Modulation of transient receptor potential Vanilloid 4-mediated membrane currents and synaptic transmission by protein kinase C

Abstract

Background: Transient receptor potential Vanilloid (TRPV) receptors are involved in nociception and are expressed predominantly in sensory neurons. TRPV1, a non-selective cation channel has been extensively studied and is responsible for inflammatory thermal hypersensitivity. In this study, the expression and function of TRPV4 have been characterized and compared with those of TRPV1.

Results: Immunohistochemical studies revealed that both TRPV1 and TRPV4 were co-expressed in dorsal root ganglion (DRG) neuronal cell bodies and in the central terminals of laminae I and II of the spinal dorsal horn (DH). In Ca2+ fluorescence imaging and whole-cell patch-clamp experiments, TRPV1- and TRPV4-mediated responses were observed in a population of the same DRG neurons. Sensitization of TRPV1 has been shown to be involved in inflammatory pain conditions. Incubation with phorbol 12, 13-dibutyrate (PDBu), a PKC activator, resulted in a significant potentiation of TRPV4 currents in DRG neurons. In TRPV4 expressing HEK 293T cells, PDBu increased 4alpha-phorbol 12, 13-didecanoate (4alpha-PDD)-induced single-channel activity in cell-attached patches, which was abrogated by bisindolylmaleimide (BIM), a selective PKC inhibitor. TRPV4 is also expressed at the central terminals of sensory neurons. Activation of TRPV4 by 4alpha-PDD increased the frequency of miniature excitatory post synaptic currents (mEPSCs) in DRG-DH neuronal co-cultures. 4alpha-PDD-induced increase in the frequency of mEPSCs was further enhanced by PDBu. The expression of TRP channels has been shown in other areas of the CNS; application of 4alpha-PDD significantly increased the mEPSC frequency in cultured hippocampal neurons, which was further potentiated by PDBu, whereas, TRPV1 agonist capsaicin did not modulate synaptic transmission.

Conclusion: These results indicate that TRPV4 and TRPV1 are co-expressed in certain DRG neurons and TRPV4 can be sensitized by PKC not only in DRG neuronal cell bodies, but also in the central sensory and non-sensory nerve terminals. Co-expression of TRPV1 and TRPV4 ion channels, their modulation of synaptic transmission and their sensitization by PKC may synergistically play a role in nociception.

Figure 1

TRPV1 and TRPV4 are co-localized in dorsal root ganglion (DRG) neurons. A. Immunostaining of TRPV1 in DRG. B. Immunostaining of TRPV4 in DRG. C. Merged images of A and B. D. Expanded merged image. Bar = 50 μm. E. Summary graph showing the expression of TRPV1, TRPV4 and the extent of co-expression (n = 4). F. Ca²⁺ fluorescence imaging showing capsaicin- and 4α-PDD-induced responses in the same neurons. G. Whole-cell current trace showing capsaicin- and 4α-PDD-mediated current responses in the same neuron.

Figure 2

Sensitization of TRPV4 and TRPV1 by PKC in cultured DRG neurons. A. Both capsaicin (100 nM)-induced and 4α-PDD (10 μM)-induced currents are potentiated by a PKC activator, PDBu (1 μM). B, C. Summary graphs showing potentiation of 4α-PDD- and capsaicin-induced current activity by PDBu (1 μM; n = 11). The asterisk (*) represents P < 0.05 as compared to control.

Figure 3

Current-voltage relationship of 4α-PDD-evoked single-channel current in TRPV4 transfected HEK 293T cells and in DRG neurons natively expressing TRPV4. A. Single-channel currents induced by 4α-PDD recorded at different membrane potentials from transfected HEK 293T cells. B. Current-voltage relationship shows that single-channel conductance is lower at negative potentials (76 ± 6 pS) as compared to positive potentials (110 ± 5 pS). C. Single-channel currents induced by 4α-PDD recorded at different membrane potentials from DRG neurons. D. Current-voltage relationship shows that the single-channel conductance is lower than that of cloned TRPV4 at negative potentials (22 ± 4 pS) as compared to positive potentials (135 ± 32 pS). The single-channel conductance contributes to the macroscopic current rectification.

Figure 4

Enhancement of TRPV4-mediated single-channel current activity by PKC. A. Single-channel currents activated by 4α-PDD (1 μM) recorded at + 60 mV from a cell-attached patch on TRPV4 transfected HEK 293T cells before and after PDBu incubation. The traces are shown at higher time resolution below and the amplitude histograms are shown on the right. B. 4α-PDD-induced single-channel currents recorded at – 60 mV before and after PDBu incubation. The traces are shown at a higher time resolution below and the amplitude histograms are shown on the right.

Figure 5

TRPV1 and TRPV4 are expressed at the central terminals and are co-localized in spinal dorsal horn. A. Immunostaining of TRPV1 in dorsal horn. B. Immunostaining of TRPV4 in dorsal horn. C. Merged image showing the extent of co-localization in yellow. Bar = 300 μm.

Figure 6

Potentiation of 4α-PDD-induced changes in synaptic transmission by PDBu in DRG-DH co-cultures. A. Application of 4α-PDD (40 μM) increases the frequency of mEPSCs and this action is enhanced by incubation with PDBu (1 μM). Synaptic currents are shown at a higher time resolution below. B. Cumulative probability plot indicates an increase in the frequency of synaptic events with 4α-PDD, which is further significantly enhanced following by PDBu incubation. C. The increase in frequency of events is not accompanied by a change in the amplitude. D. Summary graphs showing 4α-PDD-induced an increase in mEPSC frequency (n = 5) and potentiation by PDBu (n = 3). The asterisk (*) represents P < 0.05 as compared to control; (**) represents P < 0.05 as compared to 4α-PDD group.

Figure 7

Enhancement of 4α-PDD-induced increase in synaptic transmission by PDBu in cultured hippocampal neurons. A. Application of 4α-PDD (10 μM) increases the frequency of mEPSCs, which is further potentiated by PDBu. The synaptic events are shown at a higher time resolution below. B. Cumulative probability plot shows a decrease in inter-event interval. C. The increase in frequency is not accompanied by a change in the amplitude. D. Summary graph showing 4α-PDD induces an increase in mEPSC frequency and this action is enhanced by PDBu (n = 7). The asterisk (*) represents P < 0.05 as compared to control; (**) represents P < 0.05 as compared to 4α-PDD group.