It has been demonstrated that cAMP levels in the pyramidal neurons of the medial prefrontal cortex (mPFC) are altered in animal models of various neuropsychiatric disorders (1-3). cAMP may modify neuronal activity due to its role as a component in many of the transduction pathways activated by metabotropic receptors (4).

G-protein inward rectifier K+ (GIRK) channels are widely distributed in the cerebral cortex (5). They conduct K+ ions towards the interior of cells more easily than towards the exterior. Although GIRK channels have low open probability at rest, they play a role in tuning the resting membrane potential in mPFC pyramidal neurons (6-10).

GIRK channels possess protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites (6, 11, 12) thus making them susceptible to transduction systems activated by metabotropic receptors. Two types of GIRK channels, cardiac and neuronal, have been identified (6, 13). PKC activation leads to the inhibition of both types of GIRK channel-mediated currents (10, 14-17). In contrast, activation of the cAMP/PKA-dependent transduction system increases the activity of cardiac-type GIRK channels (12, 18, 19). The effects of cAMP/PKA on neuronal GIRK channel currents have not been thoroughly tested. In this study, we investigated the effects of cAMP on neuronal-type GIRK channels in mPFC pyramidal neurons.


MATERIALS AND METHODS
The experimental procedures used in this study were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Three-week-old male Wistar rats (WAG Cmd) were decapitated and their brains were placed in a cold (4°C) oxygenated solution of (mM): sucrose (234), KCl (2.5), NaH2PO4 (1), glucose (11), MgSO4 (4), HEPES-Cl [N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid)] (15), and CaCl2 (0.1). In all solutions applied in this study, the pH was adjusted to 7.4 and the osmolality to 310 mOsm/kg H2O. Coronal slices were prepared from cerebral prefrontal tissue using a vibrotome (Vibrotome 1000, Pelco International, CA).

Voltage-clamp recordings from dissociated neurons

The slices were stored at room temperature (21–22°C) in a solution containing (mM): NaCl (118), KCl (5.3), CaCl2 (1.8), MgSO4 (0.4), NaHCO3 (26), and NaH2PO4 (0.9) bubbled with 95% O2 and 5% CO2. Regions of the slices that corresponded to the mPFC in rats were dissected (2.2–3.5 mm anterior to Bregma, 3–5 mm below the upper cortical surface, and 0.6–0.9 mm from the midline (20)), then transferred to a solution bubbled with O2 that contained (mM): NaCl (135), HEPES-Cl (10), KCl (5), MgSO4 (1) and glucose (10), and incubated at 32°C with protease type XIV (1 mg/ml, Sigma Aldrich) for 18 min. Next, parts of the slices were mechanically dispersed. The suspension of neurons was transferred to a recording chamber placed on the stage of a Nikon inverted microscope (Nikon Instech Co., Ltd., Kawasaki, Kanagawa, Japan). Cells were identified under Hoffman optics (magnification 400x) according to criteria described previously (see (10) and Fig. 1B in (21)) and by other authors (22-25)). The pyramidal neurons that were selected for channel current recordings had a smooth, three-dimensional appearance, triangular shape, residual apical and basal dendrites, and a short axon at the base (10, 21).

During channel current recordings the recording chamber was perfused with a solution containing (mM): KCl (145), CaCl2 (2), MgCl2 (2), glucose (15), HEPES-Cl (10), TTX (Tetrodotoxin citrate, 0.001), and LaCl3 (0.003). PH and osmolality of the solution were 7.4 and 320 mOsm/kg, respectively. The membrane potential of the tested cells was clamped at approximately 0 mV in this external solution. The pipette solution contained (mM): potassium acetate (120), HEPES-Cl (10), MgCl2 (2), CaCl2 (2) and blockers of voltage-gated Na+ channels TTX (0.001), K+ channels (tetraethylammonium chloride, 10; 4-aminopyridine, 5) and Ih channels (ZD7288, 50 µM), pH 7.4 and osmolality 280 mOsm/kg. The reversal potential for K+ ions was close to 0 mV. Between the channel current recordings the extracellular solution contained (mM): NaCl (145), CaCl2 (2), MgCl2 (2), glucose (10), and HEPES-Cl (10). Ph and osmolality of the solution were 7.4 and 320 mOsm/kg, respectively.

