Debbie Willoughby

An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real‐time measurements of cAMP using mutant cyclic nucleotide‐gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A‐kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane‐associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co‐immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin‐associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.

Live-cell imaging of cAMP dynamics.

Spatial and temporal compartmentalization of cAMP (and its target proteins) is central to the ability of this second messenger to govern cellular activity over timescales ranging from milliseconds to several hours. Recent years have witnessed a burgeoning of methodologies that enable researchers to directly monitor rapid subcellular cAMP dynamics, which are unobtainable by traditional cAMP assays. In this review, we examine cAMP biosensors that are currently available for measuring cAMP at the single-cell level, compare their various operating principles and discuss their applications.

Ca2+ stimulation of adenylyl cyclase generates dynamic oscillations in cyclic AMP

The spatial and temporal complexity of Ca2+ signalling is central to the regulation of a diverse range of cellular processes. The decoding of dynamic Ca2+ signals is, in part, mediated by the ability of Ca2+ to regulate other second messengers, including cyclic AMP (cAMP). A number of kinetic models (including our own) predict that interdependent Ca2+ and cAMP oscillations can be generated. A previous study in Xenopus neurons illustrated prolonged, low-frequency cAMP oscillations during bursts of Ca2+ transients. However, the detection of more dynamic Ca2+ driven changes in cAMP has, until recently, been limited by the availability of suitable cAMP probes with high temporal resolution. We have used a newly developed FRET-based cAMP indicator comprised of the cAMP binding domain of Epac-1 to examine interplay between Ca2+ and cAMP dynamics. This probe was recently used in excitable cells to reveal an inverse relationship between cAMP and Ca2+ oscillations as a consequence of Ca2+-dependent activation of phosphodiesterase 1 (PDE1). Here, we have used human embryonic kidney (HEK293) cells expressing the type 8 adenylyl cyclase (AC8) to examine whether dynamic Ca2+ changes can mediate phasic cAMP oscillations as a consequence of Ca2+-stimulated AC activity. During artificial or agonist-induced Ca2+ oscillations we detected fast, periodic changes in cAMP that depended upon Ca2+ stimulation of AC8 with subsequent PKA-mediated phosphodiesterase 4 (PDE4) activity. Carbachol (10 μM) evoked cAMP transients with a peak frequency of ∼3 minute-1, demonstrating phasic oscillations in cAMP and Ca2+ in response to physiological stimuli. Furthermore, by imposing a range of Ca2+-oscillation frequencies, we demonstrate that AC8 acts as a low-pass filter for high-frequency Ca2+ events, enhancing the regulatory options available to this signalling pathway.

Direct Binding Between Orai1 and AC8 Mediates Dynamic Interplay Between Ca2+ and cAMP Signaling

A signaling complex enables the compartmentalized regulation of cyclic AMP signaling by calcium entering through a specific channel. Bound to Signal in Close Quarters Interplay between the calcium and the cyclic adenosine monophosphate (cAMP) signaling pathways is crucial to numerous physiological events. Although membrane-bound calcium-sensitive adenylyl cyclases (ACs) are sensitive to submicromolar concentrations of calcium in vitro, in cells they are highly selective in responding to store-operated calcium (SOC) entry rather than to calcium released from intracellular stores or entering the cell through ionophores. Here, Willoughby et al. used a combination of live-cell imaging techniques and biochemical approaches to resolve this conundrum and showed that AC8, which is stimulated by calcium-bound calmodulin, forms a direct protein-protein interaction with Orai1, the pore-forming component of the channel that mediates SOC entry. The existence of AC8 in a complex with SOC channels provides a mechanism for the compartmentalized regulation of cAMP signaling by specific subcellular calcium signals. The interplay between calcium ion (Ca2+) and cyclic adenosine monophosphate (cAMP) signaling underlies crucial aspects of cell homeostasis. The membrane-bound Ca2+-regulated adenylyl cyclases (ACs) are pivotal points of this integration. These enzymes display high selectivity for Ca2+ entry arising from the activation of store-operated Ca2+ (SOC) channels, and they have been proposed to functionally colocalize with SOC channels to reinforce crosstalk between the two signaling pathways. Using a multidisciplinary approach, we have identified a direct interaction between the amino termini of Ca2+-stimulated AC8 and Orai1, the pore component of SOC channels. High-resolution biosensors targeted to the AC8 and Orai1 microdomains revealed that this protein-protein interaction is responsible for coordinating subcellular changes in both Ca2+ and cAMP. The demonstration that Orai1 functions as an integral component of a highly organized signaling complex to coordinate Ca2+ and cAMP signals underscores how SOC channels can be recruited to maximize the efficiency of the interplay between these two ubiquitous signaling pathways.

Depolarization‐induced pH microdomains and their relationship to calcium transients in isolated snail neurones

Neuronal electrical activity causes only modest changes in global intracellular pH (pHi). We have measured regional pHi differences in isolated patch‐clamped neurones during depolarization, using confocal imaging of 8‐hydroxypyrene‐1,3,6‐trisulfonic acid (HPTS) fluorescence. The pHi shifts in the soma were as expected; however, substantially larger shifts occurred in other regions. These regional differences were still observed in the presence of CO2‐HCO3−, they decayed over many seconds and were associated with changes in calcium concentration. Lamellipodial HPTS fluorescence fell by 8.7 ± 1.3 % (n= 9; ∼0.1 pH unit acidification) following a 1 s depolarization to 0 mV; this was more than 4‐fold greater than the relative shift seen in the soma. Depolarization to +40 mV for 1 s caused a 46.7 ± 7.0 % increase (n= 11; ∼0.4 pH unit alkalinization) in HPTS fluorescence in the lamellipodia, more than 6‐fold that seen in the soma. Application of 5 % CO2‐20 mm HCO3− did not significantly reduce the size of the +40 mV‐evoked local pH shifts despite carbonic anhydrase activity. The pHi gradient between regions ∼50 μm apart, resulting from acid shifts, took 10.3 ± 3.1 s (n= 6) to decay by 50 %, whereas the pHi gradient resulting from alkaline shifts took only 3.7 ± 1.4 s (n= 12) to decay by 50 %. The regional rates of acidification and calcium recovery were closely related, suggesting that the acidic pH microdomains resulted from Ca2+‐H+ pump activity. The alkaline pH microdomains were blocked by zinc and resulted from proton channel opening. It is likely that the microdomains result from transmembrane acid fluxes in areas with different surface area to volume ratios. Such neuronal pH microdomains are likely to have consequences for local receptor, channel and enzyme function in restricted regions.

