Peak current values (A) and charge transfer values (B) for potassium current sensitive to apamin, iberiotoxin and clotrimazole combined, in control neuronal somata (n=13).

A. Current traces showing response to iberiotoxin (200 nM) in an individual medium-sized control neuron. Currents were elicited from HP −60mV, with 200ms square voltage command pulses, delivered every 10s from −100 to +50mV. I_Ca was inhibited by low external [Ca²⁺] and cadmium, while the cytosolic free [Ca²⁺] was clamped at 1μM. Iberiotoxin sensitive current, indicative of BK channels, was derived by subtraction of the current after the toxin from the baseline traces. B. Control medium sized DRG neuronal somata contain apamin- (n=22), iberiotoxin- (n=16) and clotrimazole (n=12)- sensitive K_(Ca) current components. I–V curves were constructed from averaged, subtracted current traces elicited using 200 ms square voltage commands every 10s from HP −60 mV, with steps from −100 mV to +50 mV. Cytosolic free [Ca²⁺] was clamped at 1μM. Only one toxin (either apamin, or iberiotoxin, or clotrimazole) was applied on each individual cell, and difference currents indicating sensitivity to this particular toxin were derived by subtracting the traces after the toxin from the baseline traces.

Bar-graph indicating the degree of inhibition of the baseline outward current (percent inhibition from baseline), at +40 mV membrane potential, by each toxin (apamin, iberiotoxin, and clotrimazole) in control, axotomized L5 and adjacent L4 DRG medium-sized neurons. Numbers of observations are for apamin are control n=22, L5 SNL n=12, L4 SNL n=9, for iberiotoxin control n=16, L5 SNL n=9, L4 SNL n=8, and for clotrimazole control n=12, L5 SNL=9 and L4 SNL n=7.

Derivation of the difference current components attributable to I_Na, I_Ca, I_K(Ca), and total I_K. A. AP recorded in current clamp mode and then replayed as voltage command stimulus in voltage clamp, from holding potential equal to this cell’s resting membrane potential. B. Whole-cell currents in response to the AP voltage command in Tyrode’s solution, and then sequentially after blocking I_Na with equimolar NMDG and 0.3μM TTX, after blocking I_K(Ca) with apamin, iberiotoxin, and clotrimazole in combination, then after blocking remaining K⁺ current with the addition of 4-AP, TEA-Cl, and glibenclamide, and finally blocking I_Ca with cadmium. All recordings were obtained sequentially from the same cell, using this individual cell’s AP as voltage command stimulus. Individual current components were derived as difference currents by subtraction of the trace after administration of the blocker from the current trace before. Total I_K (I_K(tot)) was estimated as the potassium current sensitive to all the potassium channel blockers that we used (4-AP, TEA-Cl and glibenclamide in addition to apamin, iberiotoxin and clotrimazole). This current was calculated by subtracting the current trace obtained after the addition of all these blockers, from the current recorded in the NMDG-containing Tyrode’s solution. C. Derived potassium current (I_K(Ca)) sensitive to K_(Ca) blockers (apamin, clotrimazole and iberiotoxin combined) in response to AP voltage command. Upper trace: AP recorded in current clamp mode and then replayed as voltage command stimulus in voltage clamp, from holding potential equal to this cell’s resting membrane potential (also shown in A). Lower trace: Subtracted current trace, indicative of the derived K_(Ca) current component, obtained as the difference between the current after administration of combined K_(Ca) blockers, and baseline current in Tyrode’s solution containing NMDG and TTX. Peak current as well as charge transfer (AUC) were derived digitally from this trace. Note that the peak I_(KCa) occurs during the descending limb of the AP waveform command.

Peak current (A), and charge transfer as estimated from the AUC (B), for difference current components attributable to I_Na, I_Ca, I_K(Ca), and I_K(tot), in medium-sized control neurons (C, n=13), as well as in axotomized (L5 SNL, n=14) or adjacent neurons (L4 SNL, n=14). AP waveforms were recorded in each cell in current-clamp and replayed as voltage commands to elicit currents in voltage-clamp sequentially in a time course mode. Difference currents were derived by subtraction of traces recorded by sequential application of blockers selective for each current component (see Figure 1). Peak current densities and charge transfer values were measured for each separate difference current after normalization by dividing by each cell’s capacitance. Means ± SE are shown.
*: indicate statistically significant differences with regard to cell-type main effect.
I_Na peak current and charge transfer values did not differ among control, axotomized and adjacent neuronal somata. I_Ca peak current and charge transfer also did not differ between the three cell types. However, there were statistically significant differences for peak I_K(Ca) currents and charge transfer values among different cell types (p=0.005 for peak current and p=0.029 for charge transfer), as well as for total I_K peak currents (p=0.001) and charge transfer (0.020). Post hoc analysis showed a statistically significant difference for peak I_K(Ca) between L5 SNL and L4 SNL (p=0.001), and between control and L4 SNL (p=0.031). There was also a statistically significant difference for I_K(Ca) charge transfer between L5 SNL and L4 SNL (p=0.01), as well as a marginal difference between control and L4 SNL (p=0.057). Peak total I_K also differed between L5 SNL and L4 SNL (p<0.001), and between control and L4 SNL (p=0.005). There was also a statistically significant difference for I_K(Ca) charge transfer between L5 SNL and L4 SNL (p=0.009), as well as between control and L4 SNL (p=0.025).

Subtype-specific effects of SNL on K_(Ca) currents in control, axotomized L5, and adjacent L4 DRG medium-sized neurons, as well as iberiotoxin-sensitive current in small-sized neurons. Figure 5A–C: I–V curves were constructed from subtracted current traces sensitive to apamin (A), iberiotoxin (B) and clotrimazole (C), elicited using 200 ms square voltage commands from HP −60 mV, after stepping from −100 mV to +50 mV. Cytosolic free [Ca²⁺] was clamped at 1μM. Bars indicate statistically significant differences between cell-types. Numbers of observations for apamin are control n=22, L5 SNL n=12, L4 SNL n=9, for iberiotoxin control n=16, L5 SNL n=9, L4 SNL n=8, and for clotrimazole control n=12, L5 SNL=9 and L4 SNL n=7. Figure 5D: Iberiotoxin-sensitive current densities indicating BK currents in control (n=6), axotomized L5 (n=9) and adjacent L4 DRG small-sized neurons (n=12). I–V curves were constructed from subtracted current traces sensitive to 200 nM iberiotoxin elicited using 200 ms square voltage commands from HP −60 mV, after stepping from −100 mV to +50 mV. Cytosolic free [Ca²⁺] was clamped at 1μM. Bars indicate statistically significant differences between cell-types.