Abstract
Mechanosensitive channels serve as essential sensors for cells to interact with their environment. The identity of mechanosensitive channels that underlie somatosensory touch transduction is still a mystery. One promising mechanotransduction candidate is the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel. To determine the role of TRPA1 in the generation of mechanically-sensitive currents, we used dorsal root ganglion (DRG) neuron cultures from adult mice and applied rapid focal mechanical stimulation (indentation) to the soma membrane. Small neurons (diameter <27 µm) were studied because TRPA1 is functionally present in these neurons which largely give rise to C-fiber afferents in vivo. Small neurons were classified by isolectin B4 binding.Mechanically-activated inward currents were classified into two subtypes: Slowly Adapting and Transient. First, significantly more IB4 negative neurons (84%) responded to mechanical stimulation than IB4 positive neurons (54%). Second, 89% of Slowly Adapting currents were present in IB4 negative neurons whereas only 11% were found in IB4 positive neurons. Third, Slowly Adapting currents were completely absent in IB4 negative neurons from TRPA1−/− mice. Consistent with this, Slowly Adapting currents were abolished in wild type IB4 negative neurons stimulated in the presence of a TRPA1 antagonist, HC-030031. In addition, the amplitude of Transient mechanically-activated currents in IB4 positive neurons from TRPA1−/− mice was reduced by over 60% compared to TRPA1+/+ controls; however, a similar reduction did not occur in wild-type neurons treated with HC-030031. Transfection of TRPA1 in HEK293 cells did not significantly alter the proportion or magnitude of mechanically-activated currents in HEK293 cells, indicating that TRPA1 alone is not sufficient to confer mechanical sensitivity.These parallel genetic and pharmacological data demonstrate that TRPA1 mediates the Slowly Adapting mechanically-activated currents in small-diameter IB4 negative neurons from adult mice. The TRPA1 protein may also contribute to a complex that mediates Transient mechanically-activated currents in small IB4 positive C fiber type neurons.
Highlights
Mechanotransduction is a fundamental mechanism by which virtually all cells from bacteria to specialized mammalian sensory neurons interact with their external environment
Sensory neurons express a wide variety of TRP channels that are thought to be in some manner, sensitive to mechanical force, including Transient Receptor Potential Ankyrin 1 (TRPA1), TRPV2, TRPV4, TRPC1, TRPC6 and TRPP2 [4,26]
One widely used approach is to investigate the soma of isolated dorsal root ganglion (DRG) neurons with the assumption that the soma plasma membrane mimics the peripheral terminal plasma membrane regarding the expression of the same ion channels within the same intracellular milieu
Summary
Mechanotransduction is a fundamental mechanism by which virtually all cells from bacteria to specialized mammalian sensory neurons interact with their external environment. Ionic channels that respond to mechanical force have been described in prokaryotic cells [2]. In microbes such as bacteria, mechanosensitive channels are directly activated by stretch of the membrane bilayer and are functionally redundant in that more than one type is expressed by the same cell [3]. A series of candidates for mechanotransduction have been proposed, including family members of TRP channels, ASIC channels, Na+ channels and K+ channels [4,5]. The functional redundancy of mechanosensitive channels is likely preserved in many, if not all eukaryotic cells, making the interpretation of ablation of a single protein challenging
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