Biological roles of neural J proteins.

J proteins are chief regulators of the Hsp70 family, a highly conserved family of ATPases that mediate conformational changes in a broad range of proteins. The J protein family has been the central focus of numerous prokaryote and eukaryote biologists. Common questions that arise include: How does the J protein/Hsp70 machinery support protein folding? What role do J proteins play in protein misfolding and neurodegenerative disorders? Can the J protein/ Hsp70 machinery be harnessed to provide a rational basis for recombinant protein production? The current progress that has resulted from the convergence of biochemistry with Escherichia coli and Saccharomyces cerevisiae genetics has accelerated the pace at which these questions are being elucidated. We are beginning to gain some insights into the neuronal network of J proteins. Here, we highlight recent advances in our understanding of how select J proteins harness Hsp70 s for fundamentally important conformational work in neurons.

Contribution of potential EF hand motifs to the calcium-dependent gating of a mouse brain large conductance, calcium-sensitive K(+) channel.

1. The large conductance, calcium-sensitive K(+) channel (BK(Ca) channel) is a unique member of the K(+)-selective ion channel family in that activation is dependent upon both direct calcium binding and membrane depolarization. Calcium binding acts to dynamically shift voltage-dependent gating in a negative or left-ward direction, thereby adjusting channel opening to changes in cellular membrane potential. 2. We hypothesized that the intrinsic calcium-binding site within the BK(Ca) channel alpha subunit may contain an EF hand motif, the most common, naturally occurring calcium binding structure. Following identification of six potential sites, we introduced a single amino acid substitution (D/E to N/Q or A) at the equivalent of the -z position of a bona fide EF hand that would be predicted to lower calcium binding affinity at each of the six sites. 3. Using macroscopic current recordings of wild-type and mutant BK(Ca) channels in excised inside-out membrane patches from HEK 293 cells, we observed that a single point mutation in the C-terminus (Site 6, FLD(923)QD to N), adjacent to the 'calcium bowl' described by Salkoff and colleagues, shifted calcium-sensitive gating right-ward by 50--65 mV over the range of 2--12 microM free calcium, but had little effect on voltage-dependent gating in the absence of calcium. Combining this mutation at Site 6 with a similar mutation at Site 1 (PVD(81)EK to N) in the N-terminus produced a greater shift (70--90 mV) in calcium-sensitive gating over the same range of calcium. We calculated that these combined mutations decreased the apparent calcium binding affinity approximately 11-fold (129.5 microM vs. 11.3 microm) compared to the wild-type channel. 4. We further observed that a bacterially expressed protein encompassing Site 6 of the BK(Ca) channel C-terminus and bovine brain calmodulin were both able to directly bind (45)Ca(2+) following denaturation and polyacrylamide gel electrophoresis (e.g. SDS-PAGE). 5. Our results suggest that two regions within the mammalian BK(Ca) channel alpha subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium-sensitive gating of this channel.

A catalytically inactive mutant of type I cGMP-dependent protein kinase prevents enhancement of large conductance, calcium-sensitive K+ channels by sodium nitroprusside and cGMP.

The activation of large conductance, calcium-sensitive K(+) (BK(Ca)) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BK(Ca) channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BK(Ca) channel activity 4-5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the alpha isoform of type I cGMP-dependent protein kinase (cGKI). We observed that co-transfection of cells with BK(Ca) channels and a catalytically inactive ("dead") mutant of human cGKIalpha prevented enhancement of BK(Ca) channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKIalpha supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKIalpha were found to associate with BK(Ca) channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKIalpha was able to directly phosphorylate immunoprecipitated BK(Ca) channels, suggesting that cGKIalpha-dependent phosphorylation of BK(Ca) channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKIalpha alone is sufficient to promote the enhancement of BK(Ca) channels in situ after activation of the NO/cGMP signaling pathway.

Ammonium ion enhances the calcium-dependent gating of a mammalian large conductance, calcium-sensitive K+ channel.

