Ion Channel-Based Reporters for cAMP Detection.

In the last 20 years tremendous progress has been made in the development of single cell cAMP sensors. Sensors are based upon cAMP binding proteins that have been modified to transduce cAMP concentrations into electrical or fluorescent readouts that can be readily detected using patch clamp amplifiers, photomultiplier tubes, or cameras. Here, we describe two complementary approaches for the detection and measurement of cAMP signals near the plasma membrane of cells using cyclic nucleotide (CNG) channel-based probes. These probes take advantage of the ability of CNG channels to transduce small changes in cAMP concentration into ionic flux through channel pores that can be readily detected by measuring Ca2+ and/or Mn2+ influx or by measuring ionic currents.

Chenodeoxycholic and deoxycholic acids induced positive inotropic and negative chronotropic effects on rat heart.

Bile acids are endogenous amphiphilic steroids from the metabolites of cholesterol. Studies showed that they might contribute to the pathogenesis of cardiopathy in cholestatic liver diseases. Chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) is associated with colon cancer, gallstones, and gastrointestinal disorders. However, little information is available regarding their cardiac effects. Here, we reported that CDCA (100 µM) and DCA (100 µM) significantly increased the left ventricular developed pressure of the isolated rat hearts to 122.3 ± 5.6% and 145.1 ± 13.7%, and the maximal rate of the pressure development rising and descending (± dP/dtmax) to 103.4 ± 17.6% and 124.4 ± 37.7% of the basal levels, respectively. They decreased the heart rate and prolonged the RR, QRS, and QT intervals of Langendorff-perfused hearts in a concentration-dependent manner. Moreover, CDCA and DCA increased the developed tension of left ventricular muscle and the cytosolic Ca2+ concentrations in left ventricular myocytes; these functions positively coordinated with their inotropic effects on hearts. Additionally, CDCA (150 µM) and DCA (100 µM) decreased the sinoatrial node beating rate to 80.6 ± 3.0% and 79.7 ± 0.9% of the basal rate (334.2 ± 10.7 bpm), respectively. These results were consistent with their chronotropic effects. In conclusion, CDCA and DCA induced positive inotropic effects by elevating the Ca2+ in left ventricular myocytes. They exerted negative chronotropic effects by lowering the pace of the sinoatrial node in rat heart. These results indicated that the potential role of bile acids in cardiopathy related to cholestasis.

Denatonium enhanced the tone of denuded rat aorta via bitter taste receptor and phosphodiesterase activation.

Bitter taste receptors (Tas2rs) initiate a bitter taste signaling involving the activation of taste-specific G protein gustducin and phosphodiesterases (PDEs); it leads to the decrease of cytosolic level of cyclic adenosine monophosphate (cAMP) in taste cells. Recent studies have identified the expression of Tas2rs in a variety of non-lingual tissues including vascular smooth muscle (VSM), pulmonary smooth muscle and airway smooth muscle. The current study aims to determine the expression of Tas2rs and gustducin in rat aortic smooth muscle tissue and to investigate the effect of Tas2rs agonist denatonium on the tone of isolated denuded aorta rings. Here we reported the expression of six subtypes of Tas2r mRNA and the taste receptor-associated G proteins in endothelium-denuded aorta. Immunostaining experiments showed that the protein of gustducin expressed in vascular smooth muscle cells (VSMCs). Furthermore, denatonium increased the tone of freshly isolated denuded aorta rings in a concentration-dependent manner, and the potentiation effect of denatonium was blocked by a Tas2rs antagonist adenosine 5'-monophosphate (5'-AMP), by the cAMP-hydrolyzing PDE inhibitors, and by a cAMP-synthesizing enzyme activator forskolin, respectively. The blockade of Gßγ signaling did not have a negative impact on the denatonium-induced tonic contractions. These findings suggested that the functional Tas2rs and gustducin are expressed in rat aortic smooth muscle and that denatonium might increase the smooth muscle tone through a Tas2rs signaling pathway involving the activation of PDEs.

Constitutively active PKA regulates neuronal acetylcholine release and contractility of guinea pig urinary bladder smooth muscle.

