Determinants of agonist binding affinity on neuronal nicotinic receptor beta subunits.

The alpha and beta subunits of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are thought to contribute "principal" and "complementary" components to the agonist binding site, respectively. At least six loops of amino acid sequence (A, B, and C from alpha; D, E, and F from beta) are involved. We demonstrated previously that receptors containing the beta2 subunit had consistently higher affinities for a variety of agonists than beta4-containing receptors. For example, the affinity of the alpha2beta2 receptor for epibatidine, ACh, nicotine, and dimethylphenylpiperazinium (DMPP) exceeds that of alpha2beta4 by 9-, 61-, 87-, and 120-fold, respectively. Using saturation and competition analysis of receptors formed by chimeric beta subunits coexpressed with alpha2 in Xenopus laevis oocytes, we have now identified sequence segment 54-63 (corresponding to loop D) as a major determinant of affinity for epibatidine, ACh, nicotine, and DMPP. We then analyzed a series of mutant beta2 subunits in which each residue that differs between beta2 and beta4 in this region was changed from what occurs in beta2 to what occurs in beta4. The N55S, V56I, and E63T mutations each resulted in a loss of affinity for ACh and nicotine of 3- to 4-fold, whereas the T59K mutation resulted in a 7-fold loss of ACh and nicotine affinity. These mutations had little or no effect on epibatidine and DMPP affinity. The positive charge introduced by the T59K mutation does not appear to underlie loss of agonist affinity, because a similar loss of affinity was observed when a negative charge (T59D) was introduced at this position.

Subunit-dependent modulation of neuronal nicotinic receptors by zinc.

We examined the effect of zinc on rat neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes as simple heteromers of alpha2, alpha3, or alpha4 and beta2 or beta4. Coapplication of zinc with low concentrations of acetylcholine (

Activity-dependent modulation of rod photoreceptor cyclic nucleotide-gated channels mediated by phosphorylation of a specific tyrosine residue.

Cyclic nucleotide-gated (CNG) channels are crucial for phototransduction in vertebrate rod photoreceptors. The cGMP sensitivity of these channels is modulated by diffusible intracellular messengers, including Ca2+/calmodulin, contributing to negative feedback during sensory adaptation. Membrane-associated protein tyrosine kinases and phosphatases also modulate rod CNG channels, but whether this results from direct changes in the phosphorylation state of the channel protein has been unclear. Here, we show that bovine rod CNG channel alpha-subunits (bRET) contain a tyrosine phosphorylation site crucial for modulation. bRET channels expressed in Xenopus oocytes exhibit modulation, whereas rat olfactory CNG channels (rOLF) do not. Chimeric channels reveal that differences in the C terminus, containing the cyclic nucleotide-binding domain, account for this difference. One specific tyrosine in bRET (Y498) appears to be crucial; replacement of this tyrosine in bRET curtails modulation, whereas installation into rOLF confers modulability. As the channel becomes dephosphorylated, there is an increase in the rate of spontaneous openings in the absence of ligand, indicating that changes in the phosphorylation state affect the allosteric gating equilibrium. Moreover, we find that dephosphorylation, which favors channel opening, requires open channels, whereas phosphorylation, which promotes channel closing, requires closed channels. Hence, modulation by changes in tyrosine phosphorylation is activity-dependent and may constitute a positive feedback mechanism, contrasting with negative feedback systems underlying adaptation.

Neuronal nicotinic receptor beta2 and beta4 subunits confer large differences in agonist binding affinity.