We observed that the current amplitude and activation of expressed, mouse brain large conductance, calcium-sensitive K+ channels (BKCa channels) may be reversibly enhanced following addition of low concentrations of the weakly permeant cation NH4+ to the cytoplasmic face of the channel in excised, inside-out membrane patches from HEK 293 cells. Conductance-voltage relations were left-shifted along the voltage axis by addition of NH4Cl in a concentration-dependent manner, with an EC50 of 18.5 mM. Furthermore, this effect was observed in the presence of cytosolic free calcium (approximately 1 microM), but was absent in a cytosolic bath solution containing nominally zero free calcium (e.g.. 5 mM EGTA only), a condition under which these channels undergo largely voltage-dependent gating. Recordings of single BKCa channel events indicated that NH4+ increased the channel open probability of single channel activity approximately 3-fold, but did not alter the amplitude of single channel currents. These findings suggest that the calcium-sensitive gating of mammalian BKCa channels may be modified by other ions present in cytosolic solution.

Inhibition of a mammalian large conductance, calcium-sensitive K+ channel by calmodulin-binding peptides.

The large conductance, calcium-sensitive K+ channel (BKCa channel) is a voltage-activated ion channel in which direct calcium binding shifts gating to more negative cellular membrane potentials. We hypothesized that the calcium-binding domain of BKCa channels may mimic the role played by calmodulin (CaM) in the activation of calcium-CaM-dependent enzymes, in which a tonic inhibitory constraint is removed on CaM binding. To examine such a hypothesis, we used peptides from the autoregulatory domains of CaM kinase II (CK291-317) and cNOS (the constitutive nitric oxide synthase; cNOS725-747) as probes for the calcium-dependent activation of murine BKCa channels transiently expressed in HEK 293 cells. We found that these CaM-binding peptides produced potent, time-dependent inhibition of mammalian BKCa channel current following voltage-dependent activation. Inhibition was observed in both the presence and the absence of cytosolic free calcium. Similar application of CK291-31 had no effect on either the amplitude or kinetics of voltage-dependent, macroscopic currents recorded from rabbit smooth muscle Kv1.5 potassium channels transiently expressed in HEK 293 cells. Cytosolic application of both CK291-317 and tetraethylammonium (TEA) produced an additive and non-competitive block of BKCa current. This finding suggests that the peptide-binding site is distinct (e.g. outside the pore region of the channel) from that of TEA. Our results are thus consistent with a model in which the BKCa channel's voltage-dependent gating process is under an intramolecular constraint that is relieved upon calcium binding. The intrinsic calcium sensor of the channel may thus interact with an inhibitory domain present in the BKCa channel, and by doing so, remove an inhibitory 'constraint' that permits voltage-dependent gating to occur at more negative potentials.

Enhanced activity of a large conductance, calcium-sensitive K+ channel in the presence of Src tyrosine kinase.

Large conductance, calcium-sensitive K(+) channels (BK(Ca) channels) contribute to the control of membrane potential in a variety of tissues, including smooth muscle, where they act as the target effector for intracellular "calcium sparks" and the endothelium-derived vasodilator nitric oxide. Various signal transduction pathways, including protein phosphorylation can regulate the activity of BK(Ca) channels, along with many other membrane ion channels. In our study, we have examined the regulation of BK(Ca) channels by the cellular Src gene product (cSrc), a soluble tyrosine kinase that has been implicated in the regulation of both voltage- and ligand-gated ion channels. Using a heterologous expression system, we observed that co-expression of murine BK(Ca) channel and the human cSrc tyrosine kinase in HEK 293 cells led to a calcium-sensitive enhancement of BK(Ca) channel activity in excised membrane patches. In contrast, co-expression with a catalytically inactive cSrc mutant produced no change in BK(Ca) channel activity, demonstrating the requirement for a functional cSrc molecule. Furthermore, we observed that BK(Ca) channels underwent direct tyrosine phosphorylation in cells co-transfected with BK(Ca) channels and active cSrc but not in cells co-transfected with the kinase inactive form of the enzyme. A single Tyr to Phe substitution in the C-terminal half of the channel largely prevented this observed phosphorylation. Given that cSrc may become activated by receptor tyrosine kinases or G-protein-coupled receptors, these findings suggest that cSrc-dependent tyrosine phosphorylation of BK(Ca) channels in situ may represent a novel regulatory mechanism for altering membrane potential and calcium entry.

KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart.

