Multiple alpha subunits of guanine nucleotide-binding proteins in Dictyostelium

Previous results have shown that chemotaxis and the expression of several classes of genes in Dictyostelium discoideum are regulated through a cell surface cAMP receptor interacting with guanine nucleotide-binding proteins (G proteins). We now describe cloning and sequencing of cDNAs encoding two G alpha protein subunits from Dictyostelium. The derived amino acid sequences show that they are 45% identical to each other and to G alpha protein subunits from mammals and yeast. Both cDNAs are complementary to multiple mRNAs that are differentially expressed during development. This evidence and analysis of mutants presented elsewhere suggest that they have distinct physiological functions.     

Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins.

Antisera were raised against purified subunits of regulatory GTP-binding proteins (G proteins) and against synthetic peptides that correspond to defined regions of G proteins. Peptide antisera were generated that recognized all alpha or all beta subunits from Gs, Gi, Go, and transducin; others recognized only Gs alpha or Go alpha. Such cross-reaction or complete specificity for a given alpha subunit was not obtained when purified subunits were injected. Peptide antisera were used to identify G protein subunits in selected tissue membrane preparations by immunoblots.     

Specificity of action of guanine nucleotide-binding regulatory protein subunits on the cardiac muscarinic K+ channel.

The cardiac muscarinic receptor stimulates a potassium-selective ionic current (IK.ACh) through activation of a guanine nucleotide-binding regulatory protein. Purified alpha and beta gamma subunits of the guanine nucleotide-binding regulatory protein have each been reported to open the K+ channel. We have reported that nanomolar concentrations of purified brain beta gamma subunits activated IK.ACh in chicken embryonic atrial patches. In contrast, J. Codina, A. Yatani, D. Grenet, A.M. Brown, and L. Birnbaumer [(1987) Science 236, 442-445] subsequently reported that picomolar concentrations of activated erythrocyte alpha subunits (i.e., the 40-kDa alpha subunit that the authors call alpha K) opened K+ channels in guinea pig atrial patches. In this paper, we further explore the specificity of various beta gamma and alpha subunits in embryonic chicken and neonatal rat atrial patches. Beta gamma subunits from either human placenta (beta 35 gamma) or bovine brain (beta 35,36 gamma) activated IK.ACh whereas transducin beta gamma (beta 36 gamma) did not. The beta gamma activation was consistent in rat and chicken patches [118 of 123 patches (97%)]. Beta gamma subunits opened K+ channels at concentrations greater than or equal to 200 pM and maximally activated the channel at 10 nM. Beta gamma or guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) channel activation could be reversed by alpha 41-GDP. The purified brain beta gamma preparation was contaminated with less than 0.01% unactivated alpha. The detergent (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPS), used to suspend the hydrophobic beta gamma, did not activate IK.ACh alone, with buffer, with heat-inactivated beta gamma, or with transducin beta gamma. Unactivated alpha subunits did not open K+ channels. Activated, alpha subunits purified from human erythrocytes (alpha 40-GTP[gamma-S]) or bovine brain (alpha 39-GTP[gamma-S]) at concentrations of 10 pM or higher (up to 1 nM) opened K+ channels less frequently in chicken atrial patches [5 of 27 patches (19%) and 9 of 35 patches (26%), respectively] than in rat atrial patches [5 of 11 patches (45%) and 11 of 19 patches (58%), respectively]. Negative results were not due to patch vesicle formation. Other experiments indicated that alpha and beta gamma activated the same population of channels. Activation of the channel by both beta gamma and alpha subunits implies a more complicated scheme for guanine nucleotide-binding regulatory protein action than previously proposed.     

Allosteric determinants in guanine nucleotide-binding proteins

Members of the G protein superfamily contain nucleotide-dependent switches that dictate the specificity of their interactions with binding partners. Using a sequence-based method termed statistical coupling analysis (SCA), we have attempted to identify the allosteric core of these proteins, the network of amino acid residues that couples the domains responsible for nucleotide binding and protein-protein interactions. One-third of the 38 residues identified by SCA were mutated in the G protein Gs alpha, and the interactions of guanosine 5'-3-O-(thio)triphosphate- and GDP-bound mutant proteins were tested with both adenylyl cyclase (preferential binding to GTP-Gs alpha) and the G protein beta gamma subunit complex (preferential binding to GDP-Gs alpha). A two-state allosteric model predicts that mutation of residues that control the equilibrium between GDP- and GTP-bound conformations of the protein will cause the ratio of affinities of these species for adenylyl cyclase and G beta gamma to vary in a reciprocal fashion. Observed results were consistent with this prediction. The network of residues identified by the SCA appears to comprise a core allosteric mechanism conferring nucleotide-dependent switching; the specific features of different G protein family members are built on this core.     

