Role of calcium in regulating primary sensory neuronal excitability.

The fundamental role of calcium ions (Ca(2+)) in an excitable tissue, the frog heart, was first demonstrated in a series of classical reports by Sydney Ringer in the latter part of the nineteenth century (1882a, b; 1893a, b). Even so, nearly a century elapsed before it was proven that Ca(2+) regulated the excitability of primary sensory neurons. In this chapter we review the sites and mechanisms whereby internal and external Ca(2+) can directly or indirectly alter the excitability of primary sensory neurons: excitability changes being manifested typically by variations in shape of the action potential or the pattern of its discharge.

Electrophysiological properties and chemosensitivity of acutely dissociated trigeminal somata innervating the cornea.

Adult rat sensory trigeminal ganglion neurons innervating the cornea (cTGNs) were isolated and identified following retrograde dye labeling with FM1-43. Using standard whole-cell patch clamp recording techniques, cTGNs could be subdivided by their action potential (AP) duration. Fast cTGNs had AP durations <1 ms (40%) while slow cTGNs had AP durations >1 ms and an inflection on the repolarization phase of the AP. With the exception of membrane input resistance, the passive membrane properties of fast cTGNs were different from those of slow cTGNs (capacitance: 61+/-4.5 pF vs. 42+/-2.6 pF, resting membrane potential: -59+/-0.7 mV vs. -53+/-0.9 mV, for fast and slow cTGNs respectively). Active membrane properties also differed between fast and slow cTGNs. Slow cTGNs had a higher AP threshold (-25+/-1.6 mV vs. -38+/-0.8 mV), a larger rheobase (14+/-1.9 pA/pF vs. 6.8+/-1.0 pA/pF), and a smaller AP undershoot (-56+/-1.7 mV vs. -67+/-2.5 mV). The AP overshoot, however was similar between the two types of neurons (46+/-3.1 mV vs. 48+/-4 mV). Slow cTGNs were depolarized by capsaicin (1 microM, 80%) and 60% of their APs were blocked by tetrodotoxin (TTX) (100 nM). Fast cTGNs were unaffected by capsaicin and 100% of their APs were blocked by TTX. Similarly, cTGNs were also heterogeneous with respect to their responses to exogenous ATP and 5-HT. The current work shows that cTGNs have distinctive electrophysiological properties and chemosensitivity profiles. These characteristics may mirror the distinct properties of corneal sensory nerve terminals. The availability of isolated identified cTGNs constitutes a tractable model system to investigate the biophysical and pharmacological properties of corneal sensory nerve terminals.

Substance P evokes cation currents through TRP channels in HEK293 cells.

Activation of any of the three known tachykinin receptors (NK1R, -2R, or -3R) can cause a rise in [Ca2+]i via a pertussis toxin-insensitive heterotrimeric G protein, Gq/G11, activation of phospholipase C (PLC), and a membrane depolarization. Tachykinins can depolarize neurons by two distinct mechanisms: 1) they reduce a resting K+ current in many neurons or 2) in parasympathetic and vagal primary sensory neurons, they activate a nonspecific cation current (Icat). Transient receptor potential channels (TRPC) are nonspecific cation channels that can be activated by a rise in [Ca2+]i in a PLC-dependent manner. The present work tests whether NK2R can signal TRPC. We applied standard whole cell patch-clamp recordings to HEK293 cells stably transfected with the human TRP3 channels (TRP3C), and transiently transfected with a functional NK2R-EGFP. Bath applied Substance P (SP, 1 microM) induced an Icat in the cells expressing both TRP3C and NK2R. Icat reached its peak value in approximately 3 min (195 +/- 120.0 s, mean +/- SE, n = 20), had a peak density of 11.3 +/- 3.48 pA/pF (n = 24), and was blocked by an NK2R-specific antagonist (SR48968, 100 nM). The Erev value for the SP current was 6.8 +/- 7.66 mV (n = 6), suggestive of a nonspecific cation channel. Icat was not measurable in TRP3C-expressing HEK293 cells without NK2R expression (n = 6) or in wild-type HEK293 cells with NK2R expression (n = 12). These data indicate that NK2R can be functionally coupled to TRP channels in HEK293 cells and suggest that SP-induced cation currents in vagal primary sensory neurons might be mediated by TRPC.

Inhibition of mechanical activation of guinea-pig airway afferent neurons by amiloride analogues.

1. The aim of this study was to investigate a role for Epithelial Sodium Channels (ENaCs) in the mechanical activation of low-threshold vagal afferent nerve terminals in the guinea-pig trachea/bronchus. 2. Using extracellular single-unit recording techniques, we found that the ENaC blocker amiloride, and its analogues dimethylamiloride and benzamil caused a reduction in the mechanical activation of guinea-pig airway afferent fibres. 3. Amiloride and it analogues also reduced the sensitivity of afferent fibres to electrical stimulation such that greater stimulation voltages were required to induce action potentials from their peripheral terminals within the trachea/bronchus. 4. The relative potencies of these compounds for inhibiting electrical excitability of afferent nerves were similar to that observed for inhibition of mechanical stimulation (dimethylamiloride approximately benzamil > amiloride). This rank order of potency is incompatible with the known rank order of potency for blockade of ENaCs (benzamil > amiloride >> dimethylamiloride). 5. As voltage-gated sodium channels play an important role in determining the electrical excitability of neurons, we used whole-cell patch recordings of nodose neuron cell bodies to investigate the possibility that amiloride analogues caused blockade of these channels. At the concentration required to inhibit mechanical activation of vagal nodose afferent fibres (100 microM), benzamil caused significant inhibition of voltage-gated sodium currents in neuronal cell bodies acutely isolated from guinea-pig nodose ganglia. 6. Combined, our findings suggest that amiloride and its analogues did not selectively block mechanotransduction in airway afferent neurons, but rather they reduced neuronal excitability, possibly by inhibiting voltage-gated sodium currents.