Rolf Graf

Molecular basis of regulation of ionic channels by G proteins

Publisher Summary This chapter presents primary structure of G proteins as deduced from purified proteins and cloned subunits. It also discusses their functions and presents data on direct regulation of ionic channels by G proteins. Experiments on expression of subunits, either in bacteria or by in vitro translation of messenger RNA (mRNA) synthesized from complimentary DNA (cDNA), are discussed as tools for definitive assignment of function to a given G protein. The chapter also discusses the dynamics of G protein-mediated signal transduction. The key points discussed in the chapter include the existence of two superimposed regulatory cycles in which G proteins dissociate into α plus βγ upon activation by guanosine triphosphate (GTP) and where the dissociated α subunits hydrolyze GTP. The chapter emphasizes the action of receptors to catalyze rather than regulate by allostery the activation of G proteins by GTP and also the role of subunit dissociation, without which receptors cannot act as catalysts. It also provides an overview on intramembrane networking of G protein-mediated receptor-effector coupling.

Differential stimulation of the GTPase activity of G-proteins by polylysine

Polylysine, polyornithine and, to a lesser extent, polyarginine were found to stimulate the GTPase activity of the purified recombinant alpha subunit of the human G(i)-3 transducing protein alpha i-3. Optimal stimulation of 4- to 5-fold was obtained with polylysine concentrations between 1 and 20 microM, higher concentrations being inhibitory. Polylysine at similar concentrations stimulated by 50% the GTPase of transducin (GT), the vision transducing protein, but had only a very slight effect on the GTPase of the p21 product of the H-ras protooncogene. The stimulation of the alpha i-3 GTPase caused by polylysine was due to a reduction of the apparent Km for GTP from 3.8 to 1.3 microM. The stimulation by polylysine was observed at free Mg2+ concentrations below 1 microM. These results indicate that polylysine acts in a fashion similar to mastoparan and substance P in mimicking the action of an agonist-bound receptor on G-proteins.

Multiple Roles of G Proteins in Coupling of Receptors to Ionic Channels and Other Effectors

The central role of G proteins in coupling receptors to effector systems can be best illustrated by the contents of Table 1, which lists receptors that exert their actions by interacting with a G protein, according to the general schemes shown in Fig. 1. Like receptors, which are increasing in number rapidly, effectors affected by the activated forms of G proteins are also increasing, most notably through the discovery in 1986–1987 that ionic channels form part of the family of molecules regulated by G proteins. Using cell-free systems such as provided by excision of membrane patches from cells and incorporation of plasma membrane vesicles into lipid bilayers, it was shown that ionic channels are indeed regulated by activated G proteins. Some of these channels had long before been predicted to be under the control of G-protein-coupled receptors by means other than soluble second messengers. The mechanism by which G proteins regulate some of these channels is at the very least “membrane delimited” and independent of any phosphorylation event or of changes in cytoplasmic levels of second messengers such as cAMP, Ca2+, or IP3, and is very likely due to direct interaction of the G protein α-subunit and the channel proper (for review, see Brown and Birnbaumer 1988). Our group was prominent in providing some of the initial as well as subsequent supporting data for these conclusions (Brown and Birnbaumer 1988; Yatani et al. 1987a,b,c, 1988a,b,c; Codina et al. 1987a,b, 1988; Imoto et al. 1988; Kirsch et al. 1988). Another research group has claimed that ionic channels, specifically the heart muscarinic K+ channel, now referred to as Gi-gated K+ channel (see below), may be regulated by receptors, not via a specific G protein α-subunit, but via the βγ-subunits of G proteins (Logothetis et al. 1987).