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. 2012 Jul 4:8:37.
doi: 10.1186/1744-8069-8-37.

Inflammation-induced changes in BK(Ca) currents in cutaneous dorsal root ganglion neurons from the adult rat

Xiu-Lin Zhang  1 , Lee-Peng Mok, Kwan Yeop Lee, Marcel Charbonnet, Michael S Gold
Affiliations

Inflammation-induced changes in BK(Ca) currents in cutaneous dorsal root ganglion neurons from the adult rat

Xiu-Lin Zhang et al. Mol Pain. .

Abstract

Background: Inflammation-induced sensitization of primary afferents is associated with a decrease in K(+) current. However, the type of K(+) current and basis for the decrease varies as a function of target of innervation. Because glabrous skin of the rat hindpaw is used often to assess changes in nociception in models of persistent pain, the purpose of the present study was to determine the type and extent to which K(+) currents contribute to the inflammation-induced sensitization of cutaneous afferents. Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion neurons from naïve and inflamed (3 days post complete Freund's adjuvant injection) rats were studied with whole cell and perforated patch techniques.

Results: Inflammation-induced sensitization of small diameter cutaneous neurons was associated with an increase in action potential duration and rate of decay of the afterhyperpolarization. However, no changes in voltage-gated K(+) currents were detected. In contrast, Ca(2+) modulated iberiotoxin sensitive and paxilline sensitive K(+) (BK(Ca)) currents were significantly smaller in small diameter IB4+ neurons. This decrease in current was not associated with a detectable change in total protein levels of the BK(Ca) channel α or β subunits. Single cell PCR analysis revealed a significant change in the pattern of expression of α subunit splice variants and β subunits that were consistent, at least in part, with inflammation-induced changes in the biophysical properties of BK(Ca) currents in cutaneous neurons.

Conclusions: Results of this study provide additional support for the conclusion that it may be possible, if not necessary to selectively treat pain arising from specific body regions. Because a decrease in BK(Ca) current appears to contribute to the inflammation-induced sensitization of cutaneous afferents, BK(Ca) channel openers may be effective for the treatment of inflammatory pain.