Autonomic and somatic motor neurons that innervate the urinary bladder and urethra control the highly coordinated functions of the lower urinary tract, the storage, and the emptying of urine. ACh is the primary excitatory neurotransmitter in the bladder. Here, we aimed to determine whether PKA regulates neuronal ACh release and related nerve-evoked detrusor smooth muscle (DSM) contractions in the guinea pig urinary bladder. Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation (EFS)- and carbachol-induced DSM contractions with a combination of pharmacological tools. The colorimetric method was used to measure ACh released by the parasympathetic nerves in DSM isolated strips. The pharmacological inhibition of PKA with H-89 (10 µM) increased the spontaneous phasic contractions, whereas it attenuated the EFS-induced DSM contractions. Intriguingly, H-89 (10 µM) attenuated the (primary) cholinergic component, whereas it simultaneously increased the (secondary) purinergic component of the nerve-evoked contractions in DSM isolated strips. The acetylcholinesterase inhibitor, eserine (10 µM), increased EFS-induced DSM contractions, and the subsequent addition of H-89 attenuated the contractions. H-89 (10 µM) significantly increased DSM phasic contractions induced by the cholinergic agonist carbachol. The inhibition of PKA decreased the neuronal release of ACh in DSM tissues. This study revealed that PKA-mediated signaling pathways differentially regulate nerve-evoked and spontaneous phasic contractions of guinea pig DSM. Constitutively active PKA in the bladder nerves controls synaptic ACh release, thus regulating the nerve-evoked DSM contractions, whereas PKA in DSM cells controls the spontaneous phasic contractility.

BK channel regulation by phosphodiesterase type 1: a novel signaling pathway controlling human detrusor smooth muscle function.

Large-conductance Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) function. We aimed to investigate phosphodiesterase type 1 (PDE1) interactions with BK channels in human DSM to determine the mechanism by which PDE1 regulates human urinary bladder physiology. A combined electrophysiological, functional, and pharmacological approach was applied using human DSM specimens obtained from open bladder surgeries. The perforated whole cell patch-clamp technique was used to record transient BK currents (TBKCs) and the cell membrane potential in freshly isolated human DSM cells in combination with the selective PDE1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX). Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation-induced contractions in human DSM isolated strips. Selective pharmacological inhibition of PDE1 with 8MM-IBMX (10 µM) increased TBKC activity in human DSM cells, which was abolished by subsequent inhibition of protein kinase A (PKA) with H-89 (10 µM). The stimulatory effect of 8MM-IBMX on TBKCs was reversed upon activation of muscarinic acetylcholine receptors with carbachol (1 µM). 8MM-IBMX (10 µM) hyperpolarized the DSM cell membrane potential, an effect blocked by PKA inhibition. 8MM-IBMX significantly decreased spontaneous phasic and nerve-evoked contractions of human DSM isolated strips. The results reveal a novel mechanism that pharmacological inhibition of PDE1 attenuates human DSM excitability and contractility by activating BK channels via a PKA-dependent mechanism. The data also suggest interactions between PDE1 and muscarinic signaling pathways in human DSM. Inhibition of PDE1 can be a novel therapeutic approach for the treatment of overactive bladder associated with detrusor overactivity.

Estimating the magnitude of near-membrane PDE4 activity in living cells.

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 µM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments.

Channel-based reporters for cAMP detection.

In the last 15 years, tremendous progress has been made in the development of single-cell cAMP sensors. Sensors are based upon cAMP-binding proteins that have been modified to transduce cAMP concentrations into electrical or fluorescent readouts that can be readily detected using patch clamp amplifiers, photomultiplier tubes, or cameras. Here we describe two complementary approaches for the detection and measurement of cAMP signals near the plasma membrane of cells. These probes take advantage of the ability of cyclic nucleotide-gated (CNG) channels to transduce small changes in cAMP concentrations into ionic flux through channel pores that can be readily detected by measuring Ca(2+) and/or Mn(2+) influx or by measuring ionic currents.

Constitutive PKA activity is essential for maintaining the excitability and contractility in guinea pig urinary bladder smooth muscle: role of the BK channel.