Channel currents were recorded in the cell-attached configuration using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA) at room temperature (21–23°C) using the pClamp 10 software package. The data were digitised at 20 kHz through a 4-pole low-pass Bessel filter (2 kHz) and stored on a PC computer. The data were collected for 60 s under control conditions as well as during application and washout of the tested compounds. The channel current traces were idealised with pClamp 10.0. The channel conductance, channel open probability and mean open time were measured. Single channel conductance was calculated as the slope of the best-fit line to the I-V plot in the linear regimen: Q=I/V, where Q is the channel conductance, I is the maximum channel current amplitude, and V is the membrane potential. The channel open probability (Po) was calculated as follows: Po=topen/ttotal, where topen is the total channel open time and ttotal is the analysis time. All potentials were expressed in terms of the cytoplasmic side of the patch relative to the patch pipette. The data shown in the figures were further digitised at 1 kHz.

The recording chamber was washed out (2 ml/min) with extracellular solution free of test compounds. At the same time, the tested cell was continuously washed out using tubing placed in close proximity (inside diameter 250 µm, EVH-9, Bio-Logic Science Instruments, France). The tubing delivered either the extracellular control solution or the extracellular control solution plus the test compound to the neuron.

The pipettes for voltage clamp recordings were fabricated from borosilicate glass capillaries (O.D., 1.5 mm, I.D. 0.86 mm; Harvard Apparatus, Edenbridge, UK) using a P-87 puller (Sutter Instruments, Inc., CA), and then fire-polished. The pipette resistance in the bath was 7–8 M.

The compounds ZD7288 (4-(N-ethyl-N-phenylamino)-1,2 dimethyl-6-(methylamino) pyrimidinium chloride), H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) and 8CPT-2Me-cAMP 8-(4-chlorophenylthio)-2’-O-methyladenosine-3’,5’-cyclic monophosphate sodium salt were dissolved in distilled water and stored as 10 µl stock solutions until use. 8-Br-cAMP (8-bromoadenosine-3’,5’-cyclic monophosphate) was dissolved in distilled water and stored as 100 µl stock solution. Forskolin and KT 5720 ((9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3’,2’,1’-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester) were dissolved in DMSO and stored as 10 µl stock solutions. The compounds were stored at –20°C for up to 1 month.

The majority of the chemical compounds were purchased from Sigma Aldrich. H-89 and KT 5720 were purchased from Tocris Bioscience (UK), forskolin was purchased from Ascent Scientific (UK).

Statistical analysis

All results presented throughout the paper and in the figures are shown as means ± standard errors of the mean (S.E.M.). Either the Friedman test (non-parametric repeated measures ANOVA) followed by the post-hoc Dunn test or the analysis of variance (ANOVA) followed by the Tukey-Kramer post-hoc test were used to analyse repeated measurements. GraphPad InStat software v3.06 (GraphPad Software, Inc., CA) was used for statistical analyses.


RESULTS
This study included only inwardly rectifying channel currents (n=86) with sparse resting activity (10) (Fig. 1A). The currents had a mean conductance of 31.9±1.5 pS, mean current amplitude of –2.21±0.12 pA, mean open time of 0.55±0.02 ms and mean Po of 2.0±0.17x10–3 at –75 mV (n=86). These features were identical to the properties of the GIRK channel currents recorded in the soma of mPFC pyramidal neurons (10), in the soma and dendrites of hippocampal pyramidal neurons (26) and in cardiac myocytes (27). Additionally, it was previously demonstrated that these channel currents were blocked by tertiapine and activated by baclofen (10). Therefore, it was assumed that the inward rectifier channel current we observed was likely mediated by the GIRK-like channels. During recordings also other types of potassium conductances were observed, including inward rectifier channel currents with high conductance and open probability that were probably mediated by Kir 2.x channels and various types of leak K+ channel currents. These conductances were described previously (10).

