Lin, Wei

Located in the book "Xenopus Protocols: Cell Biology and Signal Transduction" edited by Johne X Liu, the chapter, "Studying the mechanosensitivity of voltage-gated channels using oocyte patches" was written by the listed authors including Terence J. Morris (Douglas College Faculty). Book chapter: The mechanosensitivity of voltage-gated (VG) channels is of biophysical, physiological, and pathophysiological interest. Xenopus oocytes offer a critical advantage for investigating the electrophysiology of recombinant VG channels subjected to membrane stretch, namely, the ability to monitor macroscopic current from membrane patches. High-density channel expression in oocytes makes for macroscopic current in conventional-size, mechanically sturdy patches. With the patch configuration, precisely the same membrane that is voltage-clamped is the membrane subjected to on-off stretch stimuli. With patches, meaningful stretch dose responses are possible. Experimental design should facilitate within-patch comparisons wherever possible. The mechanoresponses of some VG channels depend critically on patch history. Methods for minimizing and coping with interference from endogenous voltage-dependent and stretch-activated endogenous channels are described.

During brain trauma, white matter experiences shear and stretch forces that, without severing axons, nevertheless trigger their secondary degeneration. In central nervous system (CNS) trauma models, voltage-gated sodium channel (Nav) blockers are neuroprotective. This, plus the rapid tetrodotoxin-sensitive Ca2+ overload of stretch-traumatized axons, points to "leaky" Nav channels as a pivotal early lesion in brain trauma. Direct effects of mechanical trauma on neuronal Nav channels have not, however, been tested. Here, we monitor immediate responses of recombinant neuronal Nav channels to stretch, using patch-clamp and Na+-dye approaches. Trauma constituted either bleb-inducing aspiration of cell-attached oocyte patches or abrupt uniaxial stretch of cells on an extensible substrate. Nav1.6 channel transient current displayed irreversible hyperpolarizing shifts of steady-state inactivation [availability(V)] and of activation [g(V)] and, thus, of window current. Left shift increased progressively with trauma intensity. For moderately intense patch trauma, a approximately 20-mV hyperpolarizing shift was registered. Nav1.6 voltage sensors evidently see lower energy barriers posttrauma, probably because of the different bilayer mechanics of blebbed versus intact membrane. Na+ dye-loaded human embryonic kidney (HEK) cells stably transfected with alphaNav1.6 were subjected to traumatic brain injury-like stretch. Cytoplasmic Na+ levels abruptly increased and the trauma-induced influx had a significant tetrodotoxin-sensitive component. Nav1.6 channel responses to cell and membrane trauma are therefore consistent with the hypothesis that mechanically induced Nav channel leak is a primary lesion in traumatic brain injury. Nav1.6 is the CNS node of Ranvier Nav isoform. When, during head trauma, nodes experienced bleb-inducing membrane damage of varying intensities, nodal Nav1.6 channels should immediately "leak" over a broadly left-smeared window current range.