Drugs acting upon the cyclic adenosine monophosphate/protein kinase A signalling pathway modulate memory consolidation when given late after training into rat hippocampus but not amygdala

Rats implanted bilaterally with cannulae in the CA1 region of the dorsal hippocampus or in the amygdala were trained in one-trial step-down inhibitory (passive) avoidance using a 0.4 mA footshock. At various times after training (0, 1.5, 3, 6 or 9 h for animals implanted in the hippocampus; 0 or 3 h for those implanted in the amygdala), they received infusions of 8-Br-cAMP (cyclic adenosine monophosphate) (1.25 micrograms/side), SKF38393 (7.5 micrograms/side), SCH23390 (0.5 microgram/side), norepinephrine ClH (0.3 microgram/side), timolol ClH (0.3 microgram/side), 8-HO-DPAT (2.5 micrograms/side), NAN-190 (2.5 micrograms/side), forskolin (0.5 microgram/side) or KT5720 (0.5 microgram/side). Rats were tested for retention 24 h after training. SKF38393 is an agonist and SCH23390 an antagonist at dopamine D1 receptors, timolol is a beta-adrenoceptor antagonist, 8-HO-DPAT is an agonist and NAN-190 an antagonist at 5HT1A receptors, forskolin enhances adenylyl cyclase, and KT5720 inhibits protein kinase A. When given into the hippocampus 0 h post-training, norepinephrine enhanced memory and KT5720 was amnestic. When given 1.5 h after training, all treatments were ineffective. When given 3 or 6 h post-training, 8-Br-cAMP, forskolin, SKF 38393, noradrenaline and NAN-190 caused memory facilitation, and KT5720, SCH23390, timolol and 8-HO-DPAT caused retrograde amnesia. At 9 h from training, all treatments were again ineffective. When given into the amygdala 0 or 3 h post-training all treatments were ineffective, except for noradrenaline at 0 h, which caused retrograde facilitation. The data agree with the suggestion that in the hippocampus, but not the amygdala, a cAMP/protein kinase A pathway is involved in memory consolidation at 3 and 6 h from training, and that this is regulated by D1, beta, and 5HT1A receptors. This correlates with a previous report of increased cAMP levels, protein kinase A activity and P-CREB levels at 3-6 h from training in rat hippocampus in this task. This may be taken to suggest that the hippocampus, but not the amygdala, is involved in the long-term storage of step-down inhibitory avoidance in the rat.     

Object and place memory in the macaque entorhinal cortex

Lesions of the entorhinal cortex in humans, monkeys, and rats impair memory for a variety of kinds of information, including memory for objects and places. To begin to understand the contribution of entorhinal cells to different forms of memory, responses of entorhinal cells were recorded as monkeys performed either an object or place memory task. The object memory task was a variation of delayed matching to sample. A sample picture was presented at the start of the trial, followed by a variable sequence of zero to four test pictures, ending with a repetition of the sample (i.e., a match). The place memory task was a variation of delayed matching to place. In this task, a cue stimulus was presented at a variable sequence of one to four "places" on a computer screen, ending with a repetition of one of the previously shown places (i.e., a match). For both tasks, the animals were rewarded for releasing a bar to the match. To solve these tasks, the monkey must 1) discriminate the stimuli, 2) maintain a memory of the appropriate stimuli during the course of the trial, and 3) evaluate whether a test stimulus matches previously presented stimuli. The responses of entorhinal cortex neurons were consistent with a role in all three of these processes in both tasks. We found that 47% and 55% of the visually responsive entorhinal cells responded selectively to the different objects or places presented during the object or place task, respectively. Similar to previous findings in prefrontal but not perirhinal cortex on the object task, some entorhinal cells had sample-specific delay activity that was maintained throughout all of the delay intervals in the sequence. For the place task, some cells had location-specific maintained activity in the delay immediately following a specific cue location. In addition, 59% and 22% of the visually responsive cells recorded during the object and place task, respectively, responded differently to the test stimuli according to whether they were matching or non-matching to the stimuli held in memory. Responses of some cells were enhanced to matching stimuli, whereas others were suppressed. This suppression or enhancement typically occurred well before the animals' behavioral response, suggesting that this information could be used to perform the task. These results indicate that entorhinal cells receive sensory information about both objects and spatial locations and that their activity carries information about objects and locations held in short-term memory.     