The elevation of protein kinase A (PKA) activity activates the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in urinary bladder smooth muscle (UBSM) cells and consequently attenuates spontaneous phasic contractions of UBSM. However, the role of constitutive PKA activity in UBSM function has not been studied. Here, we tested the hypothesis that constitutive PKA activity is essential for controlling the excitability and contractility of UBSM. We used patch clamp electrophysiology, line-scanning confocal and ratiometric fluorescence microscopy on freshly isolated guinea pig UBSM cells, and isometric tension recordings on freshly isolated UBSM strips. Pharmacological inhibition of the constitutive PKA activity with H-89 or PKI 14-22 significantly reduced the frequency and amplitude of spontaneous transient BK channel currents (TBKCs) in UBSM cells. Confocal and ratiometric fluorescence microscopy studies revealed that inhibition of constitutive PKA activity with H-89 reduced the frequency and amplitude of the localized Ca(2+) sparks but increased global Ca(2+) levels and the magnitude of Ca(2+) oscillations in UBSM cells. H-89 abolished the spontaneous transient membrane hyperpolarizations and depolarized the membrane potential in UBSM cells. Inhibition of PKA with H-89 or KT-5720 also increased the amplitude and muscle force of UBSM spontaneous phasic contractions. This study reveals the novel concept that constitutive PKA activity is essential for controlling localized Ca(2+) signals generated by intracellular Ca(2+) stores and cytosolic Ca(2+) levels. Furthermore, constitutive PKA activity is critical for mediating the spontaneous TBKCs in UBSM cells, where it plays a key role in regulating spontaneous phasic contractions in UBSM.

BK channel-mediated relaxation of urinary bladder smooth muscle: a novel paradigm for phosphodiesterase type 4 regulation of bladder function.

Elevation of intracellular cAMP and activation of protein kinase A (PKA) lead to activation of large conductance voltage- and Ca(2+)-activated K(+) (BK) channels, thus attenuation of detrusor smooth muscle (DSM) contractility. In this study, we investigated the mechanism by which pharmacological inhibition of cAMP-specific phosphodiesterase 4 (PDE4) with rolipram or Ro-20-1724 (C(15)H(22)N(2)O(3)) suppresses guinea pig DSM excitability and contractility. We used high-speed line-scanning confocal microscopy, ratiometric fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp techniques on freshly isolated DSM cells, along with isometric tension recordings of DSM isolated strips. Rolipram caused an increase in the frequency of Ca(2+) sparks and the spontaneous transient BK currents (TBKCs), hyperpolarized the cell membrane potential (MP), and decreased the intracellular Ca(2+) levels. Blocking BK channels with paxilline reversed the hyperpolarizing effect of rolipram and depolarized the MP back to the control levels. In the presence of H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride], a PKA inhibitor, rolipram did not cause MP hyperpolarization. Rolipram or Ro-20-1724 reduced DSM spontaneous and carbachol-induced phasic contraction amplitude, muscle force, duration, and frequency, and electrical field stimulation-induced contraction amplitude, muscle force, and tone. Paxilline recovered DSM contractility, which was suppressed by pretreatment with PDE4 inhibitors. Rolipram had reduced inhibitory effects on DSM contractility in DSM strips pretreated with paxilline. This study revealed a novel cellular mechanism whereby pharmacological inhibition of PDE4 leads to suppression of guinea pig DSM contractility by increasing the frequency of Ca(2+) sparks and the functionally coupled TBKCs, consequently hyperpolarizing DSM cell MP. Collectively, this decreases the global intracellular Ca(2+) levels and DSM contractility in a BK channel-dependent manner.

Selective inhibition of phosphodiesterase 1 relaxes urinary bladder smooth muscle: role for ryanodine receptor-mediated BK channel activation.

