Proceedings of The Physiological Society
Durham University (2010) Proc Physiol Soc 21, C11 and PC11
Defining the forces required to gate mechanosensitive channels in mammalian sensory neurons
K. Poole1, G. R. Lewin1
1. Dept. Neuroscience, Max Delbruck Center, Berlin-Buch, Germany.
Our sense of touch and mechanical pain is based on mechano-electrical transduction at the terminal endings of dorsal root ganglion (DRG) neurones innervating the skin. How this transduction works, and the molecules responsible have proven difficult to elucidate, due to inaccessibility of the nerve endings and the presumed limited quantity of transduction molecules. Sensory DRG neurones can be acutely prepared and retain mechanosensitivity when cultured on laminin, as determined using whole-cell patch-clamp during direct mechanical stimulation of the neurites or soma. However, such an approach does not allow determination of the precise forces required to gate transduction channels. In order to quantify the stimulus strengths required to gate mechanosensitive channels in these neurons, we have developed an approach using microstructured surfaces. We cast pillar arrays from PDMS, the pillar tips are coated with laminin and then DRG neurones can be cultured on top. Each pilus has a defined elasticity (2 ± 0.1 MPa) and shape (r = 0.7 µm; l = 5 µm or 8 µm), and as such a calculable spring constant. Cells were cultured on top of pili with a calculated spring constant of either 2.2 pN/nm or 9.0 pN/nm. Neurons cultured on pili with a calculated spring constant of 2.2 pN/nm did not extend neurites, in contrast to cells cultured on the stiffer pili. To apply mechanical stimuli, individual pili underneath the neurites are deflected using a piezo-driven nano-manipulator and the deflection is monitored using light microscopy. As the PDMS pili behave as light-guides, the center of the each pilus can be determined from a 2-D Gaussian fit of the intensity, allowing detection of movements as small as a couple of nanometers. The cellular response to the pili deflection is monitored using whole-cell patch-clamp to record the latency, kinetics and amplitude of mechanically gated currents. We show that pili deflections as small as 5 nm can gate the rapidly-adapting current in mechanoreceptor cells, while larger deflections (above 150 nm) are required for gating of slowly-adapting currents in nociceptors. Application of mechanical stimuli via pili deflection resulted in a lower threshold for gating of the rapidly-adapting current (5 nm), compared with application of the stimulus to the top of the neurite (minimum required movement for channel gating = 70-100 nm1). This suggests that gating occurs at the cell-substrate interface. We have also observed that the latency of activation of rapidly-adapting currents in mechanoreceptors (293 ± 1 µs, n = 31) is significantly faster (p < 0.001, Mann-Whitney test) than the latency of activation of the same type of current in nociceptors (548 ± 13 µs, n = 18). Using such a method we are currently characterising the precise physical conditions, in terms of deflection and applied force, required for mechanotransduction in sensory neurons.
Where applicable, experiments conform with Society ethical requirements