Colocalization of muscarinic acetylcholine receptors and protein kinase C gamma in rat parietal cortex

The present investigation analyzes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) and the gamma isoform of protein kinase C (PKC) in the rat parietal cortex employing the monoclonal antibodies M35 and 36G9, respectively. Muscarinic cholinoceptive neurons were most present in layers 2, 3 and 5, whereas most PKC gamma-positive cells were found in layers 2, 5 and 6. Under normal, non-stimulated conditions, approximately 58% of all muscarinic cholinoceptive neurons were immunoreactive for PKC gamma. Conversely, nearly all PKC gamma-positive neurons were M35-immunoreactive. Although both pyramidal and nonpyramidal neurons express the two types of protein, the pyramidal cell type represents the vast majority. Of all cortical neurons, the large (15-25 microns in diameter) muscarinic cholinoceptive pyramidal neurons in layer 5 express the gamma isoform of PKC most abundantly and most frequently. Approximately 96% of these cells are immunoreactive for PKC gamma. Stimulation of mAChRs by the cholinergic agonist carbachol resulted in a pronounced increase in the intensity of 36G9 immunoreactivity, which may suggest that the mAChRs are functionally linked to the colocalized PKC gamma. No change was found in the number of 36G9-immunoreactive neurons. In contrast, the number of immunocytochemically detectable muscarinic cholinoceptive neurons increased by approximately 38% after carbachol stimulation. The high degree of codistribution in cortical neurons of both transduction proteins suggests a considerable cholinergic impact upon the regulation of PKC gamma, a candidate key enzyme in cortical learning and memory mechanisms.     

A role for the Ras signalling pathway in synaptic transmission and long-term memory

Members of the Ras subfamily of small guanine-nucleotide-binding proteins are essential for controlling normal and malignant cell proliferation as well as cell differentiation. The neuronal-specific guanine-nucleotide-exchange factor, Ras-GRF/CDC25Mm, induces Ras signalling in response to Ca2+ influx and activation of G-protein-coupled receptors in vitro, suggesting that it plays a role in neurotransmission and plasticity in vivo. Here we report that mice lacking Ras-GRF are impaired in the process of memory consolidation, as revealed by emotional conditioning tasks that require the function of the amygdala; learning and short-term memory are intact. Electrophysiological measurements in the basolateral amygdala reveal that long-term plasticity is abnormal in mutant mice. In contrast, Ras-GRF mutants do not reveal major deficits in spatial learning tasks such as the Morris water maze, a test that requires hippocampal function. Consistent with apparently normal hippocampal functions, Ras-GRF mutants show normal NMDA (N-methyl-D-aspartate) receptor-dependent long-term potentiation in this structure. These results implicate Ras-GRF signalling via the Ras/MAP kinase pathway in synaptic events leading to formation of long-term memories.     

Proliferation of human and mouse astrocytes in vitro: signalling through the protein kinase C pathway

