Brain Microtubule Reconstitutionin lipid bilayer system is associated with the presence of cation-selective channel activity. 60th Annual Meeting of the Biophysical Society

RODRIGUEZ SANTOS IP; SCARINCI N; CANTIELLO HF; CANTERO MR

Congreso; 60th Annual Meeting of the Biophysical Society.; 2016

Microtubules (MTs) are unique components of the cytoskeleton formed by hollow cylindrical structures of  tubulin dimeric units. MTs play important roles in cell functions, including acting as railways for motor proteins, and separating chromosomes during cell division. MTs are also highly charged polar polyelectrolytes, capable of amplifying electrical signals. However, the actual nature of these novel electrodynamic properties of MTs remains largely unknown. Here we reconstituted MTs isolated from bovine brain (n = 72) in a lipid bilayer reconstitution system (BLM). Lipid mixture was made of a 7:3 ratio of POPC and POPE (20-25 mg/ml, Avanti Polar Lipids) in n-decane. MTs painted onto the BLM displayed electrical currents (36/72), consistent with the formation of ion channels which, in physiological K+ (140 mM), displayed cation-selective properties and a single channel conductance of 64.8 ± 6.9 pS (n = 6). In the presence of a KCl chemical gradient (150/15 mM) two main conductance of 61.4 ± 11.0 pS (n = 4), and 548 ± 45.4 pS (n =3) were determined. Occasionally, a conductance of 8.14 nS was observed. Addition of GTP (1 mM) to the cis chamber had no effect on the MT-associated channel activity. However, the ion channel properties displayed strong voltage dependence, modifying the MT channel´s mean open time but not the conductance. Addition of taxol (5 M, Paclitaxel®), an MT-stabilizer agent completely inhibited the MT channel activity. The possible structural features of the MT-generated ion channels are discussed in the context of the multiple MT assemblies present in the isolation process. The fact that MTs are capable of generating ion channel activity may be relevant for the functional properties of cells membranes, and help understand various aspects of neuronal physiology and the pathogenesis of neurodegenerative diseases. The findings offer new insights into the nonlinear behavior of the cytoskeleton.