The mechanosensitive cell line ND-C does not express functional thermoTRP channels

Abstract

The molecular basis of mechanosensation in sensory neurons has yet to be defined. We found that ND-C cells, a hybrid cell line derived from neonatal rat DRG neurons, express mechanosensitive ion channels, and provide a useful expression system for testing candidate mechanosensitive ion channels. ND-C cells retain some important features of DRG neurons such as the expression of TTX-sensitive Na⁺ and acid-activated currents as well as the ability to respond to mechanical stimulation with cationic currents sensitive to the analgesic peptide NMB1. ND-C cells do not respond to agonists of the ‘thermoTRP’ channels, suggesting that these channels are not responsible for MA currents in these cells and DRG neurons. Furthermore, transfecting ND-C cells with the candidate mechanotransducer channel TRPA1 does not increase MA current amplitudes, despite TRPA1 being functionally expressed at the plasma membrane. This correlates well with the fact that all types of MA currents can be recorded from TRPA1-negative DRG neurons.

Introduction

The mechanism by which the somatosensory neurons of the dorsal root ganglia (DRG neurons) transduce mechanical stimuli into electrical and biochemical signals remains elusive (Lumpkin and Caterina, 2007). When studied in culture, DRG neurons respond to the mechanical stimulation of their soma with the opening of non-selective cationic Gd³⁺-sensitive channels whose molecular identity has yet to be determined (Drew et al., 2002, Drew et al., 2004, Cho et al., 2002, Cho et al., 2006, Hu and Lewin, 2006, McCarter and Levine, 2006, Coste et al., 2007). These channels are insensitive to the spider stretch-activated channel blocker GsMTx4 but are blocked by the conotoxin NMB1 (Drew et al., 2007). Recent studies carried out mainly in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster have demonstrated the role of ion channels of the Transient Receptor Potential (TRP) family in mechanotransduction (Christensen and Corey, 2007). In mammals, several TRP channels were shown to be involved in mechanotransduction and/or osmosensation, amongst them are members of the TRPV family. A splice variant of the vanilloid receptor, TRPV1, was found to be essential for osmosensory transduction in the supra-optic nucleus (Sharif Naeini et al., 2006) but TRPV1 mutants display normal responses to mechanical stimuli (Caterina et al., 2000) and numerous capsaicin-insensitive DRG neurons respond to mechanical stimulation (Drew et al., 2002). Another TRPV, TRPV2, was shown to be expressed in vascular smooth muscle cells where it senses osmotic stimuli (Muraki et al., 2003). Finally, TRPV4 is expressed in dermal mechanosensory structures involved in both low and high threshold mechanosensation (Suzuki et al., 2003b) and TRPV4-null animals are less sensitive to pressure applied to their tail than their wild type counterparts (Suzuki et al., 2003a). TRPP2 (PKD2), an amiloride-sensitive channel, is involved in mechanotransduction in renal cells (Nauli et al., 2003), while TRPA1 participates in the nematode mechanosensation (Kindt et al., 2007) and may have a small effect in mammalian mechanosensation (Kwan et al., 2006). Finally, the GsMTx4-sensitive TRPC1 and C6 channels are thought to be activated by mechanical stimulation (Maroto et al., 2005) although two recent studies argue against a role for TRPC1 in mechanotransduction (Gottlieb et al., 2007, Dietrich et al., 2007). On the other hand TRPC6, which is expressed in both heart and kidney, was shown to be activated by membrane stretch (Spassova et al., 2006); TRPC6 seems to mediate the mechanical responses of ventricular myocytes (Dyachenko et al., in press), and mutations in TRPC6 are responsible for human hereditary glomerular diseases likely due to TRPC6 malfunction in the mechanosensitive renal slit diaphragm (Huber et al., 2007). Here, we reassess the possible involvement of TRP channels in mechanotransduction by studying their role in a DRG neuron-derived cell line, ND-C. We show that ND-C cells have physiological properties similar to DRG neurons and that TRP channels involved in temperature sensation in DRG neurons are not responsible for mechanosensation. We also show TRPA1 does not augment the density of endogenous MA currents when transfected in ND-C cells and TRPA1 expression in DRG neurons does not correlate with the expression of a particular type of mechanically activated (MA) current.