Cell specific distribution of guanine nucleotide-binding regulatory proteins in rat pituitary tumour cell lines

To investigate the effects of guanine nucleotide-binding regulatory proteins (G proteins) on hormonal regulation of prolactin (PRL) synthesis and secretion, the qualitative distribution of G protein alpha-subunits and their mRNAs was studied in three functionally different pituitary tumour cell lines (GH cells) and normal rat pituitary tissue. Levels of basal and modulated adenylyl cyclase (AC) and phospholipase C (PLC) activities are also included. GH cells and pituitary tissue contained various amounts of mRNAs and protein for Gs alpha, Gi-2 alpha, Gi-3 alpha and Go alpha, while mRNA for Gi-1 alpha was only detected in normal pituitary tissue. Gz alpha/Gx alpha mRNA was expressed in all pituitary cell lines as well as in pituitary tissue. Go alpha mRNA and Gz alpha/G x alpha mRNA displayed size heterogeneity. These findings may have importance in the understanding of hormone regulation of second messenger systems.     

Small guanine nucleotide-binding proteins and myocardial hypertrophy

The small (21 kDa) guanine nucleotide-binding protein (small G protein) superfamily comprises 5 subfamilies (Ras, Rho, ADP ribosylation factors [ARFs], Rab, and Ran) that act as molecular switches to regulate numerous cellular responses. Cardiac myocyte hypertrophy is associated with cell growth and changes in the cytoskeleton and myofibrillar apparatus. In other cells, the Ras subfamily regulates cell growth whereas the Rho subfamily (RhoA, Rac1, and Cdc42) regulates cell morphology. Thus, the involvement of small G proteins in hypertrophy has become an area of significant interest. Hearts from transgenic mice expressing activated Ras develop features consistent with hypertrophy, whereas mice overexpressing RhoA develop lethal heart failure. In isolated neonatal rat cardiac myocytes, transfection or infection with activated Ras, RhoA, or Rac1 induces many of the features of hypertrophy. We discuss the mechanisms of activation of the small G proteins and the downstream signaling pathways involved. The latter may include protein kinases, particularly the mitogen-activated or Rho-activated protein kinases. We conclude that although there is significant evidence implicating Ras, RhoA, and Rac1 in hypertrophy, the mechanisms are not fully understood.     

Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain.

We have cloned cDNAs encoding alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go and determined their nucleotide sequences. Purified preparations of Gi and Go alpha subunits (Gi alpha and Go alpha) from rat brain were completely digested with trypsin, and peptides were subjected to amino acid sequence analysis. By screening of a cDNA library from rat C6 glioma cells with a synthetic probe corresponding to a 17 amino acid sequence, a clone encoding the sequence of Go alpha was obtained. Then, the library was rescreened with a Go alpha cDNA probe to isolate several strongly or weakly hybridizing clones. cDNAs encoding the complete sequences of Gi alpha and Gs alpha were thus obtained. From nucleotide sequence analysis, the amino acid sequences of Gs alpha and Gi alpha were deduced; they contain 394 and 355 amino acid residues (including the initiator methionine), respectively. The calculated molecular weights for Gs alpha and Gi alpha were 45,663 and 40,499, respectively. The Go alpha clone encoded a sequence of 310 amino acid residues that lacked the NH2 terminus. The homology of the alpha subunits of Gs, Gi, Go, transducin, and ras-encoded protein is discussed.     

Deduced amino acid sequence of bovine retinal Go alpha: similarities to other guanine nucleotide-binding proteins.

A bovine retinal cDNA clone encoding the complete sequence (354 amino acids) of Go alpha, a guanine nucleotide-binding protein (G protein), was isolated by using oligonucleotide probes complementary to published sequences in two putative clones for the alpha subunit of bovine transducin (T alpha). The deduced amino acid sequence contained sequences identical to those in seven tryptic peptides (total 63 amino acids) from bovine brain Go alpha. The cDNA for bovine retinal Go alpha exhibits greater than 90% identity in both coding and 3' untranslated regions with a recently described partial cDNA clone for Go alpha from rat brain [Itoh, H., Kozasa, T., Nagata, S., Nakamura, S., Katada, T., Ui, M., Iwai, S., Ohtsuka, E., Kawasaki, H., Suzuki, K. & Kaziro, Y. (1986) Proc. Natl. Acad. Sci. USA 83, 3776-3780]. Comparison of the nucleotide and deduced amino acid sequences of the bovine Go alpha clone with those previously reported for other G proteins of bovine origin (Gs alpha, Gi alpha, and T alpha) reveals extensive regions identical to those surrounding the amino acids modified by cholera toxin and pertussis toxin. There are also marked similarities of sequence in regions of the G proteins, elongation factors, and the ras p21 gene products that are believed to be involved in guanine nucleotide binding and GTP hydrolysis.     

Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go.

Reconstitution of purified mu opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. mu opioid receptors were purified by 6-succinylmorphine AF-AminoTOYOPEARL 650M affinity chromatography and by PBE isoelectric chromatography. The purified mu opioid receptor (pI 5.6) migrated as a single Mr 58,000 polypeptide by NaDodSO4/PAGE, a value identical to that obtained by affinity cross-linking purified mu receptors. When purified mu receptors were reconstituted with purified Gi, the G protein that mediates the inhibition of adenylate cyclase, the displacement of [3H]naloxone (a mu opioid antagonist) binding by [D-Ala2,MePhe4,Gly-ol5]enkephalin (a mu opioid agonist) was increased 215-fold; this increase was abolished by adding 100 microM (guanosine 5'-[gamma-thio]triphosphate. Similar increases in agonist displacement of [3H]naloxone binding (33-fold) and its abolition by guanosine 5'-[gamma-thio]triphosphate were observed with Go, the G protein of unknown function, but not with the v-Ki-ras protein p21. In reconstituted preparations with Gi or Go, neither [D-Pen2,D-Pen5]enkephalin (a delta opioid agonist; where Pen is penicillamine) nor U-69,593 (a kappa opioid agonist) showed displacement of the [3H]naloxone binding. In addition, the mu agonist stimulated both [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding (in exchange for GDP) and the low-Km GTPase in such reconstituted preparations, with Gi and Go but not with the v-Ki-ras protein p21, in a naloxone-reversible manner. The stoichiometry was such that the stimulation of 1 mol of mu receptor led to the binding of [3H]guanosine 5'-[beta,gamma-imido]triphosphate to 2.5 mol of Gi or to 1.37 mol of Go. These results suggest that the purified mu opioid receptor is functionally coupled to Gi and Go in the reconstituted phospholipid vesicles.     

Sequence analysis of cDNA and genomic DNA for a putative pertussis toxin-insensitive guanine nucleotide-binding regulatory protein alpha subunit.

We have isolated cDNA clones from rat C6 glioma cells coding for several guanine nucleotide-binding regulatory protein (G protein) alpha subunits (G alpha). The cDNA clones were then used to isolate human chromosomal genes. Among human genomic clones isolated by cross-hybridization with the rat cDNA for the alpha subunit of the inhibitory G protein Gi2, termed Gi2 alpha, a clone designated lambda HGi62 was found to contain a sequence that is highly homologous but distinct from any of the known G alpha sequences, and we have tentatively designated this sequence Gx alpha. We have searched a rat brain cDNA library with the Gx alpha sequence and isolated a cDNA clone containing a rat sequence similar to human Gx alpha. The cDNA contained a single open reading frame of 1065 nucleotides coding for a protein of 355 amino acids with a calculated molecular weight of 40,879. The amino acid sequence of rat Gx alpha shows 66% and 40% similarity with rat Gi2 alpha and rat Gs alpha (the alpha subunit of the stimulatory G protein), respectively. By RNA blot hybridization analysis, mRNA of approximately 3.2 kilobases was detected mainly in brain. Interestingly, the deduced amino acid sequence of Gx alpha predicts that the Gx alpha protein may be refractory to modification by pertussis toxin since the cysteine residue in the fourth position from the C terminus of pertussis toxin-sensitive G alpha is replaced by isoleucine.     

Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins.     

Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.

Immobilized beta gamma subunits of GTP-binding regulatory proteins (G proteins) were used to isolate alpha subunits from solubilized membranes of bovine tissues and to separate specific alpha subunits based on their differential affinities for beta gamma subunits. The beta gamma subunits were cross-linked to omega-aminobutyl agarose. Up to 7 nmol of alpha subunit could bind to each milliliter of beta gamma-agarose and be recovered by elution with AIF4-. This affinity resin effectively separated the alpha subunits of Gi1 and Gi2 from "contaminating" alpha subunits of Go, the most abundant G protein in bovine brain, by taking advantage of the apparent lower affinity of the alpha subunits of Go for beta gamma subunits. The beta gamma-agarose was also used to isolate mixtures of alpha subunits from cholate extracts of membranes from different bovine tissues. alpha subunits of 39-41 kDa (in various ratios) as well as the alpha subunits of Gs were purified. The yields from extracts exceeded 60% for all alpha subunits examined and apparently represented the relative content of alpha subunits in the tissues. This technique can rapidly isolate and identify, from a small amount of sample, the endogenous G proteins in various tissues and cells. So far, only polypeptides in the range of 39-52 kDa have been detected with this approach. If other GTP-binding proteins interact with these beta gamma subunits, the interaction is either of low affinity or mechanistically unique from the alpha subunits isolated in this study.     

