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^1,2 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^3–6, treatment with micromolar concentrations of ATP⁴⁻ (ref. 7) and also with very small (that is non-agglutinating, non-fusogenic) amounts of Sendai virus⁸. 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.