GTP-induced tetrodotoxin-resistant Na+ current regulates excitability in mouse and rat small diameter sensory neurones

Peripheral pain thresholds are regulated by the actions of inflammatory mediators. Some act through G-protein-coupled receptors on voltage-gated sodium channels. We have found that a low-threshold, persistent tetrodotoxin-resistant Na⁺ current, attributed to Na_V1.9, is upregulated by GTP and its non-hydrolysable analogue GTP-γ-S, but not by GDP. Inclusion of GTP-γ-S (500 μ m ) in the internal solution led to an increase in maximal current amplitude of > 300 % within 5 min. In current clamp, upregulation of persistent current was associated with a more negative threshold for action potential induction (by 15–16 mV) assessed from a holding potential of −90 mV. This was not seen in neurones without the low-threshold current or with internal GDP ( P < 0.001). In addition, persistent current upregulation depolarized neurones. At −60 mV, internal GTP-γ-S led to the generation of spontaneous activity in initially silent neurones only when persistent current was upregulated. These findings suggest that regulation of the persistent current has important consequences for nociceptor excitability.     

Increased Ca2+ influx through Na+/Ca2+ exchanger during long-term facilitation at crayfish neuromuscular junctions

Intense motor neuron activity induces a long-term facilitation (LTF) of synaptic transmission at crayfish neuromuscular junctions (NMJs) that is accompanied by an increase in the accumulation of presynaptic Ca²⁺ ions during a test train of action potentials. It is natural to assume that the increased Ca²⁺ influx during action potentials is directly responsible for the increased transmitter release in LTF, especially as the magnitudes of LTF and increased Ca²⁺ influx are positively correlated. However, our results indicate that the elevated Ca²⁺ entry occurs through the reverse mode operation of presynaptic Na⁺/Ca²⁺ exchangers that are activated by an LTF-inducing tetanus. Inhibition of Na⁺/Ca²⁺ exchange blocks this additional Ca²⁺ influx without affecting LTF, showing that LTF is not a consequence of the regulation of these transporters and is not directly related to the increase in [Ca²⁺]_i reached during a train of action potentials. Their correlation is probably due to both being induced independently by the strong [Ca²⁺]_i elevation accompanying LTF-inducing stimuli. Our results reveal a new form of regulation of neuronal Na⁺/Ca²⁺ exchange that does not directly alter the strength of synaptic transmission.     

Protein kinase C mediates up-regulation of tetrodotoxin-resistant, persistent Na+ current in rat and mouse sensory neurones

The tetrodotoxin-resistant (TTX-r) persistent Na⁺ current, attributed to Na_V1.9, was recorded in small (< 25 μm apparent diameter) dorsal root ganglion (DRG) neurones cultured from P21 rats and from adult wild-type and Na_V1.8 null mice. In conventional whole-cell recordings intracellular GTP-γ-S caused current up-regulation, an effect inhibited by the PKC pseudosubstrate inhibitor, PKC19–36. The current amplitude was also up-regulated by 25 μ m intracellular 1-oleoyl-2-acetyl-sn-glycerol (OAG) consistent with PKC involvement. In perforated-patch recordings, phorbol 12-myristate 13-acetate (PMA) up-regulated the current, whereas membrane-permeant activators of protein kinase A (PKA) were without effect. PGE₂ did not acutely up-regulate the current. Conversely, both PGE₂ and PKA activation up-regulated the major TTX-r Na⁺ current, Na_V1.8. Extracellular ATP up-regulated the persistent current with an average apparent K _d near 13 μ m , possibly consistent with P2Y receptor activation. Numerical simulation of the up-regulation qualitatively reproduced changes in sensory neurone firing properties. The activation of PKC appears to be a necessary step in the GTP-dependent up-regulation of persistent Na⁺ current.