While several mitogens for astrocytes have been described, the signal transduction pathway(s) that mediates their proliferative effect remains unclear; in this report, a major role for the protein kinase C (PKC) system is suggested by several lines of evidence. Firstly, biologically active phorbol esters, 4 beta-phorbol-12,13-dibutyrate and phorbol-12-myristate-13-acetate, increase the proliferation of astrocytes as determined by [3H]thymidine incorporation or bromodeoxyuridine immunofluorescence; this effect is not reproduced by a phorbol ester that binds to PKC but does not activate it (4 alpha-phorbol-12,13-didecanoate). Secondly, 2 relatively selective inhibitors of PKC, H7 and staurosporine, attenuate the basal rate of proliferation of astrocytes in concentrations that were not cytotoxic to cells. Thirdly, mitogen-enhanced proliferation of astrocytes can be blocked by PKC inhibitors; this is observed for all astrocyte mitogens tested. Fourthly, measurements of PKC enzyme activity in astrocytes in response to serum-mitogenic factors, or to staurosporine, revealed a statistically significant correlation with proliferation rate. The mediation by PKC is not dependent on species- or age factors, since neonatal mouse or adult human astrocytes gave comparable results. The results have relevance to normal development and reactive gliosis post-injury, 2 conditions where astrocytes undergo proliferation, and to glioma growth.     

Noradrenaline release and the effect of endogenous activation of the phospholipase C/protein kinase C signalling pathway in rat atria

Coagulopathies in children after cardiopulmonary bypass (CPB) are complex. There are very limited data correlating coagulation tests with postoperative bleeding. We evaluated coagulation changes after CPB and after the administration of coagulation products to 75 children. Baseline coagulation tests were obtained and repeated after protamine administration, after transfusion of individual coagulation products, and on arrival in the intensive care unit (ICU). Regression analysis demonstrated no baseline coagulation test to predict postoperative chest tube drainage. Weight and duration of CPB were determined to be the only predictors of bleeding. Further analyses demonstrated that children <8 kg had more bleeding and required more coagulation products than children >8 kg. Postprotamine platelet count and fibrinogen level correlated independently with 24-h chest tube drainage in children <8 kg, whereas postprotamine platelet count and thrombelastographic values did so in patients weighing >8 kg. Platelet administration alone was found to restore effective hemostasis in many patients. With ongoing bleeding, cryoprecipitate improved coagulation parameters and limited blood loss. Fresh-frozen plasma administration after platelets worsened coagulation parameters and was associated with greater chest tube drainage and more coagulation product transfusions in the ICU. Objective data to guide post-CPB component therapy transfusion in children are suggested. Implications: Children <8 kg can be expected to have more severe coagulopathies, require more coagulation product transfusions, and bleed more after cardiopulmonary bypass. Correlations between coagulation tests and postoperative chest tube drainage are defined. Platelets and, if necessary, cryoprecipitate optimally restore hemostasis. Fresh-frozen plasma offers no benefits in correcting postcardiopulmonary bypass coagulopathies in children.     

Dissociation of item and order memory following parietal cortex lesions in the rat.

Rats with parietal cortex lesions were tested for both item and order memory for a list of spatial events in a probe recognition procedure. Rats with parietal cortex lesions were impaired for all events within the item memory task but had good memory for the early events within the order memory task. These data suggest a dissociation of function between item and order memory for parietal cortex damaged animals. In conjunction with previous findings with rats with medial prefrontal cortex lesions, these data suggest that item and order memory can be coded and represented independently. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Independent and exclusive modulation of cardiac delayed rectifying K+ current by protein kinase C and protein kinase A

We have recently established that local exposure to a 929.2 MHz electromagnetic near-field, used for cellular phones, does not promote rat liver carcinogenesis in a medium-term bioassay system. In the present study, a 1.439 GHz electromagnetic near-field (EMF), another microwave band employed for cellular phones in Japan, was similarly investigated. Time division multiple access (TDMA) signals for the Personal Digital Cellular (PDC) Japanese cellular telephone standard system were directed to rats through a quarter-wavelength monopole antenna. Numerical dosimetry showed that the peak SARs within the liver were 1.91-0.937 W/kg, while the whole-body average specific absorption rates (SARs) were 0.680-0.453 W/kg, when the time-averaged antenna radiation power was 0.33 W. Exposure was for 90 min a day, 5 days a week, over 6 weeks, to male F344 rats given a single dose of diethylnitrosamine (200 mg/kg, i.p.) 2 weeks previously. At week 3, all rats were subjected to a two-thirds partial hepatectomy. At week 8, the experiment was terminated and the animals were killed. Carcinogenic potential was scored by comparing the numbers and areas of the induced glutathione S-transferase placental form (GST-P)-positive foci in the livers of exposed (48) and sham-exposed rats (48). Despite increased serum levels of corticosterone, adrenocorticotropic hormone (ACTH) and melatonin, the numbers and the areas of GST-P-positive foci were not significantly altered by the exposure. These findings clearly indicated that local body exposure to a 1.439 GHz EMF, as in the case of a 929.2 MHz field, has no promoting effect on rat liver carcinogenesis in the present model.     

