Mutation of a conserved glutamine residue does not abolish desensitization of acid-sensing ion channel 1

Desensitization is a common feature of ligand-gated ion channels, although the molecular cause varies widely between channel types. Mutations that greatly reduce or nearly abolish desensitization have been described for many ligand-gated ion channels, including glutamate, GABA, glycine, and nicotinic receptors, but not for acid-sensing ion channels (ASICs) until recently. Mutating Gln276 to a glycine (Q276G) in human ASIC1a was reported to mostly abolish desensitization at both the macroscopic and the single channel levels, potentially providing a valuable tool for subsequent studies. However, we find that in both human and chicken ASIC1, the effect of Q276G is modest. In chicken ASIC1, the equivalent Q277G slightly reduces desensitization when using pH 6.5 as a stimulus but desensitizes, essentially like wild-type, when using more acidic pH values. In addition, steady-state desensitization is intact, albeit right-shifted, and recovery from desensitization is accelerated. Molecular dynamics simulations indicate that the Gln277 side chain participates in a hydrogen bond network that might stabilize the desensitized conformation. Consistent with this, destabilizing this network with the Q277N or Q277L mutations largely mimics the Q277G phenotype. In human ASIC1a, the Q276G mutation also reduces desensitization, but not to the extent reported previously. Interestingly, the kinetic consequences of Q276G depend on the human variant used. In the common G212 variant, Q276G slows desensitization, while in the rare D212 variant desensitization accelerates. Our data reveal that while the Q/G mutation does not abolish or substantially impair desensitization as previously reported, it does point to unexpected differences between chicken and human ASICs and the need for careful scrutiny before using this mutation in future studies.     

Spermine regulates N-methyl-D-aspartate receptor desensitization.

The action of the endogenous polyamine spermine on NMDA-induced responses (in the presence of glycine) was evaluated in cultured spinal cord neurons under voltage- and concentration-clamp conditions. Spermine potentiated NMDA-induced responses in a dose-dependent manner. It was more effective in potentiating steady-state currents (i.e., desensitized response) than the peak phase of the response, indicating that the degree of desensitization was reduced in the presence of the polyamine. Kinetic analysis revealed that the desensitization onset rate, but not recovery rate, was affected by spermine. The effect was voltage independent and was seen in thoroughly dialyzed cells, in which desensitization becomes independent of glycine. Spermine potentiation showed fast on-off kinetics, and intracellular spermine, loaded in the recording pipette, did not occlude potentiation by extracellularly applied spermine. These results are consistent with the existence of a modulatory site for polyamines in the extracellular domain of the NMDA receptor, the activation of which potentiates NMDA receptor function by regulating its desensitization kinetics.     

Cell-free heterologous desensitization of adenylyl cyclase in S49 lymphoma cell membranes mediated by cAMP-dependent protein kinase

We have examined the cell-free heterologous desensitization of adenylyl cyclase in plasma membrane preparations from S49 wild-type (WT) and kin- cells (which lack cAMP-dependent protein kinase) incubated with purified catalytic subunit of cAMP-dependent protein kinase (cA.PKc). cA.PKc caused a rapid (t1/2 = 40 s) decrease in the hormone responsiveness of adenylyl cyclase in the WT membrane preparations that mimicked the intact cell heterologous desensitization; that is, there was an increase in the Kact for both epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase induced at the receptor level because neither forskolin- nor NaF-stimulated activity was affected. The desensitization was independent of agonist occupancy of the receptor, and the effects were blocked both by the active fragment (amino acids 5-22) of the specific inhibitor of cA.PK and by p[NH]ppA. cA.PKc treatment of kin- membranes resulted in a heterologous desensitization that resembled the effects of WT adenylyl cyclase, with the exception that forskolin-stimulated activity was also reproducibly decreased by 24%. cA.PKc had no effect on WT membranes isolated from cells that had previously undergone maximal heterologous desensitization during treatment with 10 microM forskolin. In contrast, cA.PKc-induced heterologous desensitization of kin- membranes was additive with the epinephrine-induced homologous desensitization of intact cells. Cell-free desensitizations were reversed by incubation of membranes with cA.PKc and ADP, conditions that drive the kinase reaction backward. The similarities of our cell-free cA.PKc-mediated heterologous desensitization of adenylyl cyclase with the intact cell desensitization support our hypothesis that heterologous desensitization of the WT lymphoma cells is mediated by cA.PK via a mechanism independent of homologous desensitization.     