The large conductance voltage- and Ca(2+)-activated K(+) (BK) channel is a major regulator of detrusor smooth muscle (DSM) excitability and contractility. Recently, we showed that nonselective phosphodiesterase (PDE) inhibition reduces guinea pig DSM excitability and contractility by increasing BK channel activity. Here, we investigated how DSM excitability and contractility changes upon selective inhibition of PDE type 1 (PDE1) and the underlying cellular mechanism involving ryanodine receptors (RyRs) and BK channels. PDE1 inhibition with 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX; 10 µM) increased the cAMP levels in guinea pig DSM cells. Patch-clamp experiments on freshly isolated DSM cells showed that 8MM-IBMX increased transient BK currents and the spontaneous transient hyperpolarization (STH) frequency by ∼2.5- and ∼1.8-fold, respectively. 8MM-IBMX hyperpolarized guinea pig and human DSM cell membrane potential and significantly decreased the intracellular Ca(2+) levels in guinea pig DSM cells. Blocking BK channels with 1 µM paxilline or inhibiting RyRs with 30 µM ryanodine abolished the STHs and the 8MM-IBMX inhibitory effects on the DSM cell membrane potential. Isometric DSM tension recordings showed that 8MM-IBMX significantly reduced the spontaneous phasic contraction amplitude, muscle force integral, duration, frequency, and tone of DSM isolated strips. The electrical field stimulation-induced DSM contraction amplitude, muscle force integral, and duration were also attenuated by 10 µM 8MM-IBMX. Blocking BK channels with paxilline abolished the 8MM-IBMX effects on DSM contractions. Our data provide evidence that PDE1 inhibition relaxes DSM by raising cellular cAMP levels and subsequently stimulates RyRs, which leads to BK channel activation, membrane potential hyperpolarization, and decrease in intracellular Ca(2+) levels.

A-kinase anchoring proteins regulate compartmentalized cAMP signaling in airway smooth muscle.

A-kinase anchoring proteins (AKAPs) have emerged as important regulatory molecules that can compartmentalize cAMP signaling transduced by ß2-adrenergic receptors (ß(2)ARs); such compartmentalization ensures speed and fidelity of cAMP signaling and effects on cell function. This study aimed to assess the role of AKAPs in regulating global and compartmentalized ß(2)AR signaling in human airway smooth muscle (ASM). Transcriptome and proteomic analyses were used to characterize AKAP expression in ASM. Stable expression or injection of peptides AKAP-IS or Ht31 was used to disrupt AKAP-PKA interactions, and global and compartmentalized cAMP accumulation stimulated by ß-agonist was assessed by radioimmunoassay and membrane-delineated flow through cyclic nucleotide-gated channels, respectively. ASM expresses multiple AKAP family members, with gravin and ezrin among the most readily detected. AKAP-PKA disruption had minimal effects on whole-cell cAMP accumulation stimulated by ß-agonist (EC(50) and B(max)) concentrations, but significantly increased the duration of plasma membrane-delineated cAMP (τ=251±51 s for scrambled peptide control vs. 399±79 s for Ht31). Direct PKA inhibition eliminated decay of membrane-delineated cAMP levels. AKAPs coordinate compartmentalized cAMP signaling in ASM cells by regulating multiple elements of ß(2)AR-mediated cAMP accumulation, thereby representing a novel target for manipulating ß(2)AR signaling and function in ASM.

All preconditioning-related G protein-coupled receptors can be demonstrated in the rabbit cardiomyocyte.

G protein-coupled receptors for adenosine (A(1), A(3), A(2A), and A(2B)), bradykinin (B(1)) and opioids (δ) are all involved in the mechanism of ischemic preconditioning. Although the heart is comprised of many tissue types, it has been assumed that preconditioning's protective signaling occurs in the cardiomyocyte. We critically tested that hypothesis by testing for the presence of each of these receptors in isolated adult rabbit ventricular myocytes that had been transfected with cyclic nucleotide-gated (CNG) ion channels. Because subsarcolemmal cyclic adenosine monophosphate (cAMP) opens the CNG channels, we could monitor cAMP levels within a single cardiomyocyte by measuring channel current with a patch pipette. The presence of a receptor would be confirmed if we could alter cAMP in the cell with a selective agonist to the receptor being studied. Superfusion with the ß-adrenergic G(s)-coupled receptor agonist isoproterenol (50 nmol/L) transiently increased cAMP levels and, therefore, channel current. Pretreatment with selective agonists to A(1) or A(3) adenosine receptors (ARs) that are G(i)-coupled markedly attenuated the response to isoproterenol, indicating inhibition of adenylyl cyclase by increased G(i) activity. Agonists to bradykinin or δ-opioid receptors also attenuated isoproterenol's response. A(2A)AR and A(2B)AR are G(s)-coupled. The A(2A)AR-selective agonist CGS21680 increased current through CNG channels but only in the presence of phosphodiesterase (PDE) inhibitors, indicating low surface receptor activity and high intracellular PDE activity. As we previously reported, BAY 60-6583, an A(2B)AR-selective agonist which mimics preconditioning's protection in rabbit heart, neither increased nor decreased membrane current in transfected cardiomyocytes, suggesting the absence or a markedly limited number of A(2B)AR in the sarcolemma. However, reverse transcription polymerase chain reaction (RT-PCR) of purified cardiomyocytes yielded an A(2B)AR band, implying that rabbit cardiomyocytes do indeed express A(2B)AR. These data reveal that all receptors reported to be involved in ischemic preconditioning do exist on or within the cardiomyocyte.

