Involvement of guanine nucleotide-binding protein in the gating of Ca2+ by receptors

Abstract

The introduction of impermeant aqueous solutes into individual cells by microinjection has long been established but the difficulties of manipulating the cytosol composition of large populations of microscopic cells have only recently been overcome. Successful techniques include a dielectric breakdown procedure, treatment with micromolar concentrations of ATP4- (ref. 7) and also with very small (that is nonagglutinating, non-fusogenic) amounts of Sendai virus. So far, attention has been concentrated on the behaviour of the cells (generally their response to applied Ca2+ buffers) at the time when the membrane permeability lesions are open, and thus cytosol and external medium are in contact. I now report a novel technique for monitoring the state of molecular solute permeability in cell membranes and show that the lesions generated by ATP4- in the membrane of mast cells can be closed within seconds of adding Mg2+ so that a cycle of permeabilization and resealing can be used to explore the effect of foreign compounds trapped in the cytosol of effectively intact cells. I show that non-hydrolysable GTP analogues, introduced into the cytosol of mast cells, cause them to undergo exocytotic secretion in response to addition of extracellular Ca2+. This finding is discussed in the light of previous experience relating guanine nucleotide regulatory proteins as intermediaries between receptors and the transducers which they control.