Agonist-induced long-term desensitization of the human prostacyclin receptor

Phosphorylation of the human prostacyclin (PGI(2)) receptor (hIP-R) by diacylglycerol-regulated protein kinase C (PKC) has been reported to be responsible for its rapid desensitization in HEK293 cells. In this study we demonstrate, that human fibroblasts reveal a much slower hIP-R desensitization kinetics, which was neither affected by stimulation nor inhibition of PKC by either phorbol 12-myristate-13-acetate or GF-109203X suggesting a different cellular mechanism. Although agonist-promoted sequestration of a C-terminally green fluorescent protein-tagged hIP-R was demonstrated, it did not account for the long-term desensitization. Concanavalin A did not abolish, but accelerated receptor desensitization kinetics. Resensitization of hIP-R involved receptor recycling and/or de novo synthesis of receptor protein, depending on the duration of prior desensitization. This is the first study investigating the mechanisms of hIP-R desensitization in intact human cells naturally expressing hIP-R. Our data suggest, that a hitherto unknown mechanism of hIP-R long-term desensitization, which is independent of receptor phosphorylation by conventional and novel type PKC isoforms or endocytosis, is a key event in regulating the cellular responsiveness to PGI(2).     

Variation of glycine-activated conductivity of the membranes of spinal cord neurons in the chick embryo in culture

The method of leading off whole-cell currents is used to study the neuron responses elicited by applications of glycine at different stages of culturing of the chick embryo dissociated spinal cord. For two types of cells described earlier [4] differently directed shifts of the concentration dependence curves of the transmitter effect are shown to be characteristic. "Mature" cells, surviving several days in culture and represented by motoneurons, typically show a lowered sensitivity to glycine. The sensitivity of little-differentiated neurons increases more than tenfold by the end of the second week of culturing. The desensitization kinetics of the glycine-activated current also slows down with age. Blocking of the spontaneous electrical activity during development prevented these changes, and the presence of agonist in the culture medium did not affect them. Analogous patterns of formation of transmitter sensitivity are assumed to be exhibited by differentiating neurons of the intact nervous system.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 580–587, September–October, 1991.     

Cl(-) concentration changes and desensitization of GABA(A) and glycine receptors

Desensitization of ligand-gated ion channels plays a critical role for the information transfer between neurons. The current view on γ-aminobutyric acid (GABA)(A) and glycine receptors includes significant rapid components of desensitization as well as cross-desensitization between the two receptor types. Here, we analyze the mechanism of apparent cross-desensitization between native GABA(A) and glycine receptors in rat central neurons and quantify to what extent the current decay in the presence of ligand is a result of desensitization versus changes in intracellular Cl(-) concentration ([Cl(-)](i)). We show that apparent cross-desensitization of currents evoked by GABA and by glycine is caused by changes in [Cl(-)](i). We also show that changes in [Cl(-)](i) are critical for the decay of current in the presence of either GABA or glycine, whereas changes in conductance often play a minor role only. Thus, the currents decayed significantly quicker than the conductances, which decayed with time constants of several seconds and in some cells did not decay below the value at peak current during 20-s agonist application. By taking the cytosolic volume into account and numerically computing the membrane currents and expected changes in [Cl(-)](i), we provide a theoretical framework for the observed effects. Modeling diffusional exchange of Cl(-) between cytosol and patch pipettes, we also show that considerable changes in [Cl(-)](i) may be expected and cause rapidly decaying current components in conventional whole cell or outside-out patch recordings. The findings imply that a reevaluation of the desensitization properties of GABA(A) and glycine receptors is needed.     

The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism.