A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin.

BACKGROUND AND PURPOSE: A(2B) adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). However, the underlying mechanism(s) and signalling pathway(s) remain undefined. EXPERIMENTAL APPROACH: HEK 293 cells stably transfected with human A(2B) adenosine receptors (HEK-A(2B) ) and isolated adult rabbit cardiomyocytes were used to assay phosphorylation of ERK by Western blot and cation flux through cAMP-gated channels by patch clamp methods. Generation of reactive oxygen species (ROS) by mitochondria was measured with a fluorescent dye. KEY RESULTS: In HEK-A(2B) cells, the selective A(2B) receptor agonist Bay 60-6583 (Bay 60) increased ERK phosphorylation and cAMP levels, detected by current through cAMP-gated ion channels. However, increased cAMP or its downstream target protein kinase A was not involved in ERK phosphorylation. Pertussis toxin (PTX) blocked ERK phosphorylation, suggesting receptor coupling to G(i) or G(o) proteins. Phosphorylation was also blocked by inhibition of PI3K (with wortmannin) or of ERK kinase (MEK1/2, with PD 98059) but not by inhibition of NO synthase (NOS). In cardiomyocytes, Bay 60 did not affect cAMP levels but did block the increased superoxide generation induced by rotenone, a mitochondrial complex I inhibitor. This effect of Bay 60 was inhibited by PD 98059, wortmannin or PTX. Inhibition of NOS blocked superoxide production because NOS is downstream of ERK. CONCLUSION AND IMPLICATIONS: Activation of A(2B) adenosine receptors reduced superoxide generation from mitochondrial complex I through G(i/o) , ERK, PI3K, and NOS, all of which have been implicated in ischaemic preconditioning.

Roles of GRK and PDE4 activities in the regulation of beta2 adrenergic signaling.

An important focus in cell biology is understanding how different feedback mechanisms regulate G protein-coupled receptor systems. Toward this end we investigated the regulation of endogenous beta(2) adrenergic receptors (beta2ARs) and phosphodiesterases (PDEs) by measuring cAMP signals in single HEK-293 cells. We monitored cAMP signals using genetically encoded cyclic nucleotide-gated (CNG) channels. This high resolution approach allowed us to make several observations. (a) Exposure of cells to 1 muM isoproterenol triggered transient increases in cAMP levels near the plasma membrane. Pretreatment of cells with 10 muM rolipram, a PDE4 inhibitor, prevented the decline in the isoproterenol-induced cAMP signals. (b) 1 muM isoproterenol triggered a sustained, twofold increase in phosphodiesterase type 4 (PDE4) activity. (c) The decline in isoproterenol-dependent cAMP levels was not significantly altered by including 20 nM PKI, a PKA inhibitor, or 3 muM 59-74E, a GRK inhibitor, in the pipette solution; however, the decline in the cAMP levels was prevented when both PKI and 59-74E were included in the pipette solution. (d) After an initial 5-min stimulation with isoproterenol and a 5-min washout, little or no recovery of the signal was observed during a second 5-min stimulation with isoproterenol. (e) The amplitude of the signal in response to the second isoproterenol stimulation was not altered when PKI was included in the pipette solution, but was significantly increased when 59-74E was included. Taken together, these data indicate that either GRK-mediated desensitization of beta2ARs or PKA-mediated stimulation of PDE4 activity is sufficient to cause declines in cAMP signals. In addition, the data indicate that GRK-mediated desensitization is primarily responsible for a sustained suppression of beta2AR signaling. To better understand the interplay between receptor desensitization and PDE4 activity in controlling cAMP signals, we developed a mathematical model of this system. Simulations of cAMP signals using this model are consistent with the experimental data and demonstrate the importance of receptor levels, receptor desensitization, basal adenylyl cyclase activity, and regulation of PDE activity in controlling cAMP signals, and hence, on the overall sensitivity of the system.

