An efficient method for infection of adrenal chromaffin cells using the Semliki Forest virus gene expression system

We have expanded the use of the Semliki Forest virus (SFV) by infecting chr omaffin tells with synaptic proteins at high efficiency. Using the SFV gene expression system, up to 40 % of cultured bovine chromaffin cells express the protein of interest within 12-48 h after infection. In order to learn a bout the basic physiological properties of infected cells, we performed mem brane capacitance measurements using the whole-cell patch-clamp technique a nd monitored catecholamine release with amperometry. We found that chromaff in cells infected with green fluorescent protein (GFP) were comparable to c ontrol cells in intracellular calcium concentrations ([Ca2+](i)), leak curr ents and cell sizes, In response to depolarization, calcium currents were e licited and the cells secreted catecholamine, Comparison of the calcium cur rent amplitude and the size of the readily releasable pool of vesicles reve aled a small decrease in these parameters compared to control cells, The re filling kinetics after pool depletion, however, were not altered, Overexpre ssed munc13-1 translocates to the plasma membrane in response to phorbol es ters, an effect that is also observed in fibroblasts transfected with conve ntional methods. Thus, the use of the SFV gene expression system to infect chromaffin cells represents a major improvement in infection efficiency com pared to other methods. It opens up new opportunities to introduce synaptic proteins into chromaffin cells and study their role in secretion.