Isolectin IB4 conjugated to FITC (IB4) was used to discriminate bladder sensory neurons of C- and A delta origin. Patch-clamp studies showed that 100% (0/15) of the bladder sensory neurons labeled with IB4 have TTX-resistant (TTX-R) action potentials. Thus, to assess gene expression in sensory neurons with TTX-S action potentials (A delta origin), we collected IB4(-) bladder sensory neurons under fluorescent light with an oil-filled glass pipette plugged to a nanoinjector. We examined gene expression for voltage-gated K+ channel subunits Kv1.1, Kv2.1, Kv2.2, Kv3.4, Kv4.1, Kv7.2, Kv7.3, Kv9.1 and the large-conductance Ca2+-activated K+ channel (BK), and for TTX-S voltage-gated Na+ channel subunits Nav1.1, Nav1.3, Nav1.6 and Nav1.7, which are known to be present in A delta sensory neurons. Gene expression analysis showed a significant upregulation of mRNA levels for subunits Kv2.2 and Kv9.1 in IB4(-) bladder sensory neurons from rats with bladders transduced with AdCldn2, when compared to controls (AdGFP). Kv2.2 is a pore forming subunit expressed in somas and axons that constitute the neuronal outward delayed rectifier K+ (Ik) current and associates with silent Kv subunits (9.1, 9.2, 9.3). No significant difference in mRNA expression for Nav subunits was observed between bladder sensory neurons harvested from rats transduced with AdGFP or AdCldn2.
To determine whether the hyperexcitability seen in sensory neurons with TTX-S action potential from rats transduced with AdCldn2 reflects changes in the activity of Kv2.2/9.1 channels, we measured whole-cell K+ currents before and after treatment with guangxitoxin-1E (GxTx-1E), a selective blocker of Kv2 channels. GxTx-1E-sensitive currents were 58+/-15 pA/pF (n=14) and 8+/-2 pA/pF (n=16) in sensory neurons from rats transduced with AdCldn2 and AdGFP, respectively (p<0.01). Moreover, we observed a 3-fold increase in TTX-S Na+ currents in sensory neurons from rats transduced with AdCldn2, when compared to controls. Significantly, GxTX-1E reduced repetitive firing in response to electrical stimulation in IB4(-) neurons from rats transduced with AdCldn2 (Fig.1).
Figure 1. Guanxitoxin-1E inhibits repetitive firing of bladder sensory neurons from rats transduced with AdCldn2. A and B, Representative tracings of action potential firing in response to suprathreshold electrical stimulation for bladder sensory neurons from rats transduced with AdGFP (A) or AdCldn2 (B) before (upper panel) and after (lower panel) the addition of 100 nM GxTX-1E. C, Stimulus response relationships for bladder sensory neurons from rats transduced with AdGFP or AdCldn2. Action potentials were evoked by the injection of depolarizing current pulses. The number of spikes evoked in response to stimuli of increased intensity for each neuron were computed before and after GxTX-1E (100 nM)(n = 22–27, * p<0.01 GFP vs Cldn2, # p<0.05 and ## p< 0.01 for GFP or Cldn2 +/- GxTX-1E, Student’s t-test).