance-voltage (G-V, filled circles) and steady-state inactivation (I-V, open circles) relationships for hNav1.2 with and without hβ2. Pre-toxin values are shown in black and post-toxin in red. Fit values can be found in Figure 3—source data 1. (c) Dot plot comparing hβ2 mutations by ability to prevent ProTx-II inhibition of hNav1.2. Black circles represent individual oocytes; vertical axis shows percent of inhibition by ProTx-II at peak conductance. Blue lines represent a 95% confidence interval. hβ2 mutations are presented underneath the horizontal axis and label the lanes below in (d). Statistical significance (p<0.01) is indicated by an asterisk. (d) Western blot against the C-terminal myc-tag of hβ2. No signal is seen in the negative control but is observed for the WT hβ2 and all mutants, both in whole cell (filled circle) and surface (open circle) fractions.

DOI: http://dx.doi.org/10.7554/eLife.10960.007

Figure 3—source data 1. Table providing values for fits of the data presented in Figure 3 and Figure 3—figure supplement 2.
G-V and SSI relationship data were fitted by a Boltzmann curve. V1/2 provides the midpoint voltage of the calculated curve (in mV) and Vc the unit-less slope, with standard error of the mean (SEM). Right column shows peak conductance after toxin treatment as a fraction of untreated peak conductance with the upper and lower bounds of the 95% confidence interval in parentheses, reflecting the data displayed in the dot plots.
DOI: http://dx.doi.org/10.7554/eLife.10960.008
elife-10960-fig3-data1.docx (16.2KB, docx)
DOI: 10.7554/eLife.10960.008

Figure 3.

Figure 3—figure supplement 1. ProTx-II does not bind directly to β4.

Figure 3—figure supplement 1.

Isothermal calorimetry (ITC) experiments showing titration of 400 μM WT hβ4 extracellular domain into 40 μM ProTx-II (left) and into buffer (right). No significant heat differences are detected.
DOI: http://dx.doi.org/10.7554/eLife.10960.009
Figure 3—figure supplement 2. G-V and SSI relationships for hβ2 mutants.

Figure 3—figure supplement 2.

(a) Conductance-voltage (G-V, filled circles) and steady-state inactivation (SSI, open circles) relationships before 10nM ProTx-II addition are indicated in black and red in the presence of toxin. (b) Representative traces at -15 mV (holding potential of -90 mV) for the corresponding graphs seen in (a). Scale bar is 10 ms along horizontal axis and displayed nA along the vertical axis. (c) Western blot showing that the C72A C75A hβ2 mutant is expressed in whole cell (filled circle) and surface (open circle) fractions either alone (middle column) or with hNav1.2 (right column). The hβ2 subunit was detected using an antibody against a C-terminal myc-tag.
DOI: http://dx.doi.org/10.7554/eLife.10960.010