We used equilibrium binding analysis to characterize the agonist binding properties of six different rat neuronal nicotinic receptor subunit combinations expressed in Xenopus laevis oocytes. The alpha4beta2 receptor bound [3H]cytisine with a Kdapp of 0.74 +/- 0. 14 nM. The rank order of Kiapp values of additional nicotinic ligands, determined in competition assays, was cytisine < nicotine < acetylcholine < carbachol < curare. These pharmacological properties of alpha4beta2 expressed in oocytes are comparable to published values for the high affinity cytisine binding site in rat brain (alpha4beta2), demonstrating that rat neuronal nicotinic receptors expressed in X. laevis oocytes display appropriate pharmacological properties. Use of [3H]epibatidine allowed detailed characterization of multiple neuronal nicotinic receptor subunit combinations. Kdapp values for [3H]epibatidine binding were 10 pM for alpha2beta2, 87 pM for alpha2beta4, 14 pM for alpha3beta2, 300 pM for alpha3beta4, 30 pM for alpha4beta2, and 85 pM for alpha4beta4. Affinities for six additional agonists (acetylcholine, anabasine, cytisine, 1, 1-dimethyl-4-phenylpiperazinium, lobeline, and nicotine) were determined in competition assays. The beta2-containing receptors had consistently higher affinities for these agonists than did beta4-containing receptors. Particularly striking examples are the affinities displayed by alpha2beta2 and alpha2beta4, which differ in 1,1-dimethyl-4-phenylpiperazinium, nicotine, lobeline, and acetylcholine affinity by 120-, 86-, 85-, and 61-fold, respectively. Although smaller differences in affinity could be ascribed to different alpha subunits, the major factor in determining agonist affinity was the nature of the beta subunit.

Differential roles for disulfide bonds in the structural integrity and biological activity of kappa-Bungarotoxin, a neuronal nicotinic acetylcholine receptor antagonist.

kappa-Bungarotoxin, a kappa-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. kappa-Bungarotoxin is a potent, selective, and slowly reversible antagonist of alpha3 beta2 neuronal nicotinic acetylcholine receptors. kappa-Bungarotoxin is structurally related to the alpha-neurotoxins, such as alpha-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (alpha1 beta1 gamma delta) and the homopentameric neuronal type receptors (alpha7, alpha8, and alpha9). Like the kappa-neurotoxins, the long alpha-neurotoxins contain the same five conserved disulfide bonds, while the short alpha-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in kappa-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors.

Glycosylation within the cysteine loop and six residues near conserved Cys192/Cys193 are determinants of neuronal bungarotoxin sensitivity on the neuronal nicotinic receptor alpha3 subunit.

Neuronal bungarotoxin (NBT) is a highly selective, slowly reversible, competitive antagonist of the alpha3beta2 neuronal nicotinic receptor. Contributions to NBT sensitivity are made by both the alpha3 and beta2 subunits. We used a chimeric alpha subunit to demonstrate that the entire alpha3 contribution lies within sequence segment 84-215. Construction and analysis of a series of mutant alpha3 subunits identified seven amino acid residues (Thr143, Tyr184, Lys185, His186, Ile188, Gln198, Ser203) within this region that contribute to NBT sensitivity. Changing Thr143 to lysine, as in alpha2, resulted in a approximately 1000-fold loss of NBT sensitivity. The effect on NBT sensitivity of changing each of the other six residues ranged from 1.8- to 40.5-fold. More extensive mutagenesis demonstrated that Thr143 serves as part of the consensus sequence for glycosylation at N141, and it is this glycosylation that is the determinant of NBT sensitivity. Only serine could substitute for threonine to maintain full NBT sensitivity, and changing Asn141 to alanine resulted in a approximately 300-fold loss of NBT sensitivity. The chimera alpha2-181-alpha3, containing all identified determinants except the glycosylation site, formed receptors insensitive to 300 nM NBT. Installation of threonine to complete the glycosylation consensus site in this chimera conferred NBT sensitivity only 10-fold less than that of wild-type alpha3beta2. These seven determinants of NBT sensitivity are located in close proximity to a series of conserved residues that are common features of all nicotinic receptor binding sites.

Differential inhibition by alpha-conotoxin-MII of the nicotinic stimulation of [3H]dopamine release from rat striatal synaptosomes and slices.

