The internal surface of the urinary bladder is lined by the urothelium, a barrier-forming epithelium that restricts the passage of ions and metabolic products from the urine into the bladder interstitium. Urothelial abnormalities, which range from mucosal ulcerations, urothelial ruptures and widening of the space between urothelial cells to denuded epithelium, have been reported in patients with interstitial cystitis/bladder pain syndrome (IC/BPS). We previously showed the overexpression of the tight-junction associated protein claudin-2 (Cldn2) in the rat urothelium reproduces the cardinal features of IC/BPS including increased urothelial permeability to ions, inflammation in the bladder mucosa and lamina propria, increased voiding frequency and pelvic allodynia [1, 2]. These changes were associated to an increase in the excitability (sensitization) of bladder sensory neurons with tetrodotoxin-sensitive (TTX-S) action potentials, which are considered of A delta origin. 
     Voltage-gated Na+ and K+ channels are responsible for the generation, propagation and termination of action potentials in neurons. We put forth the hypothesis that the hyperexcitability seen in TTX-S sensory neurons from rats with increased urothelial permeability reflects changes in the activity of voltage-gated Na+ and K+ channels.

Female Sprague-Dawley rats (250-300 g) were used throughout. In situ transduction of umbrella cells was accomplished via direct intravesical instillation of adenovirus coding for GFP (AdGFP) or Cldn2 (AdCldn2). Bladder afferent neurons were labeled by injecting Dil in the bladder wall. Lumbosacral (L6-S2) dorsal root ganglion (DRG) neurons were isolated with collagenase/trypsin and plated on coverslips coated with ornithine and laminin. Taqman probes were employed to assess gene expression by qPCR in acutely isolated bladder sensory neurons. Neuronal excitability and ion channel function was assessed with the patch-clamp technique and a set of pharmacological tools.

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).

Our data indicate that urothelial barrier dysfunction increases the expression and activity of Kv2.2 and the activity of TTX-S voltage-gated Na+ channels in bladder sensory neurons with TTX-S action potentials. These results suggest that increased activity of voltage-gated Na+ and K+ channels contributes to the hyperexcitability we see in this population of bladder sensory neurons from rats transduced with AdCldn2. Current-clamp experiments showed that inhibition of Kv2.2 by GxTX-1E significantly reduced action potential firing in response to suprathreshold stimulation in this population of bladder sensory neurons. This result is in good agreement with previous electrophysiological studies that showed that Kv2 activity is necessary for sustained firing of hippocampal pyramidal, trapezoid body and substantia nigra dopaminergic neurons.

Although aberrant bladder afferent signaling is considered to play an important role in symptoms generation in IC/BPS, little is known about the sensory pathways that are involved in this process and the mechanisms that promote afferent sensitization. We previously showed that the overexpression of Cldn2 in the rat urothelium reproduces the cardinal features of IC/BPS by a mechanism that involves A delta fiber afferents [2]. The data presented in this report suggest that the functional changes seen in rats transduced with AdCldn2 reflect in part changes in ion channels that control neuronal excitability. These findings provide novel insights into the molecular mechanism that cause neuronal hyperexcitability in the face of urothelial barrier dysfunction and reveal novel potential targets to treat hypersensitive bladder disorders.

Montalbetti, N., et al., Increased urothelial paracellular transport promotes cystitis. Am J Physiol Renal Physiol, 2015. 309(12): p. F1070-81.
Montalbetti, N., et al., Urothelial Tight Junction Barrier Dysfunction Sensitizes Bladder Afferents. eNeuro, 2017. 4(3).