The multifunctional Ca++/calmodulin-dependent protein kinase II (CaM kinase) mediates Ca++-induced augmentation of L-type Ca++ current (ICa); therefore it may act as a proarrhythmic signaling molecule during early afterdepolarizations (EADs) due to ICa. To investigate the hypothesis that ICa-dependent EADs are favored by CaM kinase activation EADs were induced with clofilium in isolated rabbit hearts. All EADs were rapidly terminated with ICa antagonists. Hearts were pretreated with the CaM kinase inhibitor KN-93 or the inactive analog KN-92 (0.5 microM) for 10 min before clofilium exposure. EADs were significantly suppressed by KN-93 (EADs present in 4/10 hearts) compared to KN-92 (EADs present in 10/11 hearts) (P =.024). There were no significant differences in parameters favoring EADs such as monophasic action potential duration or heart rate in KN-93- or KN-92-treated hearts. CaM kinase activity in situ increased 37% in hearts with EADs compared to hearts without EADs (P =.015). This increase in CaM kinase activity was prevented by pretreatment with KN-93. In vitro, KN-93 potently inhibited rabbit myocardial CaM kinase activity (calculated Ki 100 microM). The actions of KN-93 and KN-92 on ICa and other repolarizing K+ currents did not explain preferential EAD suppression by KN-93. These data show a novel association between CaM kinase activation and EADs and are consistent with the hypothesis that the ICa and CaM kinase activation both contribute to EADs in this model.

Distinct voltage-dependent gating behaviours of a swelling-activated chloride current in human epithelial cells.

1. The swelling-activated chloride current is critical in the homeostatic regulatory volume decrease (RVD) of both excitable and non-excitable cells. Although not activated by voltage, it displays kinetic behaviour similar to voltage-gated Shaker-type potassium currents. We have studied the voltage-dependent properties of this current in single T84 human cell line epithelial cells using whole-cell patch clamp methodology. 2. An external anion permeability sequence of I- > Cl- > methanesulphonate (MeSO3-) was observed for the swelling-activated current. Extracellular application of the chloride channel blocker DIDS (100 microM) resulted in approximately 50% block of the current in a voltage-dependent manner. 3. At positive membrane potentials, the swelling-activated chloride current undergoes time-dependent inactivation. Following such inactivation, recovery of both the inward and outward components of the macroscopic current was found to be voltage dependent. The time constants describing these two individual recovery processes were identical over a range of membrane potentials. In addition, the magnitude of current recovery was directly dependent upon the degree prior inactivation of current at positive voltage. 4. We further observed that the swelling-activated current undergoes a form of steady-state, voltage-dependent inactivation that appears to differ from the inactivation observed at positive potentials. This steady-state inactivation occurred over the physiological voltage range, with a membrane potential at half-maximal inactivation (V1/2) of -72 mV, and differed from the time-dependent inactivation observed at positive membrane potentials, which occurred with a V1/2 of 40 mV. These observations demonstrate two distinct forms of voltage-dependent inactivation, probably reflecting two separate gating processes at the level of the channel. 5. These latter properties are thus anticipated to regulate voltage-dependent chloride efflux under cell swelling conditions and further influence RVD and membrane excitability in cells generating action potentials.

A non-selective cation current activated via the multifunctional Ca(2+)-calmodulin-dependent protein kinase in human epithelial cells.

1. Activation of macroscopic membrane currents by intracellular calcium ([Ca2+]i) signalling pathways was examined in human T84 epithelial cells, a model secretory cell line. 2. Elevation of [Ca2+]i by either the calcium ionophore A23187 (1 microM) or the cholinergic agonist carbachol, led to the transient activation of both a chloride and cation current in single voltage clamped cells. The channels underlying the cation conductance were found to be equally permeable to external Na+, K+ and Cs+, but impermeable to the large organic cations tetraethylammonium and N-methyl-D-glucamine (NMDG). These observations indicate that the cation channels are non-selective with respect to monovalent cations. 3. Persistent activation of both the chloride and non-selective cation currents by [Ca2+]i was observed following inhibition of cellular phosphatase activity by the phosphatase inhibitor microcystin LR or the ATP analogue ATP gamma S. This finding strongly suggests the presence of a phosphorylation event in the calcium-dependent activation pathway for both currents. 4. Intracellular dialysis with peptide inhibitors of the multifunctional Ca(2+)-calmodulin-dependent protein kinase (CaM kinase) blocked the activation of both the chloride and cation conductances by elevated [Ca2+]i. Dialysis with an inactive control peptide had no effect on the activation of either current. CaM kinase thus appears to be critically involved in the calcium-dependent activation of both the chloride and cation currents in these cells. 5. Associated with the whole-cell cation conductance were macroscopic tail currents observed at the chloride reversal potential. The distinct kinetic properties of these tail currents were used as a biophysical 'signature' of the whole-cell conductance. 6. In excised, inside-out membrane patches, [Ca2+]i activated single cation channel activity. These channels had a mean conductance of 20 pS, were impermeable to NMDG, and their mean open probability increased at positive membrane potentials. The properties of these single channel events thus closely resemble those reported previously for calcium-activated cation channels in epithelia. 7. Using a novel 'tail current' voltage clamp protocol in excised membrane patches, we observed that ensemble averages of single cation channel events reproduced the behaviour and kinetic properties of the macroscopic tail currents of the calcium-activated cation conductance. This finding provides evidence that the observed single channel events probably underlie the macroscopic cation current recorded from intact cells. 8. The results from this study demonstrate that CaM kinase mediates the calcium-dependent activation of both a chloride and a non-selective cation current in human T84 epithelial cells. Using single channel recordings, we believe we have identified the corresponding whole-cell current for the 20-40 pS calcium-activated cation channel activity reported previously in epithelia and other cell preparations. Physiologically, a calcium-activated inward cation current would allow sodium influx in association with calcium-dependent electrolyte and protein secretion. Thus CaM kinase-dependent activation of cation channels may serve as a co-ordinated influx pathway to balance the efflux and influx of osmotically active solutes as part of an overall cell volume regulatory mechanism.

Multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca(2+)-induced enhancement of the L-type Ca2+ current in rabbit ventricular myocytes.

The intracellular mechanism underlying the Ca(2+)-induced enhancement of the L-type Ca2+ current (ICa) was examined in adult rabbit cardiac ventricular myocytes by using patch-clamp methodology. Internal Ca2+ was elevated by flash photolysis of the Ca2+ chelator Nitr 5, and intracellular Ca2+ levels were simultaneously monitored by Fluo 3 fluorescence. Flash photolysis of Nitr 5 produced a rapid (< 1-second) elevation of internal Ca2+, which led to enhancement (39% to 51% above control) of the peak inward Ca2+ current after a delay of 20 to 120 seconds. Internal dialysis of myocytes with synthetic inhibitory peptides derived from the pseudosubstrate (peptide 273-302) and calmodulin binding (peptide 291-317) regions within the regulatory domain of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) blocked enhancement of ICa produced by elevation of internal Ca2+ but not that produced by beta-adrenergic stimulation. These inhibitory peptides also had no effect on the elevation of internal Ca2+ produced by flash photolysis of Nitr 5. A pseudosubstrate inhibitory peptide derived from protein kinase C had no significant effect on Ca(2+)-dependent enhancement of ICa. We conclude that CaM kinase mediates the Ca(2+)-induced enhancement of ICa in mammalian cardiac myocytes by a mechanism likely involving direct phosphorylation of the L-type Ca2+ channel complex or an associated regulatory protein.

Cardiac alpha 1-adrenoceptors stimulate a high-affinity GTPase activity in sarcolemmal membranes from rabbit atrial and ventricular myocytes.

The interaction between cardiac alpha 1-adrenoceptors and GTP-binding regulatory proteins was characterized in isolated rabbit cardiac myocytes (thereby avoiding interference by other cell types present in the myocardium) by examining the alpha 1-adrenergic stimulation of GTPase activity in sarcolemma-enriched membrane fractions. Stimulation of membrane-associated GTPase activity in both atrial and ventricular myocyte preparations by the alpha 1-adrenergic agonists 1-noradrenaline and methoxamine (in the presence of propranolol) was observed to be both linear with time and saturable. alpha 1-adrenergic stimulation did not change the Km for GTP (0.14-0.21 microM), but increased the Vmax by 39% and 72% above basal levels in atrial and ventricular membranes, respectively. Stimulation of GTPase activity by alpha 1-agonists occurred in a concentration-dependent fashion and was blocked in the presence of the alpha-adrenoceptor antagonists phentolamine and prazosin, but not yohimbine. Prior treatment of myocytes with pertussis toxin had no effect on the alpha 1-adrenergic stimulation of GTPase activity, but inhibited stimulation by muscarinic-receptor activation with carbachol. Finally, photoaffinity labelling of an approximately 75-kDa membrane-bound protein with [alpha-32P]GTP was enhanced in the presence of the alpha 1-agonist methoxamine and abolished by addition of excess non-labelled GTP, suggesting that this GTP-binding protein may interact with cardiac alpha 1-adrenoceptors; a similar GTP-binding protein which may be coupled to alpha 1-adrenoceptors has been reported in rat liver plasma membranes (Im, M. J. & Graham, R. M. (1990) J. Biol. Chem. 265, 18944-18951).