The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, alpha-conotoxin-MII has been reported to inhibit potently and selectively the rat alpha3beta2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (+/-)-anatoxin-a-evoked release of [3H]dopamine from rat striatal synaptosomes and slices. Alpha-conotoxin-MII (112 nM) completely blocked acetylcholine-evoked currents of alpha3beta2 nAChRs expressed in Xenopus oocytes (IC50 = 8.0 +/- 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nM alpha-conotoxin-MII. On perfused striatal synaptosomes and slices, alpha-conotoxin-MII dose-dependently inhibited [3H]dopamine release evoked by 1 microM (+/-)-anatoxin-a with IC50 values of 24.3 +/- 2.9 and 17.3 +/- 0.1 nM, respectively. The dose-response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by alpha-conotoxin-MII (112 nM) was 44.9 +/- 5.4% for synaptosomes and 25.0 +/- 4.1% for slices, compared with an inhibition by 10 microM mecamylamine of 77.9 +/- 3.7 and 88.0 +/- 2.1%, respectively. These results suggest the presence of presynaptic alpha3beta2-like nAChRs on striatal dopaminergic terminals, but the incomplete block of (+/-)-anatoxin-a-evoked [3H]dopamine release by alpha-conotoxin-MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an alpha-conotoxin-MII-insensitive nAChR.

Determinants of specificity for alpha-conotoxin MII on alpha3beta2 neuronal nicotinic receptors.

The competitive antagonist alpha-conotoxin-MII (alpha-CTx-MII) is highly selective for the alpha3beta2 neuronal nicotinic receptor. Other receptor subunit combinations (alpha2beta2, alpha4beta2, alpha3beta4) are >200-fold less sensitive to blockade by this toxin. Using chimeric and mutant subunits, we identified amino acid residues of alpha3 and beta2 that participate in determination of alpha-CTx-MII sensitivity. Chimeric alpha subunits, constructed from the alpha3 and alpha4 subunits, as well as from the alpha3 and alpha2 subunits, were expressed in combination with the beta2 subunit in Xenopus laevis oocytes. Chimeric beta subunits, formed from the beta2 and beta4 subunits, were expressed in combination with alpha3. Determinants of alpha-CTx-MII sensitivity on alpha3 were found to be within sequence segments 121-181 and 181-195. The 181-195 segment accounted for approximately half the difference in toxin sensitivity between receptors formed by alpha2 and alpha3. When this sequence of alpha2 was replaced with the corresponding alpha3 sequence, the resulting chimera formed receptors only 26-fold less sensitive to alpha-CTx-MII than alpha3beta2. Site-directed mutagenesis within segment 181-195 demonstrated that Lys185 and Ile188 are critical in determination of sensitivity to toxin blockade. Determinants of alpha-CTx-MII sensitivity on beta2 were mapped to sequence segments 1-54, 54-63, and 63-80. Site-directed mutagenesis within segment 54-63 of beta2 demonstrated that Thr59 is important in determining alpha-CTx-MII sensitivity.

Heterogeneity of nicotinic receptor class and subunit mRNA expression among individual parasympathetic neurons from rat intracardiac ganglia.

Neurons have the potential to form thousands of distinct neuronal nicotinic receptors from the eight alpha and three beta subunits that currently are known. In an effort to determine how much of this potential complexity is realized among individual neurons, we examined the nicotinic pharmacological and biophysical properties and receptor subunit mRNA expression patterns in individual neurons cultured from rat epicardial ganglia. Analysis of the whole-cell pharmacology of these neurons showed a diversity of responses to the agonists acetylcholine, nicotine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium, suggesting that a heterogeneous population of nicotinic receptor classes, or subtypes, is expressed by individual neurons. Single-channel analysis demonstrated three distinct conductances (18, 24, and 31 pS), with patches from different neurons containing different combinations of these channel classes. We used single-cell RT-PCR to examine nicotinic acetylcholine receptor (nAChR) subunit mRNA expression by individual neurons. Although mRNAs encoding all eight neuronal nAChR subunits for which we probed (alpha 2-alpha 5, alpha 7, beta 2-beta 4) were present in multicellular cultures, we found that individual epicardial neurons express distinct subsets of these nAChR subunit mRNAs. These results suggest that individual epicardial neurons express distinct arrays of nAChR subunits and that these subunits may assemble into functional receptors with distinct and variable subunit composition. This variable receptor subunit expression provides an explanation for the diversity of pharmacological and single-channel responses we have observed in individual neurons.