Treatment of intact hepatocytes with glucagon, TH-glucagon [( 1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), angiotensin or vasopressin led to a rapid time- and dose-dependent loss of the glucagon-stimulated response of the adenylate cyclase activity seen in membrane fractions isolated from these cells. Intracellular cyclic AMP concentrations were only elevated with glucagon. All ligands were capable of causing both desensitization/loss of glucagon-stimulated adenylate cyclase activity and stimulation of inositol phospholipid metabolism in the intact hepatocytes. Maximally effective doses of angiotensin precluded any further inhibition/desensitizing action when either glucagon or TH-glucagon was subsequently added to these intact cells, as has been shown previously for the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) [Heyworth, Wilson, Gawler & Houslay (1985) FEBS Lett. 187, 196-200]. Treatment of intact hepatocytes with these various ligands caused a selective loss of the glucagon-stimulated adenylate cyclase activity in a washed membrane fraction and did not alter the basal, GTP-, NaF- and forskolin-stimulated responses. Angiotensin failed to inhibit glucagon-stimulated adenylate cyclase activity when added directly to a washed membrane fraction from control cells. Glucagon GR2 receptor-stimulated adenylate cyclase is suggested to undergo desensitization/uncoupling through a cyclic AMP-independent process, which involves the stimulation of inositol phospholipid metabolism by glucagon acting through GR1 receptors. This action can be mimicked by other hormones which act on the liver to stimulate inositol phospholipid metabolism. As the phorbol ester TPA also mimics this process, it is proposed that protein kinase C activation plays a pivotal role in the molecular mechanism of desensitization of glucagon-stimulated adenylate cyclase. The site of the lesion in desensitization is shown to be at the level of coupling between the glucagon receptor and the stimulatory guanine nucleotide regulatory protein Gs, and it is suggested that one or both of these components may provide a target for phosphorylation by protein kinase C.     

A possible molecular mechanism for beta-receptor desensitization: experiments and hypotheses

β-Receptor desensitization in intact NRK-S cells and in a crude membrane preparation derived from these cells was found not to involve methylation, cAMP dependent phosphorylation, Ca⁺⁺ dependent phosphorylation or ADP ribosylation, but is absolutely dependent on ATP in cell-free systems. Also, the desensitized state could not be relieved by protein phosphatases or alkaline phosphatases. Depletion of intact cells from ATP by prolonged incubation with 2-deoxy-glucose and NaN₃ did not inhibit the rapid onset of desensitization by incubating the cells with β-agonists. In order to rationalize the two seemingly contradictory findings, namely, the absolute requirement of ATP in the cell-free desensitization system and the inability to reverse the desensitized state by a variety of phosphatases, we propose that the uncoupling reaction responsible involve for establishing the desensitized state does not Our working hypothesis suggests instead, that the uncoupling reaction involves a covalent modification of the receptor protein and is catalyzed by a receptor modulator protein which is under the control of the ATP dependent phosphorylation.     

Investigation of the alpha(1)-glycine receptor channel-opening kinetics in the submillisecond time domain.

The activation and desensitization kinetics of the human alpha(1)-homooligomeric glycine receptor, which was transiently expressed in HEK 293 cells, were studied with a 100-microseconds time resolution to determine the rate and equilibrium constants of individual receptor reaction steps. Concentration jumps of the activating ligands glycine and beta-alanine were initiated by photolysis of caged, inactive precursors and were followed by neurotransmitter binding, receptor-channel opening, and receptor desensitization steps that were separated along the time axis. Analysis of the ligand concentration-dependence of these processes allows the determination of 1) the rate constants of glycine binding, k(+1) approximately 10(7) M(-1) s(-1), and dissociation, k(-1) = 1900 s(-1); 2) the rates of receptor-channel opening, k(op) = 2200 s(-1), and closing, k(cl) = 38 s(-1); 3) the receptor desensitization rate, alpha = 0.45 s(-1); 4) the number of occupied ligand binding sites necessary for receptor-channel activation and desensitization, n >/= 3; and 5) the maximum receptor-channel open probability, p(0) > 0.95. The kinetics of receptor-channel activation are insensitive to the transmembrane potential. A general model for glycine receptor activation explaining the experimental data consists of a sequential mechanism based on rapid ligand-binding steps preceding a rate-limiting receptor-channel opening reaction and slow receptor desensitization.     