Activation of alpha 1-adrenoceptors modulates the inwardly rectifying potassium currents of mammalian atrial myocytes.

The selective alpha 1-adrenergic agonist methoxamine (10(-4)-10(-3) M), in the presence of propranolol (10(-6) M), can reduce both the inwardly rectifying K+ background current (IK1) and the muscarinic cholinergic receptor-activated K+ current (IK,ACh) in rabbit atrial myocytes resulting in action potential prolongation during the final phase of repolarization and a depolarization of the resting membrane potential. The reduction of these K+ currents(s) by alpha 1-adrenoceptor stimulation was insensitive to pre-treatment of atrial myocytes with pertussis toxin (0.15-0.5 micrograms/ml) and was irreversible following intracellular dialysis with the non-hydrolysable guanosine triphosphate (GTP) analogue, Gpp(NH)p (1-5 x 10(-3) M). Neither the protein kinase C (PKC) inhibitors, 1((5-isoquinolinesulphonyl)-2-methylpiperoxine (H-7) (5 x 10(-5) M) and staurosporine (1 x 10(-7) M), nor "downregulation" of PKC by prolonged phorbol ester exposure (5 x 10(-7) M, for 7-8 h) had an effect on the alpha 1-adrenergic modulation of this K+ current. Under cell-attached patch-clamp conditions, bath application of methoxamine reversibly decreased acetylcholine-induced single-channel activity, thus confirming the observed reduction of the ACh-induced current under whole-cell voltage clamp. These results demonstrate that the alpha 1-adrenoceptor, once activated, can reduce current through two different inwardly rectifying K+ channels in rabbit atrial myocytes. These current changes are mediated via a pertussis toxin-insensitive GTP-binding protein, and do not appear to involve the activation of PKC.

Alpha 1-adrenoceptors reduce background K+ current in rabbit ventricular myocytes.

1. Ventricular myocytes were isolated by enzymatic dispersion of adult rabbit hearts, and voltage clamped using the whole-cell variation of the patch clamp technique. Experiments were carried out at either 35 degrees C or room temperature (21-23 degrees C). 2. In the presence of 10(-3) M-4-aminopyridine to block the transient outward K+ current, and 10(-6) M-propranolol to block beta-adrenoceptors, the alpha 1-adrenergic agonist methoxamine produced action potential prolongation, and a small depolarization of the diastolic membrane potential. Under voltage clamp conditions, methoxamine decreased the magnitude of the inward rectifier K+ current, IK1, in both the inward and outward directions. This effect was dose dependent (10(-5)-10(-3) M) and fully reversible upon wash-out of the agonist. 3. The neurotransmitter noradrenaline (10(-6)-2 x 10(-5) M), in the presence of propranolol (10(-6) M), also reduced IK1 in ventricular cells, and this effect was blocked by the specific alpha 1-adrenoceptor antagonist prazosin. 4. The alpha 1-adrenoceptor-mediated decrease in IK1 in ventricular myocytes was not affected by pre-incubation of the cells with 0.5 micrograms/ml pertussis toxin (8-10 h, 30-32 degrees C). This result suggests that in rabbit ventricular cells, the alpha 1-modulation of IK1 occurs via a pertussis toxin-insensitive guanine nucleotide-binding regulatory protein. 5. These observations demonstrate that IK1 in ventricular myocytes can be modulated by cardiac alpha 1-adrenoceptors. The resulting changes in action potential repolarization and diastolic membrane potential may have significant effects on cardiac performance.

Intracellular mechanisms for alpha 1-adrenergic regulation of the transient outward current in rabbit atrial myocytes.