Stimulatory and inhibitory regulation of calcium-activated potassium channels by guanine nucleotide-binding proteins.

The regulation of membrane ion channels by guanine nucleotide-binding proteins (G proteins) has been described in numerous tissues. This regulation has been shown to involve the membrane-delimited stimulatory action of G proteins on ion channels. We now show that single calcium-activated potassium channels (KCa channels) in airway smooth muscle cells are both stimulated and inhibited by G proteins in membrane patches. We demonstrate that the beta-adrenergic agonist isoproterenol stimulates channel activity via the alpha subunit of the stimulatory G protein of adenylyl cyclase, Gs, and that channel opening is inhibited by the action of the muscarinic agonist methacholine, acting via a pertussis toxin-sensitive G protein. Isoproterenol stimulated and methacholine inhibited channel activity in the same outside-out patches when GTP was present at the cytosolic surface of the patch. In inside-out patches, addition of GTP and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) augmented channel activity when isoproterenol was included in the patch pipette, and inhibited channel activity when methacholine was included in the pipette. Consistent with these results, in the presence of GTP[gamma S], the alpha subunit of Gs (alpha s.GTP[gamma S] complex) opened KCa channels in a dose-dependent manner, whereas in the presence of guanosine 5'-[beta-thio]diphosphate, alpha s had no effect. By contrast, application of activated alpha i or alpha o proteins did not inhibit channel activity in inside-out patches, indicating that channel inhibition is more complex than a simple alpha subunit/channel interaction, similar to the complex inhibitory regulation of adenylyl cyclase. These results suggest that hormonal regulation of KCa channels shares substantial features with the regulation of adenylyl cyclase and demonstrate that a single ion channel may serve as the regulatory target for the membrane-delimited action of stimulatory and inhibitory G proteins. Moreover, they demonstrate a potentially important functional pathway by which beta-adrenergic and other Gs-linked receptors stimulate relaxation of smooth muscle, independent of cAMP-dependent protein phosphorylation.     

Antibody specific to the alpha subunit of the guanine nucleotide-binding regulatory protein Go: developmental appearance and immunocytochemical localization in brain.

A polyclonal rabbit antibody (9120) against the alpha subunit of the "other" guanine nucleotide-binding protein Go (alpha o) was raised against a synthetic alpha o peptide fragment (Asp-Gly-Ile-Ser-Ala-Ala-Lys-Asp-Val) attached to a branched core system. Antiserum 9120, at a final dilution of 1:400, can detect alpha o in as little as 0.2 microgram of Go on immunoblots, and, at a final dilution of 1:20,000, can detect alpha o in 1 microgram of Go on immunoblots. Antiserum and affinity-purified antibody are specific to alpha o. No cross-reactivity was detected to the alpha subunits of the stimulatory or inhibitory guanine nucleotide-binding regulatory proteins or of transducin (alpha s, alpha i, or alpha T) or to the beta or gamma subunits. Immunoblots revealed a high density of alpha o in rat brain and lung membrane preparations, but other tissues (such as adipose tissue, heart, erythrocytes, and liver) have no detectable alpha o. Developmental studies showed that alpha o in rat brain was low before birth, increased after birth, and reached the full adult level at 4 weeks of age. In contrast, ADP-ribosylation of 40-kDa proteins increased for up to 1 week and then decreased. Immunocytochemistry revealed that alpha o was localized to somatic and synaptic membranes in rat brain, whereas little or no alpha o was detected in the cytoplasm of neuronal cell bodies. Our observations suggest that Go in brain might have a role in membrane signal transduction at synaptic and extrasynaptic sites.     

Methylation and demethylation reactions of guanine nucleotide-binding proteins of retinal rod outer segments.

Retinal transducin was previously shown to be farnesylated on its gamma subunit. This farnesylation reaction on a cysteine residue near the carboxyl terminus is followed by peptidase cleavage at the cysteine. Thus the modified cysteine becomes the carboxyl terminus. It is shown here that the free carboxyl group can be methylated by an S-adenosyl-L-methionine-dependent methyltransferase associated with the rod outer segment membranes. This process can be inhibited by S-adenosyl-L-homocysteine and sinefungin. Moreover, synthetic N-acetyl-S-farnesyl-L-cysteine, but not N-acetyl-L-cysteine, is a substrate for the enzyme. Rapid demethylation of N-acetyl-S-farnesyl-L-cysteine methyl ester can be observed in the membranes. Transducin is also enzymatically demethylated by the rod outer segment membranes. Moreover, the 23- to 29-kDa small G proteins are methylated and demethylated in this system. These data suggest that methylation/demethylation may play a regulatory role in visual signal transduction.     

