FIGURE 2.

Functional expression of α7 nAChRs and β4-containing nAChRs in different rat DRG neuronal subclasses. After two ACh pulses,10 μM BuIA was applied to the bath as indicated by a short, black horizontal bar. The ACh-responsive neurons were blocked irreversibly by BuIA. After block by BuIA, 1 μM PNU-120596 (PNU) was applied to the bath solution for the duration of the experiment, as indicated by a long, gray horizontal bar. After application of PNU, ACh pulses revealed a second class of DRG neurons that were previously unresponsive to ACh in the absence of PNU. ACh responses in this second subclass of DRG neurons were blocked completely and reversibly by 200 nM ArIB[V11L;V16D]. (A) Traces of responses from selected neurons in an experimental trial conducted without VGCC blockers. (B) Traces of responses from selected neurons in an experimental trial conducted in the presence of VGCC blockers, as indicated by a long, open horizontal bar. The cocktail of VGCC blockers consisted of 200 nM ω-conotoxin GVIA to block N-type calcium channels, 200 nM ω-conotoxin MVIIC to block P/Q-type calcium channels, and 1 μM nicardipine to block L-type calcium channels. This protocol eliminated the shoulders observed for ACh-elicited calcium signals in the β4-expressing subclass, suggesting that those shoulders were caused by co-activation of nAChRs and VGCCs. This protocol also confirms that the signals observed in (B) are from calcium influx through nAChRs. At the end of this experiment, a pulse of 25 mM [K⁺]_o (K) was followed by application of 300 nM capsaicin (C), to demonstrate that these responses were from viable neurons (a subset of DRG neurons respond to capsaicin). The top trace demonstrates the response from an additional neuronal subclass that expresses both α7 nAChRs and β4-containing nAChRs.