Multiple determinants of dihydro-beta-erythroidine sensitivity on rat neuronal nicotinic receptor alpha subunits.

Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro-beta-erythroidine. Both alpha and beta subunits participate in determining sensitivity to this antagonist. The alpha subunit contribution to dihydro-beta-erythroidine sensitivity is illustrated by comparing the alpha 4 beta 4 receptor and the alpha 3 beta 4 receptor, which differ in sensitivity to dihydro-beta-erythroidine by approximately 120-fold. IC50 values for blocking alpha 4 beta 4 and alpha 3 beta 4, responding to EC20 concentrations of acetylcholine, were 0.19 +/- 0.06 and 23.1 +/- 10.2 microM, respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric alpha subunits containing portions of the alpha 4 and alpha 3 subunits. These chimeras were coexpressed with beta 4, allowing pharmacological characterization. We found determinants of dihydro-beta-erythroidine sensitivity to be distributed throughout the N-terminal extracellular domain of the alpha subunit. These determinants were localized to sequence segments 1-94, 94-152, and 195-215. Loss of determinants within segment 1-94 had the largest effect, decreasing dihydro-beta-erythroidine sensitivity by 4.3-fold.

Determinants of competitive antagonist sensitivity on neuronal nicotinic receptor beta subunits.

We constructed a series of chimeric and mutant neuronal nicotinic acetylcholine receptor beta subunits to map amino acid residues that determine sensitivity to competitive antagonists. The beta 2 and beta 4 subunits form pharmacologically distinct receptors when expressed in combination with the alpha 3 subunit in Xenopus oocytes. At equipotent acetylcholine concentrations, alpha 3 beta 2 is 56-fold more sensitive to blockage by dihydro-beta-erythroidine than is alpha 3 beta 4. The alpha 3 beta 2 combination is also sensitive to long-term blockade by neuronal bungarotoxin, whereas alpha 3 beta 4 is not. Pharmacological analysis of receptors formed by chimeric beta subunits reveals that amino acid residues that determine both dihydro-beta-erythroidine and neuronal bungarotoxin sensitivity are located within several sequence segments. The major determinant of sensitivity to both competitive antagonists is located between residues 54 and 63. A minor determinant of sensitivity to both antagonists lies between residues 1 and 54, whereas a minor determinant of NBT sensitivity lies between residues 74 and 80. Within region 54-63 of beta 2, mutant beta 2 subunits were used to identify threonine 59 as a residue critical in determining competitive antagonist sensitivity. Changing threonine 59 to lysine, as occurs in beta 4, causes a 9-fold decrease in dihydro-beta-erythroidine sensitivity and a 71-fold decrease in neuronal bungarotoxin sensitivity. Changing polar threonine 59 to negatively charged aspartate causes a 2.5-fold increase in neuronal bungarotoxin sensitivity and has no effect on dihydro-beta-erythroidine sensitivity.

Mapping of ligand binding sites of neuronal nicotinic acetylcholine receptors using chimeric alpha subunits.

We constructed a series of chimeric neuronal nicotinic acetylcholine (ACh) receptor (nAChR) alpha subunits to map the location of amino acid residues that determine the pharmacological properties of these receptors. The alpha 2 and alpha 3 subunits form pharmacologically distinct nAChRs upon expression, in combination with the beta 2 subunit, in Xenopus oocytes. The alpha 2 beta 2 subunit combination is insensitive to the nicotinic antagonist neuronal bungarotoxin (NBT) and is much more sensitive to nicotine than to ACh. In contrast, the alpha 3 beta 2 subunit combination is potently inhibited by NBT and is much less sensitive to nicotine than to ACh. Chimeric subunits were constructed by replacing portions of alpha 2 or alpha 3 with the analogous portion of the other alpha subunit. Pharmacological analysis of receptors formed by these chimeric subunits, in combination with beta 2, revealed that amino acid residues involved in determining NBT sensitivity were located within sequence segments 84-121, 121-181, and 195-215. Amino acid residues that determine agonist sensitivity were located within sequence segments 1-84 and 195-215. Within region 195-215, we used site-directed mutagenesis to demonstrate the importance of Gln-198 of alpha 3 (proline in alpha 2) in determining both the antagonist sensitivity and the agonist sensitivity of neuronal nAChRs.

Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors.

Two properties were found to distinguish neuronal from muscle nicotinic acetylcholine receptors (nAChRs). First, neuronal nAChRs have a greater Ca2+ permeability. The high Ca2+ flux through neuronal nAChRs activates a Ca(2+)-dependent Cl- conductance, and the Ca2+ to Cs+ permeability ratio (PCa/PCs) is 7 times greater for neuronal than for muscle nAChRs. A second difference between the receptor types is that neuronal nAChRs are potently modulated by physiological levels of external Ca2+. Neuronal nAChR currents are enhanced by external Ca2+ in a dose-dependent manner. The results indicate that changes in extracellular Ca2+ modulate neuronal nAChRs and may modulate cholinergic synapses in the CNS. Also, activation of neuronal nAChRs produces a significant influx of Ca2+ that could be an important intracellular signal.

Both alpha- and beta-subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors.

A family of genes has been identified that encodes subunits of nicotinic acetylcholine receptors (nAChRs) and is expressed in the nervous system. Functional neuronal nAChRs can be expressed in Xenopus oocytes by injection of RNA encoding 1 of 2 different beta-subunits (beta 2, beta 4) in pairwise combination with RNA encoding 1 of 3 different alpha-subunits (alpha 2, alpha 3, alpha 4). We examined the sensitivity of these 6 different alpha- beta-subunit combinations to the nicotinic agonists ACh, nicotine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium (DMPP). Each subunit combination displayed a distinct pattern of sensitivity to these 4 agonists. The alpha 2 beta 2 combination was 5-fold more sensitive to nicotine than to acetylcholine, while the alpha 3 beta 2 combination was 17-fold less sensitive to nicotine than to ACh, and the alpha 3 beta 4 combination was equally sensitive to both nicotine and ACh. nAChRs composed of alpha 2, alpha 3, or alpha 4 in combination with beta 2 were 14-100-fold less sensitive to cytisine than to ACh. In contrast, nAChRs composed of alpha 2, alpha 3, or alpha 4 in combination with beta 4 were 3-17-fold more sensitive to cytisine than to ACh. The alpha 2 beta 2, alpha 3 beta 2, and alpha 3 beta 4 combinations were each equally sensitive to DMPP and ACh, while the alpha 2 beta 4, alpha 4 beta 2, and alpha 4 beta 4 combinations were 4-24-fold less sensitive to DMPP than to ACh. We also demonstrated that these differences are neither a consequence of variation in the relative amounts of RNA injected nor an artifact of oocyte expression. The oocyte system can accurately express ligand-gated ion channels because mouse muscle nAChRs expressed in oocytes display pharmacological properties similar to those reported for these receptors expressed on BC3H-1 cells. We conclude that both the alpha- and the beta-subunits contribute to the pharmacological characteristics of neuronal nAChRs.

The postsynaptic 43K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes.

Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of Mr 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Coinjection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5-1.5 microns diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis.

Neurotoxins distinguish between different neuronal nicotinic acetylcholine receptor subunit combinations.