Cytoskeletal effects of rho-like small guanine nucleotide-binding proteins in the vascular system

Rho-like small GTPases, with their main representatives (Rho, Rac, and Cdc42), have been recognized in the past decade as key regulators of the F-actin cytoskeleton. Rho-like small GTPases are now known to play a major role in vascular processes caused by changes in the actin cytoskeleton, such as smooth muscle cell contraction, endothelial permeability, platelet activation, and leukocyte migration. Data are now accumulating regarding the involvement of Rho GTPases in vascular disorders associated with vascular remodeling, altered cell contractility, and cell migration. The unraveling of signal transduction pathways used by the Rho-like GTPases revealed many upstream regulators and downstream effector molecules, and their number is still growing. An important action of Rho, Rac, and Cdc42 is their ability to regulate the phosphorylation status of the myosin light chain, a major regulator of actin-myosin interaction. Present knowledge of the Rho-like small GTPases has resulted in the development of promising new strategies for the treatment of many vascular disorders, including hypertension, vasospasms, and vascular leakage.     

Proximal tubular epithelial cells possess a novel 42-kilodalton guanine nucleotide-binding regulatory protein

The proximal tubule of the kidney represents an important location where adenylate cyclase regulates salt and water transport; yet a detailed characterization of the distribution and classification of guanine nucleotide-binding protein (G protein) and adenylate cyclase is lacking. We used purified brush border (20-fold) and basolateral membranes (14-fold) to characterize parathyroid hormone- and G protein-regulated adenylate cyclase and G-protein distribution. Adenylate cyclase was predominantly localized to basolateral membranes, while the 46-kDa alpha subunit of the stimulatory G protein (Gs) was 2-fold higher in brush border membranes than in basolateral membranes. The alpha subunit of the inhibitory G protein (Gi; 41 kDa) was equally distributed on immunoblotting but was 2-fold higher in brush border membranes than in basolateral membranes on radiolabeling with pertussis toxin. A 42-kDa cholera toxin substrate that cross-reacted with antisera to the common alpha subunit of G proteins and to Gs on immunoblotting and that was not immunoprecipitated with two Gi antisera was the most abundant alpha subunit and comprised approximately 1% of the total membrane proteins. These observations suggest that G proteins are important regulators of proximal tubular transport independent of adenylate cyclase.     

Angiotensin II-induced changes in guanine nucleotide binding and regulatory proteins.

Systemic infusion of angiotensin II, a potent agonist, using doses that are initially subpressor, eventually produces sustained blood pressure elevation and reductions in glomerular capillary ultrafiltration coefficient characterized by enhanced signal transduction to angiotensin II and other agonists. In this setting, there is a significant increased affinity of angiotensin II binding to smooth muscle and glomerular mesangial receptors and enhanced sensitivity and magnitude of angiotensin II-induced decrements in cyclic AMP. Since G proteins are important modulators of binding and signal transduction, the present studies were designed to test the hypothesis that differences in the relative amounts of G proteins may be present and have accounted for differences observed. G proteins were identified and quantitated by isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radiolabeling in the presence of activated toxins with [gamma-32P]NAD+, immunoprecipitation, and immunoblotting. A 168% and 465% increase in pertussis toxin-catalyzed ADP ribosylation of alpha 40-41 was found in angiotensin II-treated groups over control groups for glomerular and mesenteric membranes, respectively. Immunoblotting revealed a 250% and 35% increase in the levels of the Gi isoforms alpha i-2 and alpha i-3, respectively, and a decrease of 53% in alpha i-1 from the angiotensin II-treated group. No differences were observed in cholera toxin labeling or immunoblotting of Gs. These results demonstrate multiple mechanisms whereby angiotensin-induced signal transduction can be modulated involving both the receptors and G proteins. These observed differences in G proteins in systemic and renal vasculature accompanying angiotensin II infusion suggest the possibility of a regulatory role in the pathophysiology of angiotensin II-induced hypertension and renal disease.     

Cytosolic Na+ controls and epithelial Na+ channel via the Go guanine nucleotide-binding regulatory protein.

In tight Na+-absorbing epithelial cells, the fate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-beta-S, pertussis toxin, and antibodies against the alpha-subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.