The absence of desensitization in the beta adrenergic receptors of turkey reticulocytes and erythrocytes and its possible origin.

β-adrenergic receptors on turkey erythrocytes and on turkey reticulocytes do not undergo desensitization subsequent to the exposure of the cells to the β-agonist, l-isoproterenol. The increase in the membrane fluidity of the turkey reticulocytes by depleting it of cholesterol does not modify this property of the cells. However, exposure of intact turkey reticulocytes, but not of mature erythrocytes, to dl-propranolol induced a 100% increase in the number of β-adrenergic receptors on the cell surface. This increase is not inhibited by the protein biosynthesis inhibitor cycloheximide. The possible origin of the lack of desensitization in turkey red cells to β-agonists and of the propranolol induced exposure of cryptic receptors is discussed.     

Hydroxylated residues influence desensitization behaviour of recombinant alpha3 glycine receptor channels

The human glycine receptor subunit alpha3 exists in two splice variants (alpha3K/L), with alpha3L bearing an additional segment of 15 amino acids within the cytoplasmic TM3-4 loop. Homomeric alpha3K glycine receptors show faster desensitization than alpha3L receptors. Ion channel properties were compared of alpha3L, alpha3K, and of the triple mutant alpha3LDeltaOH = alpha3L(T358A/Y367F/S370A), where hydroxyl functions of the spliced insert had been removed by site-directed mutagenesis. Upon recombinant expression in HEK 293 cells, patch-clamp recording experiments revealed that removal of hydroxyl functions primarily affected receptor desensitization. The fraction of non-desensitizing current was 68 +/- 13% for alpha3L, 21 +/- 13% for alpha3K, and 48 +/- 16% for alpha3LDeltaOH. Desensitization time constants at saturating glycine concentration were 8.4 +/- 2.8 s, 1.9 +/- 2.3 s, and 2.8 +/- 0.4 s, for alpha3L, alpha3K, and the triple mutant alpha3LDeltaOH, respectively. In contrast, single-channel and whole-cell properties were similar for all three constructs. Thus, ion channel activation, desensitization, and conductance properties are independently controlled by distinct structural elements. Hydroxyl functions within the M3-4 loop of the glycine receptor alpha3 subunit are crucial, but not exclusive, determinants of receptor desensitization.     

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.     

Homologous desensitization of beta-adrenergic receptors in lymphoma cells is not altered by the inactivation of Ni (Gi), the inhibitory guanine nucleotide regulatory protein.

We had previously demonstrated that the cyc- mutant of S49 wild-type lymphoma cells both desensitizes and undergoes a sequestration-internalization of the beta-receptor in response to short-term treatment with adrenaline. The cyc- mutant of S49 wild-type lymphoma cells lacks the alpha s subunit of the stimulatory coupling protein Ns, but has fully functional Ni, the inhibitory component of the regulatory complex. This suggested that functional Ns was not required for desensitization. To examine the role of Ni in desensitization, both S49 wild-type and cyc- cells were treated with islet-activating protein under conditions that led to over 85% attenuation of Ni function in S49 wild-type cells and approx. 50% attenuation of Ni function in cyc- cells. This treatment had no effect on the adrenaline-induced desensitization of adenylate cyclase or the sequestration event measured by the apparent movement of beta-adrenergic receptors to a light-vesicle fraction. Further, the desensitization event, which occurs before the sequestration event, observable only in intact cells, was also not altered by islet-activating-protein pretreatment of S49 wild-type cells. The data suggest that a functional Ni is not required for desensitization in the S49 lymphoma cells.     

Long-term regulation of muscarinic acetylcholine receptors on cultured nerve cells.