Small Molecule AKAP-Protein Kinase A (PKA) Interaction Disruptors That Activate PKA Interfere with Compartmentalized cAMP Signaling in Cardiac Myocytes

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

AKAP79/150 Interacts with AC8 and Regulates Ca2+-dependent cAMP Synthesis in Pancreatic and Neuronal Systems

Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca2+. However, whether AKAPs play a role in the control of AC activity by Ca2+ is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca2+-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca2+ events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca2+-stimulated cAMP production.

Palmitoylation Targets AKAP79 Protein to Lipid Rafts and Promotes Its Regulation of Calcium-sensitive Adenylyl Cyclase Type 8

PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca2+ entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE. In this study, we have addressed the role of palmitoylation of AKAP79 using a combination of pharmacological, mutagenesis, and cell biological approaches. We reveal that AKAP79 is palmitoylated via two cysteines in its N-terminal region. This palmitoylation plays a key role in targeting the AKAP to lipid rafts in HEK-293 cells. Mutation of the two critical cysteines results in exclusion of AKAP79 from lipid rafts and alterations in its membrane diffusion behavior. This is accompanied by a loss of the ability of AKAP79 to regulate SOCE-dependent AC8 activity in intact cells and decreased PKA-dependent phosphorylation of raft proteins, including AC8. We conclude that palmitoylation plays a key role in the targeting and action of AKAP79. This novel property of AKAP79 adds an unexpected regulatory and targeting option for AKAPs, which may be exploited in the cellular context.

Electrically evoked dendritic pH transients in rat cerebellar Purkinje cells

Our aim was to test the hypothesis that depolarization‐induced intracellular pH (pHi) shifts in restricted regions (dendrites) of mammalian neurones might be larger and faster than those previously reported from the cell soma. We used confocal imaging of the pH‐sensitive dye, HPTS, to measure pH changes in both the soma and dendrites of whole‐cell patch‐clamped rat cerebellar Purkinje cells. In the absence of added CO2‐HCO3−, depolarization to +20 mV for 1 s caused large (≈0.14 pH units) and fast dendritic acid shifts, whilst the somatic acidifications were significantly smaller (≈0.06 pH units) and slower. The pHi shifts were smaller in the presence of 5 % CO2‐25 mm HCO3−‐buffered saline (≈0.08 pH units in the dendrites and ≈0.03 pH units in the soma), although a clear spatiotemporal heterogeneity remained. Acetazolamide (50 μM) doubled the size of the dendritic acid shifts in the presence of CO2‐HCO3−, indicating carbonic anhydrase activity. Removal of extracellular calcium or addition of the calcium channel blocker lanthanum (0.5 mm) inhibited the depolarization‐evoked acid shifts. We investigated more physiological pHi changes by evoking modest bursts of action potentials (≈10 s duration) in CO2‐HCO3−‐buffered saline. Such neuronal firing induced an acidification of ≈0.11 pH units in the fine dendritic regions, but only ≈0.03 pH units in the soma. There was considerable variation in the size of the pHi shifts between cells, with dendritic acid shifts as large as 0.2‐0.3 pH units following a 10 s burst of action potentials in some Purkinje cells. We postulate that these large dendritic pHi changes (pH microdomains) might act as important signals in synaptic function.

Capacitative Ca2+ Entry via Orai1 and Stromal Interacting Molecule 1 (STIM1) Regulates Adenylyl Cyclase Type 8

Capacitative Ca2+ entry (CCE), which occurs through the plasma membrane as a result of Ca2+ store depletion, is mediated by stromal interacting molecule 1 (STIM1), a sensor of intracellular Ca2+ store content, and the pore-forming component Orai1. However, additional factors, such as C-type transient receptor potential (TRPC) channels, may also participate in the CCE apparatus. To explore whether the store-dependent Ca2+ entry reconstituted by coexpression of Orai1 and STIM1 has the functional properties of CCE, we used the Ca2+-calmodulin stimulated adenylyl cyclase type 8 (AC8), which responds selectively to CCE, whereas other modes of Ca2+ entry, including those activated by arachidonate and the ionophore ionomycin, are ineffective. In addition, the Ca2+ entry mediated by previous CCE candidates, diacylglycerol-activated TRPC channels, does not activate AC8. Here, we expressed Orai1 and STIM1 in HEK293 cells and saw a robust increment in CCE, and a proportional increase in CCE-stimulated AC8 activity. Inhibitors of the CCE assembly process ablated the effects on cyclase activity in both AC8-overexpressing HEK293 cells and insulin-secreting MIN6 cells endogenously expressing Ca2+-sensitive AC isoforms. AC8 is believed to be closely associated with the source of CCE; indeed, not only were AC8, Orai1, and STIM1 colocalized at the plasma membrane but also all three proteins occurred in lipid rafts. Together, our data indicate that Orai1 and STIM1 can be integral components of the cAMP and CCE microdomain associated with adenylyl cyclase type 8.