Regulation of rat neuronal voltage-dependent calcium channels by endogenous p21-ras

Influx of calcium through voltage-dependent calcium channels (VDCCs) has been implicated in the processes of cell growth and differentiation. Various signalling proteins, including nerve growth factor (NGF), p21-ras and src tyrosine kinases, have been suggested to have a role in the regulation of neuronal VDCCs. Using the whole-cell patch-clamp technique we have investigated the role of endogenous p21-ras in the regulation of VDCCs in primary cultured dorsal root ganglion (DRG) neurons obtained from neonatal rats. Neutralization of endogenous p21-ras by microinjection of p21-ras antibody (Y13-259) reduced the maximum peak barium current, I-max, whereas microinjection of oncogenic p21-K-ras increased the current. Thus, endogenous p21-ras is involved in the tonic regulation of calcium currents in these cells. Intracellular application of a phosphopeptide, Trk 490, which prevents the binding of the adaptor protein she to the activated NGF receptor, so blocking p21-ras activation, reduced I-max. Similarly deprivation of NGF by overnight incubation in NGF-free medium also reduced I-max. Together, these results suggest that NGF receptor tyrosine kinase activation of p21-ras is likely to be involved in the tonic regulation of VDCCs in DRG neurons. Deprivation of NGF combined with microinjection of p21-ras antibody (Y13-259), however, caused an even greater reduction of I-max. Thus, NGF activation can only partially explain the regulation of these currents by endogenous p21-ras. Src tyrosine kinases have been suggested to activate p21-ras. In DRG neurons, microinjection of purified src tyrosine kinase, pp60(c-src), increased I-max in these cells. However, co-microinjection of pp60(c-src) with Y13-259 antibody prevented the increase in I-max, implying that pp60(c-src) can also regulate calcium currents via the activation of endogenous p21-ras. Further support for the involvement of tyrosine kinases in VDCC regulation was provided by the application of the general tyrosine kinase inhibitor, genistein, which also reduced I-max. Thus, VDCCs in rat DRG neurons appear to be tonically up-regulated by endogenous p21-ras. This effect appears largely to involve NGF receptor tyrosine kinase activation of p21-ras. in addition, src tyrosine kinase may also regulate VDCCs, possibly via p21-ras.