Incubation of mouse neuroblastoma cells (clone 1LE-115) with the muscarinic agonist, carbachol, resulted in a time-dependent desensitization to muscarinic receptor-mediated cyclic GMP formation and a decrease in the number and affinity of muscarinic receptors as measured by the binding to intact cells of the muscarinic antagonist, [³H]quinuclidinyl benzilate (QNB). The decrease in responsiveness to cyclic GMP formation reached a maximum after a 15-minute exposure to 1 mM carbachol (short-term desensitization) whereas changes in [³H] QNB binding became apparent only after a one hour exposure and reached a maximum after about 12 hrs (long-term desensitization). Recovery of sensitivity after short-term desensitization was rapid, suggesting that this process may involve a conformational change in the muscarinic receptor. In contrast, recovery after long-term desensitization was prolonged and could be slowed by the inhibition of protein synthesis. These results indicate that different cellular mechanisms are involved in the short-term and long-term desensitization of muscarinic receptors.     

Desensitization of NMDA receptor channels is modulated by glutamate agonists

Two distinct forms of desensitization have been characterized for N-methyl-D-aspartate (NMDA) receptors. One form results from a weakening of agonist affinity when channels are activated whereas the other form of desensitization results when channels enter a long-lived nonconducting state. A weakening of glycine affinity upon NMDA receptor activation has been reported. Cyclic reaction schemes for NMDA receptor activation require that a concomitant affinity shift should be observed for glutamate agonists. In this study, measurements of peak and steady-state NMDA receptor currents yielded EC50 values for glutamate that differed by 1.9-fold, but no differences were found for another agonist, L-cysteine-S-sulfate (LCSS). Simulations show that shifts in EC50 values may be masked by significant degrees of desensitization resulting from channels entering a long-lived nonconducting state. Simulations also show that a decrease in the degree of desensitization with increasing agonist concentration is a good indicator for the existence of desensitization resulting from a weakening of agonist affinity. Both glutamate and LCSS exhibited this trend. An affinity difference of three- to eightfold between high-and low-affinity agonist-binding states was estimated from fitting of dose-response data with models containing both types of desensitization. This indicates that activation of NMDA receptors causes a reduction in both glutamate and glycine affinities.     

Reappearance of beta-adrenergic receptors after isoproterenol treatment in intact C6-cells

The reappearance of beta-adrenergic receptors in C6-glioma cells after desensitization with isoproterenol was studied using the antagonist [3H]CGP-12177. Reappearance had the following properties: (a) it occurred in intact cells only, (b) it was temperature dependent, (c) it required an Na+/H+ gradient, low intracellular Ca2+ activity, and (d) it required ATP, and (e) intact lysosomes. The results suggest endocytosis and recycling of the beta-adrenergic receptor after agonist treatment.     

Phorbol ester causes desensitization of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line

The murine Leydig tumor cell line, MLTC-1, contains gonadotropin receptors and a gonadotropin-responsive adenylate cyclase system that became refractory (desensitized) when exposed to human chorionic gonadotropin (hCG). MLTC-1 cells also contain phorbol ester receptors with a Kd of 53 nM for [3H]phorbol dibutyrate. Exposing cells to 12-O-tetradecanoyl phorbol 13-acetate (TPA) also causes desensitization of the hCG response. TPA-induced desensitization was similar to hCG-induced desensitization by every criteria tested. Both TPA- and hCG-induced desensitization caused approximately 50% loss of the hormone response within 30 min. Neither TPA or hCG altered receptor affinity for hCG. The dose response of adenylate cyclase to hCG or GTP in isolated membranes was not affected by either hCG- or TPA-induced desensitization. Similarly the dose response to hCG of cAMP accumulation in intact cells was not altered by desensitization with hCG or TPA. It was determined that MLTC-1 cells have Ca2+/phospholipid-dependent protein kinase activity that displayed a dose-dependent response to TPA. The concentration of TPA required to activate the protein kinase was similar to that required for desensitization. Phorbol esters that were unable to activate protein kinase C were also unable to desensitize MLTC-1 cells. The protein kinase from MLTC-1 cells was also activated by diacylglycerol. In addition, diacylglycerols caused desensitization of the hCG response. TPA- and diacylglycerol-induced desensitization is probably mediated by protein kinase C, and the similarities between hCG- and TPA-induced refractoriness suggests a convergence of mechanisms at some point of MLTC-1 cell desensitization.     