Fig. 1. Effects of cAMP on GIRK-like channel currents in mPFC pyramidal neurons (* - p<0.05, ** - p<0.01, ns - not significant). [A] (a) Channel currents recorded at different membrane potentials. (b) The segment of the trace recorded at –50 mV is extended and shown with an overlapping idealised trace generated by pClamp 10.0. c. The I/V relationship of the channel currents. [A] Mean open probability (Po) of the channel current before and during forskolin (10 µM, a) and 8-Br-cAMP (100 µM, b) application, and after washout of the compounds. [B] Mean open probability of channel current before and during KT5720 (0.5 µM) application and after washout (a), and before and during H-89 (10 µM) application and after washout (b). [C] Mean open probability of channel current before and during KT5720 (0.5 µM) alone and together with the application of forskolin (10 µM) (a). Mean open probability of channel current before, during and after washout of 8CPT-2Me-cAMP (10 µM) (b).

In order to test whether cAMP modulates this GIRK-like channel current, forskolin (10 µM), a membrane-permeable activator of adenylyl cyclase that augments cytosolic cAMP levels, was applied. Forskolin administered for 9 min significantly decreased GIRK-like channel activity. Po decreased by 61% from the control, from 2.3±0.4 x 10–3 to 0.9±0.2 x 10–3 (n=13, p<0.0042, Friedman test Fr =10.9 followed by the Dunn test, p<0.01). After 12 min of forskolin washout, the Po of the GIRK-like channel current was 1.6±0.9 x 10–3 (Dunn test, p>0.05, Fig. 1Ba). To validate this effect, a membrane-permeable cAMP analogue, 8-Br-cAMP (100 µM), was applied for 9 min. 8-Br-cAMP decreased the Po of the GIRK-like channels by 83%, from 1.8±0.4 x 10–3 to 0.3±0.1 x 10–3(n=7, p=0.0004, ANOVA F(2, 20) =16.58, Tukey-Kramer post-hoc test p<0.001). Activity did not recover after the 9 min washout (Po =0.4±0.1 x 10–3, Tukey-Kramer post-hoc test p>0.05, Fig. 1Bb).

The obtained results indicate that cAMP leads to inhibition of the GIRK-like channel current.

We then attempted to assess the molecular mechanism linking cAMP to the GIRK channels. PKA belongs to the cellular effectors of cAMP. The application of the membrane-permeable and irreversible PKA inhibitor KT5720 (0.5 µM) significantly increased the Po of the tested channels. Po increased from 2.1±0.7 x 10–3 (control) to 2.9±1.2 x 10–3 after 9 min of KT5720 application (n=8, p=0.02, ANOVA(2, 23) =4.98, Tukey-Kramer test p<0.05). The effect was not abolished after the 9 min washout (n=8, Po=3.0±0.8 x 10–3, Tukey-Kramer test, p>0.05, Fig. 1Ca). To confirm this finding, another membrane-permeable PKA inhibitor, H-89, was applied at concentrations of 5 and 10 µM. H-89 at 5 µM did not affect the Po of the GIRK-like channels. In this case, Po was 2.2±0.6 x 10–3 in control conditions, 2.3±0.7 x 10–3 after 6 min of H-89 application and 2.4±0.6 x 10–3 after 6 min of washout (n=10, p>0.05, Friedman test Fr =1.6). However, the application of H-89 at a higher concentration (10 µM) significantly increased GIRK-like channel activity. The Po was 2.4±0.55 x 10–3 under control conditions and 4.2±1.25 x 10–3 after 9 min of H-89 application (Friedman test, n=6, p=0.029 Fr=7, Dunn test p<0.05). Activity partially recovered after a 15 min washout (3.3±0.98 x 10–3, n=6, Dunn test P>0.05, Fig. 1Cb). These data indicate that PKA can modulate the activity of the GIRK channels.

Therefore, we tried to establish whether GIRK-like channel activity inhibition by cAMP is abolished by a prior PKA blockade. First, the irreversible kinase A inhibitor KT5720 (0.5 µM) was applied for 6 min. As expected, GIRK-like channel activity increased after PKA inhibition. Po rose from 1.3±0.18 x 10–3 (control) to 2.3±0.4 x 10–3 after 9 min of KT5720 application (n=5, ANOVA(2, 14), p=0.0176, Tukey-Kramer post test, p<0.05). After application of the PKA inhibitor alone, we delivered KT5720 together with forskolin (adenylyl cyclase activator) for 9 min. The application of forskolin, despite PKA inhibition, still decreased Po by 52% to 1.2±0.3 x 10–3 (n=5, Tukey-Kramer post test, p<0.05, Fig. 1Da). This finding suggested that GIRK-like channel current activity might be inhibited by cAMP also in a PKA-independent manner.