Neuronal and muscle nicotinic acetylcholine receptor subunit combinations expressed in Xenopus oocytes were tested for sensitivity to various neurotoxins. Extensive blockade of the alpha 3 beta 2 neuronal subunit combination was achieved by 10 nM neuronal bungarotoxin. Partial blockade of the alpha 4 beta 2 neuronal and alpha 1 beta 1 gamma delta muscle subunit combinations was caused by 1,000 nM neuronal bungarotoxin. The alpha 2 beta 2 neuronal subunit combination was insensitive to 1,000 nM neuronal bungarotoxin. Nearly complete blockade of all neuronal subunit combinations resulted from incubation with 2 nM neosurugatoxin, whereas 200 nM neosurugatoxin was required for partial blockade of the alpha 1 beta 1 gamma delta muscle subunit combination. The alpha 2 beta 2 and alpha 3 beta 2 neuronal subunit combinations were partially blocked by 10,000 nM lophotoxin analog-1, whereas complete blockade of the alpha 4 beta 2 neuronal and alpha 1 beta 1 gamma delta muscle subunit combinations resulted from incubation with this concentration of lophotoxin analog-1. The alpha 1 beta 1 gamma delta muscle subunit combination was blocked by the alpha-conotoxins G1A and M1 at concentrations of 100 nM. All of the neuronal subunit combinations were insensitive to 10,000 nM of both alpha-conotoxins. Thus, neosurugatoxin and the alpha-conotoxins distinguish between muscle and neuronal subunit combinations, whereas neuronal bungarotoxin and lophotoxin analog-1 distinguish between different neuronal subunit combinations on the basis of differing alpha subunits.

Nicotine receptors in the mammalian brain.

Nicotine is a drug of abuse that presumably exerts its psychoactive effect through its interactions with nicotine binding sites in the central nervous system. Among its potential sites of action are the neuronal nicotinic acetylcholine receptors and the neuronal alpha-bungarotoxin binding sites. In this review we focus on the neuronal nicotinic acetylcholine receptors, their diversity, distribution, and functions as nicotine receptors or as mediators of synaptic transmission in the mammalian brain. We find that the complexity characteristic of the gene family encoding the subunits of these receptors is reflected both in the pattern of expression of the genes and in the pharmacological diversity of the expressed receptors.

Differential tissue expression and developmental regulation of guanine nucleotide binding regulatory proteins and their messenger RNAs in rat heart.

The expression and developmental regulation of the alpha and beta subunits of the guanine nucleotide binding regulatory proteins, Gi and Go, were examined in rat atria and ventricles. Protein levels were determined by quantitative immunoblot analysis using affinity purified monospecific antibodies. Northern blot and dot blot analyses were used to characterize and quantitate relative amounts of mRNA encoding these G protein subunits. The concentrations of Go alpha, Gi alpha, and beta subunit protein were found to be greater in adult atrial than in adult ventricular membranes (5.2-, 1.5-, and 2.8-fold, respectively). A corresponding 3.4-fold difference in Go alpha mRNA level was also observed, as well as a 1.3-fold difference in Gi alpha-3 mRNA level. No difference was seen between the amount of beta, Gi alpha-1, Gi alpha-2 mRNA in adult atria and adult ventricles. Comparison of neonatal and adult tissues revealed a developmental decrease in ventricular Gi alpha protein and Gi alpha-2 mRNA levels (70 and 47%, respectively). Developmental decreases were also observed in the amount of mRNA encoding beta and Go alpha in ventricles (47 and 61%, respectively), and beta and Gi alpha-2 in atria (40 and 36%, respectively), while a developmental increase in atrial Gi alpha-3 mRNA levels was observed (57%). These results demonstrate differences in the expression of G protein subunits in rat atria and ventricles, as well as regulation of the levels of these subunits during cardiac development.

Chronic membrane depolarization regulates the level of the guanine nucleotide binding protein Go alpha in cultured neuronal cells.

Chronic membrane depolarization results in an increase in muscarinic acetylcholine receptor (mAChR) number in N1E-115 neuroblastoma cells. Because the mAChR interacts with the guanine nucleotide binding regulatory (G) proteins, Gi and Go, the effect of chronic membrane depolarization on the levels of subunits of these G proteins was examined. Quantitation of G protein subunit levels was performed using affinity-purified, monospecific antibodies in a quantitative immunoblot assay. Incubation with 50 microM veratridine (VTN), an activator of voltage-sensitive Na+ channels, induced a 48 +/- 15% increase in the level of the alpha subunit of Go. The effect of VTN was blocked by tetrodotoxin. On removal of VTN, the level of Go alpha decreased to control levels within 24 h. The levels of the alpha subunit of Gi and the common beta subunit were not affected by VTN treatment. These results show that in N1E-115 cells, the level of the alpha subunit of Go is regulated in a manner similar to the level of mAChR in response to chronic membrane depolarization.