Beta-adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg2+

Treatment with low physiological concentrations of epinephrine (5-50 nM) rapidly desensitizes beta-adrenergic stimulation of cAMP formation in S49 wild-type (WT) lymphoma cells. Previous attempts to detect this early phase of desensitization in cell-free assays of adenylate cyclase (EC 4.6.1.1) after intact cell treatment were unsuccessful. We have now found that reducing the Mg2+ concentrations in the adenylate cyclase assays to less than 1.0 mM unmasked this rapid phase of desensitization of the WT cells, and that high Mg2+ concentrations (5-10 mM) largely obscured the desensitization. Submillimolar Mg2+ conditions also revealed a two- to threefold decrease in the affinity of epinephrine binding to the beta-adrenergic receptor after desensitization with 20 nM epinephrine. Detection of 4 beta-phorbol 12-myristate 13-acetate (PMA) desensitization of the WT beta-adrenergic receptor was also dependent on low Mg2+ as measured either by the decrease in epinephrine stimulation of adenylate cyclase or by the reduction in the affinity of epinephrine binding. Unexpectedly, when cyc- cells were pretreated with 50 nM epinephrine, the beta-adrenergic stimulation of reconstituted adenylate cyclase was not desensitized. The characteristics of the Mg2+ effect on epinephrine- and PMA-induced desensitizations suggest a similar mechanism of action with the most likely events being phosphorylations of the beta-adrenergic receptors. Our data indicate that cAMP-dependent protein kinase (EC 2.7.1.37) may play a role in the desensitization caused by low epinephrine concentrations inasmuch as this phase of desensitization did not occur in the cyc-. For the PMA-induced desensitization, the phosphorylation may be mediated by protein kinase C (EC 2.7.1.37).     

On the mechanism of isoproterenol-induced desensitization of adenylate cyclase in cultured differentiated hepatocytes

The adenylate cyclase of cultured differentiated RL-PR-C hepatocytes is desensitized to 1-isoproterenol by exposure to this beta-agonist. Virtually complete desensitization occurred by 60 min (intact cells) or 30 min (isolated plasma membranes). Isoproterenol was maximally effective at 10 micrometers, although substantial desensitization occurred at isoproterenol concentrations as low as 10 nM. Protein synthesis was not required for desensitization. Recovery from desensitization under tissue culture conditions was only 25% complete by 24 h. Maximum desensitization was accompanied by only a modest 35% decrease in binding sites (as determined by binding assays with [3H]dihydroalprenolol), with no change in binding affinity. Adenylate cyclase desensitized to 1-isoproterenol responded normally to guanine nucleotides and to fluoride, suggesting that the regulatory and catalytic proteins were not the sites of the desensitization "defect'. Using N-ethylmaleiimide to inactive the regulatory and catalytic proteins, and dicyclohexylcarbodiimide to inactivate the beta-adrenergic receptor, of intact hepatocytes, various heterologous cell fusion hybrids were produced, and their adenylate cyclases tested for responsiveness to 1-isoproterenol; only hybrids containing "desensitized' receptor failed to respond to isoproterenol. These results suggest that the mechanism of desensitization to isoproterenol involves only the receptor component of the receptor-regulatory protein(s)-adenylate cyclase complex, and that the receptors are reduced in number and/or ability to interact with the regulatory protein as a result of the desensitization process.     

Temperature Dependence of Muscarinic Acetylcholine Receptor Activation, Desensitization, and Resensitization

Abstract: The effects of temperature on muscarinic acetylcholine receptor activation, desensitization, and resensitization were studied with the use of intact mouse neuroblastoma cells (clone N1E-115), which have muscarinic receptors that mediate cyclic GMP synthesis. Below 15-20°C, activation or desensitization of muscarinic receptors by carbamylcholine and recovery from desensitization (caused by carbamylcholine at 37°C) did not occur. Above these temperatures, the apparent rates of receptor-mediated cyclic GMP synthesis, desensitization, and recovery of sensitivity increased as the incubation temperature was increased. Arrhenius plots of the data yielded activation energies of 25, 14, and 23 kcal.mol⁻¹ for activation, desensitization, and resensitization, respectively. These data suggest that a certain degree of membrane phospholipid fluidity is required for these processes to occur.