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Figures

Figure 1
Figure 1
Persistent inflammation of the hindpaw results in an increase in the excitability of cutaneous neurons that is associated with an increase in action potential duration. Excitability was assessed in acutely dissociated DRG neurons retrogradely labeled from the glabrous skin of the hindpaw harvested from naïve and inflamed (CFA) rats. Depolarizing current injection was used to determine action potential (AP) threshold (A), rheobase (B) and the response to suprethreshold current injection (C, D), which was injected at intensities 1, 2, and 3 times rheobase. The voltage traces in C, are typical of the pooled data plotted in D. As indicated in D, pooled data for all panels are from 10 neurons from naïve and 23 neurons from inflamed rats. The number of action potentials evoked at 2 and 3x rheobase in neurons from inflamed rats is significantly greater than that in neurons from naïve rats. Error bars for data from naïve neurons are smaller than the symbol. Inset: The slope of the stimulus response function (SRF) is significantly greater for neurons from inflamed rats than that for neurons from naïve rats. E. Typical action potentials from naïve (black trace) and inflamed (gray trace) rats evoked in response to a 4 ms current injection are overlayed with the average action potential duration indicated for each. These values are significantly different (p < 0.05). F. The afterhyperpolarization (AHP) following the action potentials shown in E are plotted to illustrate the trend toward a decrease in the AHP duration in neurons from inflamed rats. The full amplitude of the action potential is clipped in these traces to facilitate visualization of the AHP. Average values are indicated next to each trace, and these differences are significantly different. * is p < 0.05 and ** is p < 0.01
Figure 2
Figure 2
There is no detectable influence of inflammation on voltage-gated K+(Kv) currents in cutaneous DRG neurons. A. Typical Kv currents evoked in a cutaneous DRG neuron from a naïve rat with the inactivation protocol shown below the current traces. A significant fraction of total current (a) was subject to steady-state inactivation (b, inactivatable), leaving a sustained, non-inactivatable current (c). The potential at which steady-state inactivation was complete was used as the prepulse potential with which inactivatable current was separated from non-inactivatable current. B. The voltage-dependence of total Kv current activation, determined from conductance-voltage (GV) plots, was comparable in neurons from naïve and inflamed (CFA) rats. Similarly, the steady-state inactivation curves from these two groups of neurons were also comparable. Complete data sets were only collected on a subpopulation of the total number of neurons studied. C. The current density (peak current at +60 mV) of the inactivatable fraction of the total current was comparable in neurons from naïve and inflamed (CFA) rats, whether data were collected from neurons contralateral (CFA Contra) or ipsilateral (CFA Ipsi) to the site of inflammation. D. Similar results were obtained with the analysis of current density of the non-inactivatable fraction of the total Kv current in cutaneous DRG neurons
Figure 3
Figure 3
Inflammation is associated with a significant decrease in BKCacurrents in cutaneous DRG neurons. A. Outward currents were evoked before (Total) and after the application of iberiotoxin (IbTx, 100 nM), with the voltage protocol shown below the Total current traces. The toxin sensitive current (the bottom set of current traces) was obtained by digitally subtracting current evoked in the presence of IbTx from that evoked prior to its application. B. Pooled current voltage (I-V) data from cutaneous neurons from naïve and inflamed (CFA) rats highlight the significant decrease in BKCa current density in neurons from inflamed rats. Note, pooled data are only from neurons in which BKCa current was detectable (> 200 pA at +60 mV). ** is p <0.01
Figure 4
Figure 4
The biophysical properties of BKCacurrents are altered in neurons from inflamed rats. BKCa currents from naïve (A) and inflamed (B, CFA) rats were isolated as described in Figure 3. C. To illustrate differences in the rate of current activation, current evoked at +30 mV in the traces shown in A and B have been scaled relative to peak current and overlaid. The time constant for current activation was used to quantify the rise time. D. Pooled data indicate that this difference in activation rate is significant for currents isolated with IbTx and paxilline. E. Deactivation rate was determined from tail currents evoked at −90 mV following BKCa current activation at +30 mV. While there was no apparent difference in the deactivation rate of currents isolated with IbTx between naïve and inflamed neurons (not shown), currents isolated with paxilline appeared to deactivate more slowly in neurons from inflamed rats. Inset: example of IbTx sensitive tail currents used to assess deactivation rate. F. Pooled deactivation rate data (at -90 mV) for BKCa currents isolated with IbTx and paxilline from naive and inflamed rats. ** is p < 0.01
Figure 5
Figure 5
Inflammation was associated with no detectable change in total BKCachannel subunit protein levels. Total protein was extracted from L4 and L5 ganglia from naïve and inflamed rats ipsilateral (I) or contralateral (C) to the site of CFA injection. A. Example of a western blot of the BKCa α subunit. A sample of total protein from the uterus of a naïve rat was used as a loading control to enable comparisons between blots. Ispilateral and contralateral in the naïve rats were simply left and right, respectively, as all CFA injections in the inflamed group were made in the left hindpaw. GAPDH was used as a loading control. B. Example of western blots for BKCa β subunits 2, 3, and 4. The uterus protein sample was again used to enable comparisons between blots. Pooled data for relative levels of α (C), and β (D) subunit revealed no significant differences between groups
Figure 6
Figure 6
Inflammation-induced changes in the pattern of BKCaα-subunit splice variant and β subunit expression. A. Example of splice variants at the X4 site of the BKCa α subunit detected in mRNA harvested from single cutaneous DRG neurons. Each lane is the PCR product from a single neuron. The “zero insert” bottom band is dominant in all neurons. However, the larger inserts were more common in neurons from inflamed (CFA) than naïve rats. B. Pooled data for the expression of splice variants at the X4 site from 4 naïve and 4 inflamed rats were analyzed with a two-way ANOVA, which revealed a significant influence of inflammation (p < 0.05), but no interaction between inflammation and splice variant. C. Pooled data for β subunit expression from 4 naïve and 4 inflamed rats was also analyzed with a two-way ANOVA, which also revealed a significant influence of inflammation (p < 0.05), but no interaction between inflammation and subunit expression
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