Another pathway by which cAMP may regulate the activity of the ionic channels involves the activation of a guanine nucleotide exchange factor, Epac (28). While there are no known inhibitors of Epac, activators are available. The effect of a specific activator of Epac, 8CPT-2Me-cAMP at 10 µM, on GIRK-like channel activity was tested. After 6 min of Epac activator delivery, Po decreased from 2.83±1.2 x 10–3 to 1.61±0.47 x 10–3 and subsequently recovered after 15 min of washout (n=5, 2.4±0.52, Friedman test, p=0.0085, Fr =8.4, Fig. 1Db). Therefore, Epac may be a potential candidate for mediating the effects of cAMP on GIRK-like channels in mPFC pyramidal neurons.


DISCUSSION
The purpose of this study was to clarify the effect of the intracellular second messenger, cAMP, on GIRK-like channel currents in mPFC pyramidal neurons.

We found that GIRK-like channel activity was inhibited during application of adenylyl cyclase activator, forskolin, or membrane permeable cAMP analogue, 8-bromo-cAMP. Both procedures increased the concentration of cAMP in the cytoplasm and could potentially activate protein kinase A. The application of the PKA inhibitors KT5720 and H-89 led to an increase in GIRK-like channel current activity. These results strongly suggest that the cAMP/PKA system in neurons modulates GIRK-like channel current activity.

Despite application of the PKA inhibitor (KT5720, 0.5 µM), the activation of adenylyl cyclase by forskolin still led to an inhibition of GIRK-like channel current activity. This result may be interpreted as insufficient inhibition of PKA by KT5720, as other authors applied KT5720 in concentrations ranging from 0.1 µM to a few micromoles in order to fully suppress PKA activity (29-32). It may also indicate that another transduction pathway, independent of PKA, is activated by cAMP. It was demonstrated that cAMP activates another cAMP-dependent protein, i.e. an exchange protein directly activated by cAMP (Epac). Epac transduces diverse cellular actions of the cAMP independently of the PKA (28). The results of our study indicate that the application of a specific activator of Epac, 8CPT-2Me-cAMP, also inhibits GIRK-like channel current activity.

The obtained results demonstrate that cAMP inhibits GIRK-like channel currents and that the inhibitory effect is presumably mediated by both PKA and Epac.

Previous studies have shown that activation of the cAMP/PKA transduction system increased the open probability of cardiac-type GIRK channels (12, 18, 19). Cardiac and neuronal GIRK channels have different structures. The neuronal channels are heteromultimers composed of GIRK1 and GIRK2 subunits, while the cardiac types are composed of GIRK1 and GIRK4 subunits (6, 13). Therefore, differences in channel structure might be responsible for the difference in their susceptibility to cAMP.

The working memory is a system for temporarily storing and managing information required to carry out learning, reasoning and comprehension (33). The proper functioning of the working memory is compromised during metabolic encephalopathies (34), mental (35) and neurodegenerative diseases (36). Prolonged depolarisation and the resulting persistent activity of the mPFC pyramidal neurons are both considered to be an electrophysiological substrate of the working memory (37). At present it is unclear how the cAMP-linked transduction systems affect the working memory. Aujla and Beninger (38) found that the cAMP/PKA transduction system supports the working memory; in contrast, Taylor et al. (39) suggested that this transduction system impairs the working memory.

Our results suggest that an increase in the concentration of cAMP leads to a decrease in GIRK-like channel activity. In turn, inhibition of the GIRK-like channels can induce mPFC pyramidal neuron depolarisation (10) similarly to the inhibition of other types of potassium channels (40). There are several G-protein-coupled receptors which control cellular function by adenylyl cyclase (41-46). Activation of these receptors may thus reinforce prolonged depolarisation and sustained activity in mPFC pyramidal neurons. This mechanism may promote the working memory.

Acknowledgements: This study was supported by the Ministry of Science and Higher Education (Warsaw, Poland) grant no. NN401 076537 and the Ministry of Science and Higher Education (Warsaw, Poland) grant no. NN401 030037.

Conflict of interests: None declared.


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