Cellular mechanisms underlying prostaglandin-induced transient cAMP signals near the plasma membrane of HEK-293 cells.

We have previously used cyclic nucleotide-gated (CNG) channels as sensors to measure cAMP signals in human embryonic kidney (HEK)-293 cells. We found that prostaglandin E(1) (PGE(1)) triggered transient increases in cAMP concentration near the plasma membrane, whereas total cAMP levels rose to a steady plateau over the same time course. In addition, we presented evidence that the decline in the near-membrane cAMP levels was due primarily to a PGE(1)-induced stimulation of phosphodiesterase (PDE) activity, and that the differences between near-membrane and total cAMP levels were largely due to diffusional barriers and differential PDE activity. Here, we examine the mechanisms regulating transient, near-membrane cAMP signals. We observed that 5-min stimulation of HEK-293 cells with prostaglandins triggered a two- to threefold increase in PDE4 activity. Extracellular application of H89 (a PKA inhibitor) inhibited stimulation of PDE4 activity. Similarly, when we used CNG channels to monitor cAMP signals we found that both extracellular and intracellular (via the whole-cell patch pipette) application of H89, or the highly selective PKA inhibitor, PKI, prevented the decline in prostaglandin-induced responses. Following pretreatment with rolipram (a PDE4 inhibitor), H89 had little or no effect on near-membrane or total cAMP levels. Furthermore, disrupting the subcellular localization of PKA with the A-kinase anchoring protein (AKAP) disruptor Ht31 prevented the decline in the transient response. Based on these data we developed a plausible kinetic model that describes prostaglandin-induced cAMP signals. This model has allowed us to quantitatively demonstrate the importance of PKA-mediated stimulation of PDE4 activity in shaping near-membrane cAMP signals.

A functional interaction of sodium and calcium in the regulation of NMDA receptor activity by remote NMDA receptors.

The NMDA receptor is an important subtype glutamate receptor that acts as a nonselective cation channel highly permeable to both calcium (Ca2+) and sodium (Na+). The activation of NMDA receptors produces prolonged increases of intracellular Ca2+ concentration ([Ca2+]i) and thereby triggers downstream signaling pathways involved in the regulation of many physiological and pathophysiological processes. Previous studies have focused on how Ca2+ or Na+ affects NMDA receptor activity in isolation. Specifically, [Ca2+]i increase may downregulate NMDA channels and thus is considered an important negative feedback mechanism controlling NMDA receptor activity, whereas an increase in intracellular Na+ concentration ([Na+]i) may upregulate NMDA channel activity. Thus so that the activity-dependent regulation of NMDA receptors and neuroplasticity may be further understood, a critical question that has to be answered is how an individual NMDA receptor may be regulated when both of these ionic species flow into neurons during the same time period via neighboring activated NMDA receptors. Here we report that the gating of a NMDA channel is regulated by the activation of remote NMDA receptors via a functional Na+-Ca2+ interaction and that during the activation of NMDA receptors Na+ influx potentiates Ca2+ influx on one hand and overcomes Ca2+-induced inhibition of NMDA channel gating on the other hand. Furthermore, we have identified that a critical increase (5 +/- 1 mM) in [Na+]i is required to mask the effects of Ca2+ on NMDA channel gating in cultured hippocampal neurons. Thus cross talk between NMDA receptors mediated by a functional Na+-Ca2+ interaction is a novel mechanism regulating NMDA receptor activity.