1. The intracellular mechanism(s) underlying the decrease of a transient outward K+ current (It) induced by alpha 1-adrenergic agonists was studied in isolated adult rabbit atrial myocytes using whole-cell voltage clamp and cell-attached patch clamp techniques. Experiments were carried out at 22-23 degrees C. 2. Application of the specific alpha 1-adrenergic agonist, methoxamine, produced a decrease in It which was irreversible after the non-hydrolysable GTP analogues, GTP gamma S and Gpp(NH)p, had been introduced into cells via the recording micropipette. 3. Pre-treatment of cells with 0.1-0.15 microgram/ml pertussis toxin (PT) for 8-9 h at 30-34 degrees C did not prevent the alpha 1-induced decrease in It. Yet, this protocol, as measured by the PT-catalysed incorporation of [32P]ADP-ribose in membrane-associated 40 and 41 kDa proteins, effectively caused the ADP-ribosylation of approximately 70% of the PT-sensitive GTP-binding proteins (i.e. Gi) in these treated cells. After taking into account the proportion of non-viable cells (20-30%), the effectiveness of this treatment probably approaches 100% in the viable myocytes from which electrophysiological recordings were made. 4. Cell-attached patch recordings showed that bath application of methoxamine altered the single-channel events underlying It by decreasing their opening probability. Averaged currents from ensemble single-channel openings recorded in the presence of 0.2 mM-methoxamine outside the patch reproduced the features of alpha 1-adrenergic modulation of the macroscopic It observed during whole-cell voltage clamp measurements. This observation provides evidence for the involvement of a diffusible intracellular second messenger in the alpha 1-adrenergic modulation of It. 5. The protein kinase C (PKC) activators, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetylglycerol (OAG) increased It, when included in the bath perfusate, whereas the inactive analogues, 4 alpha-phorbol and 4 alpha-phorbol 12,13-didecanoate, had no effect on It. 6. Exposure of cells to the PKC inhibitors, staurosporine and H-7, either by bath superfusion or intracellularly, via the recording micropipette, did not block the decrease in It produced by methoxamine. 7. Prolonged stimulation of atrial myocytes for 7-9 h at 22 degrees C with 500 nM-PMA produced a 'down-regulation' of endogenous PKC activity, as well as a physical loss of the immunoreactive enzyme, as measured by an in vitro assay, and an anti-PKC monoclonal antibody, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic acetylcholine receptors in the sino-atrial node and right atrium of bovine heart.

Muscarinic acetylcholine receptors were identified by the specific binding of [H](-)quinuclidinylbenzilate [( 3H](-)QNB) and [3H]oxotremorine-M [( 3H]Oxo-M), to membranes isolated from the sino-atrial (SA) node and right atrium (RA) of bovine heart. The density of [3H](-)QNB binding sites was greater in the SA node compared to the RA. Specific [3H](-)QNB binding was saturable and occurred to a single population of binding sites in both regions. The binding of antagonists, as assessed by competition with [3H](-)QNB, also occurred to a single population of sites; the binding affinities of all antagonists were similar in either region. Agonist competition curves, except for McN-A-343, were complex and computer analyses indicated that agonists bound to at least two populations of binding sites that differed in affinity. The proportion of high-affinity agonist binding sites was consistently greater in the SA nodal, relative to the RA membranes, while the affinity of the high-affinity agonist binding sites to a given agonist was essentially similar in either region. The high-affinity binding of [3H]Oxo-M was saturable and occurred to a single population of sites. The maximal binding of [3H]Oxo-M in the SA nodal membranes was higher than in the RA membranes. Guanine nucleotides and N-ethylmaleimide (NEM) markedly decreased [3H]Oxo-M binding; NEM did not appear to influence guanine nucleotide-dependent decrease in [3H]Oxo-M binding. Phospholipase A2 decreased both [3H](-)QNB and [3H]Oxo-M specific binding, the latter being affected to a greater extent. Phospholipase C also decreased [3H](-)QNB and [3H]Oxo-M binding, although to a lesser degree compared to phospholipase A2. Either lipase, however, increased the guanine nucleotide-sensitive agonist binding. Analysis of [3H](-)QNB binding to microsomal subfractions showed that binding sites were enriched in the light plasma membrane fractions that were also enriched in pertussis toxin sensitive guanine nucleotide binding proteins.

Pertussis toxin-sensitive G-proteins in the sino-atrial node and right atrium of bovine heart.

Three apparently distinct pertussis toxin (PTX)-sensitive substrates, with Mrs of 39, 40 and 41 kDa, were identified in membranes prepared from the sino-atrial (SA) node and right atrium of bovine heart. Based on their biochemical characterization, the effects of guanine nucleotides/MgCl2 on their PTX-catalyzed [32P]ADP ribosylation, and the PTX-induced decrease in radiolabelled agonist high-affinity binding to muscarinic acetylcholine receptors present in these membranes, we tentatively identify these proteins as the alpha-subunits of the G0 and Gi subtypes of G-proteins. These results indicate that PTX alters the G-protein modulation of SA nodal and atrial muscarinic acetylcholine receptors by disrupting at least one of a group of PTX-sensitive G-proteins present in these tissues.