cAMP and beta gamma subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells

Mitogen-activated protein kinases (MAPKs) are activated by a variety of extracellular stimuli, including agonists for G protein-coupled receptors. Using transient transfection of COS-7 cells, we have studied the stimulation of a hemagglutinin-tagged p44mapk (p44HA-mapk) by receptors coupled to Gs, Gq, and Gi. Agonists that act via all three G proteins stimulated p44HA-mapk activity. A constitutively activated alpha s mutant, forskolin, and a cAMP analog also increased p44HA-mapk activity, indicating that cAMP in COS-7 cells, in contrast to other cell types, activates the MAPK pathway. Similarly, a constitutively activated alpha q mutant, overexpression of phospholipase C-beta 2, and a phorbol ester also stimulated p44HA-mapk, suggesting that Gq-coupled receptors stimulate the MAPK pathway by increasing phosphatidylinositol turnover and probably stimulating protein kinase C. In COS-7 cells, in contrast to Rat-1 cells, mutationally activated alpha i did not stimulate the MAPK pathway. G protein beta and gamma subunits, overexpressed together, did activate p44HA-mapk; this finding suggests that in COS-7 cells Gi-coupled receptors may stimulate the MAPK pathway through beta gamma. These unexpected results in COS-7 cells show that G proteins and second messengers regulate the MAPK pathway differently in different cell types.     

Two forms of spatial memory: A double dissociation between the parietal cortex and the hippocampus in the rat.

Two variants of a continuous recognition training procedure were designed in order to query 2 forms of spatial memory. A continuous reinforcement condition (reflecting perceptual memory) and a differential reinforcement condition (reflecting episodic-like memory) were used to test rats on a 12-arm radial maze. After total hippocampal lesions, rats demonstrated intact performance on the continuous reinforcement condition, but impaired performance on the differential reinforcement condition. After parietal lesions, rats demonstrated the reverse pattern of performance: impaired performance on the continuous reinforcement condition and intact performance on the differential reinforcement condition. Thus, a double dissociation appears to exist between parietal cortex and hippocampus for the continuous reinforcement condition (reflecting perceptual memory) versus the differential reinforcement condition (reflecting episodic memory) for spatial location information. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A quantitative autoradiographic and electrophysiological study of the reinnervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions

The post-lesion proliferation of contralateral enthorhinal afferents which occurs in response to ipsilateral entorhinal lesions was quantitatively analyzed with autoradiographic and electrophysiological techniques. In both cases, the extent of the crossed projection to the dentate granule cells was quantified on the basis of a contralateral/ipsilateral (C/I) ratio. Autoradiographic measures of grain density in the entorhinal terminal field indicates that the very sparse crossed entorhinal projection in intact animals proliferates approximately 6-fold following unilateral entorhinal lesions (on the basis of an increased C/I ratio of grain density in animals with long standing unilater entorhinal lesions). Furthermore, the total number of grains in the entorhinal terminal zone (obtained by subtracting background from non-terminal regions) also increases approximately 6-fold, indicating that compression of the neuropil cannot be the factor responsible for the increased grain density. These increases in the anatomical extent of the crossed projection as a consequence of unilateral entorhinal lesions are also reflected electrophysiologically. In operated animals, the C/I ratio of the extracellular population EPSP (a measure of the synaptic current generated by the crossed projections) also increase 5-8 fold. In addition, while in normal animals, no population spikes are observed following stimulation of the contralateral entorhinal area (indicating an absence of synchronous grnaule cell discharge in response to contralateral entorhinal input), such population spikes are quite prominent in the reinnervated dentate gyrus, indicating a large increase in the effective synaptic drive of the proliferated crossed projections.     

Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication

The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.     

A comparison between the effects of medial septal lesions and entorhinal cortex lesions on performance of nonspatial working memory tasks and reversal learning

Two studies explored the relationship between cognitions and long-term symptoms in adult child sexual abuse (CSA) survivors. In Study 1, an American sample of 43 survivors completed questionnaires assessing attributional style and dysfunctional beliefs in cognitive themes affected by victimization, as well as measures of posttraumatic symptoms. Survivors' attributions of negative events were more internal, stable, and global than those of 29 comparison subjects without a history of CSA. However, only the globality scale was significantly related with severity of long-term symptoms. High correlations between dysfunctional beliefs concerning safety, trust, esteem, or intimacy, and posttrauma symptoms were found. The latter finding was replicated in Study 2 with a German sample of 35 CSA survivors, even when controlling for frequency of abuse.     

Opposing effects on modulation of angiogenesis by protein kinase C and cAMP-mediated pathways

The role of cAMP-mediated pathway in modulating angiogenesis was investigated. We have shown previously that activators of protein kinase C (PKC) caused a marked increase in angiogenesis, while the specific inhibitor of PKC, Ro 318220 suppressed angiogenesis. Here we show that forskolin, which activates adenylate cyclase and elevates the intracellular levels of cAMP, and the Sp-diastereomer of adenosine cyclic-3',5'-monophosphothioate (Sp-cAMPS), caused a dose-dependent suppression of collagenous protein biosynthesis and angiogenesis in the chick chorioallantoic membrane system (CAM). The opposite modulation of angiogenesis by activators of PKC and elevated cAMP levels was further confirmed by the suppression of 4 beta-phorbol-12-myristate-13-acetate (4 beta-PMA)-stimulated angiogenesis by either forskolin or Sp-cAMPS. On the contrary, the Rp-diastereomer of adenosine cyclic-3',5'-monophosphothioate (Rp-cAMPS), which antagonises endogenous cAMP biochemical actions, had no effect on angiogenesis alone and did not suppress 4 beta-PMA stimulated angiogenesis. However, Rp-cAMPS antagonised the effect of forskolin and Sp-cAMPS on 4 beta-PMA induced angiogenesis. Similar results were obtained in the human umbilical vein endothelial cell tube formation assay. In this system, the PKC inhibitor, Ro 318220, caused a dose-dependent inhibition of 4 beta-PMA reversed this effect. Also, forskolin and Sp-cAMPS caused an inhibition in tube formation. These results indicate that increased levels of intracellular cAMP have a negative effect in normal angiogenesis and cause a large reduction of the promotion of angiogenesis resulting from PKC activation.     

Effects of hippocampal and parietal cortex lesions on memory for egocentric distance and spatial location information in rats

To assess the working memory system for egocentric distance and place information, delayed matching-to-sample (DMTS) go/no-go tasks were run for each rat. To assess the reference memory system, and to serve as a control for nonmemory deficits, successive discrimination go/no-go tasks were then conducted using the same rats. Rats with hippocampal, but not parietal cortex, lesions were impaired relative to controls in the working memory (DMTS) task for both egocentric distance and place information, although the deficit observed in the working memory task for egocentric distance information by rats with hippocampal lesions was mild. Neither hippocampal nor parietal cortex lesioned rats were impaired relative to controls in the reference memory (successive discrimination) task for either cue. The hippocampus appears to be involved in working memory for egocentric distance and in spatial location information, whereas the parietal cortex is not.     

