INTEGRIN-MEDIATED NEURITE OUTGROWTH IN NEUROBLASTOMA-CELLS DEPENDS ONTHE ACTIVATION OF POTASSIUM CHANNELS

Electrical signals elicited by integrin interaction with ECM component s and their role in neurite outgrowth were studied in two clones (N1 a nd N7) isolated from 41A3 murine neuroblastoma cell line. Although the two clones similarly adhered to fibronectin (FN) and vitronectin (VN) , this adhesion induced neurite outgrowth in N1 but not in N7 cells. P atch clamp recordings in whole cell configuration showed that, upon ad hesion to FN or VN but not to platelet factor 4 (PF4), N1 cells underg o a marked (is-approximately-equal-to 20 mV) hyperpolarization of the resting potential (V(rest)) that occurred within the first 20 min afte r cell contact with ECM, and persisted for approximately 1 h before re verting to the time zero values. This hyperpolarization was totally ab sent in N7 cells. A detailed analysis of the molecular mechanisms invo lved in N1 and N7 cell adhesion to ECM substrata was performed by usin g antibodies raised against the FN receptor and synthetic peptides var iously competing with the FN or VN binding to integrin receptors (GRGD SP and GRGESP). Antibodies, as well as GRGDSP, abolished adhesion of N 1 and N7 clones to FN and VN, revealing a similar implication of integ rins in the adhesion of these clones to the ECM proteins. However, the se anti-adhesive treatments, while ineffective on V(rest), of N7 cells , abolished in N1 cells the FN- or VN-induced hyperpolarization and ne urite outgrowth, that appeared therefore strictly associated and integ rin-mediated phenomena. The nature of this association was deepened th rough a comparative analysis of the integrin profiles and the ion chan nels of N1 and N7 cells. The integrin immunoprecipitation profile resu lted very similarly in the two clones, with only minor differences con cerning the alphaV containing complexes. Both clones possessed Ca2+ an d K+ delayed rectifier (K(DR)) channels, while only N1 cells were endo wed with inward rectifier K+ (K(IR)) channels. The latter governed the V(rest), and, unlike K(DR) channels, were blocked by Ba2+ and Cs+. By moving patched cells in contact with FN-coated beads, it was shown th at K(IR) channel activation was responsible for the FN-mediated hyperp olarization of V(rest). Treatment with Pertuxis toxin (PTX) abolished this hyperpolarization and neurite outgrowth, indicating that a G prot ein is interposed between integrins and K(IR) channels and that the ac tivation of these channels is required for neuritogenesis. In fact, th e block of K(IR) channels by Cs+ abolished both hyperpolarization and neurite outgrowth, provided that the cation was supplied during the fi rst two hours after N1 cell contact with FN. This Cs+-sensitive commit ment time for neuritogenesis coincided with the time length of the FN- induced hyperpolarization.