Presynaptic modulation of cholecystokinin release by protein kinase C in the rat hippocampus

The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK-8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC-activating phorbol ester 4beta-phorbol 12,13-dibutyrate (beta-PDBu) dose dependently (5-5,000 nM) increased CCK-8 release in a strictly Ca2+dependent way. This effect was observed only when synaptosomes were stimulated with the K+(A) channel blocker 4-aminopyridine (4-AP; 1 mM) but not with KCl (10-30 mM). The PDBu-induced exocytosis of CCK-8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin-C and was not mimicked by alpha-PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK-8 exocytosis. Beta-PDBu (50-100 nM) also stimulated the 4-AP-evoked Ca2+-dependent release of the classic transmitter GABA, which co-localizes with CCK-8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid did not stimulate, but instead inhibited, both the CCK-8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of co-localizing neurotransmitters via modulation of presynaptic K+ channels in rat hippocampus.     

A visuo-somatomotor pathway through superior parietal cortex in the macaque monkey: cortical connections of areas V6 and V6A

This report addresses the connectivity of the cortex occupying middle to dorsal levels of the anterior bank of the parieto-occipital sulcus in the macaque monkey. We have previously referred to this territory, whose perimeter is roughly circumscribed by the distribution of interhemispheric callosal fibres, as area V6, or the 'V6 complex'. Following injections of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA-HRP) into this region, we examined the laminar organization of labelled cells and axonal terminals to attain indications of relative hierarchical status among the network of connected areas. A notable transition in the laminar patterns of the local, intrinsic connections prompted a sub-designation of the V6 complex itself into two separate areas, V6 and V6A, with area V6A lying dorsal, or dorsomedial to V6 proper. V6 receives ascending input from V2 and V3, ranks equal to V3A and V5, and provides an ascending input to V6A at the level above. V6A is not connected to area V2 and in general is less heavily linked to the earliest visual areas; in other respects, the two parts of the V6 complex share similar spheres of connectivity. These include regions of peripheral representation in prestriate areas V3, V3A and V5, parietal visual areas V5A/MST and 7a, other regions of visuo-somatosensory association cortex within the intraparietal sulcus and on the medial surface of the hemisphere, and the premotor cortex. Subcortical connections include the medial and lateral pulvinar, caudate nucleus, claustrum, middle and deep layers of the superior colliculus and pontine nuclei. From this pattern of connections, it is clear that the V6 complex is heavily engaged in sensory-motor integration. The specific somatotopic locations within sensorimotor cortex that receive this input suggest a role in controlling the trunk and limbs, and outward reaching arm movements. There is a secondary contribution to the brain's complex oculomotor circuitry. That the medial region of the cortex is devoted to tightly interconnected representations of the sensory periphery, both visual and somatotopic-which are routinely stimulated in concert-would appear to be an aspect of the global organization of the cortex which must facilitate multimodal integration.     

Dissecting cAMP binding domain A in the RIalpha subunit of cAMP-dependent protein kinase. Distinct subsites for recognition of cAMP and the catalytic subunit

The two gene-duplicated cAMP binding domains in the regulatory subunits of cAMP dependent protein kinase are each comprised of an A helix, an eight-stranded beta-barrel, and a B and C helix (1). The A domain is required for high affinity binding to C, while the B domain regulates access to the A domain. Using a combination of a yeast two-hybrid screen coupled with deletion analysis, cAMP binding domain A of RI was dissected into two structurally and functionally distinct subsites, one that binds cAMP and another that binds the C subunit. The minimum stable subdomain required for binding to C in the 1-3 micromolar range is composed of residues 94-169, while residues 236-244, mapped to the C helix of cAMP binding domain A, were defined as a second surface necessary for high affinity (5-10 nanomolar) binding to C. This portion of the C helix, due to its position directly between the two subsites, serves as a molecular switch for either a cAMP-bound conformation or a C-bound conformation and can thus modulate interactions of cAMP binding domain A with cAMP, with C, and with cAMP binding domain B.