PKA-mediated inhibition of a novel K⁺ channel underlies the slow after-hyperpolarization in enteric AH neurons

Abstract

Postspike after-hyperpolarizations (AHPs) control the excitability of neurons and are important in shaping firing patterns. The duration of some of these events extends to tens of seconds and they can render neurons inexcitable for much of their time course. While consensus is strong that the medium duration (< 1 s AHPs are mediated by the opening of small conductance Ca²⁺-activated K⁺ channels, the K⁺ channels mediating slow AHPs (> 5 s in a subset of enteric (AH) neurons) have an intermediate unit conductance (IK_Ca). Using whole-cell and excised-patch recording, we have demonstrated that the cAMP-protein kinase A (PKA) pathway regulates the activity of these channels. In whole-cell mode, forskolin (0.003–1 μm) inhibited the current underlying the slow AHP (I_sAHP) by 90 %, and this was partially sensitive to inhibition of PKA with internal Rp-cAMPS (500 μm). Rp-cAMPS alone increased the current following break-in and caused a 20 mV hyperpolarization, suggesting that PKA maintains slow AHP channels in the closed state. Internal perfusion of the inhibitory peptide PKI_5–24 slightly increased the I_sAHP and opposed the inhibitory action of forskolin. Internal perfusion of the catalytic subunit of PKA (PKA_cat) suppressed the I_sAHP by 50 % without affecting membrane potential or action potential configuration. In inside-out patches containing IK_Ca-like channels, PKA_cat decreased the open probability of IK_Ca-like channels while alkaline phosphatase activated them. These results suggest that the IK_Ca-like channels that underlie the slow AHP in myenteric AH neurons are subject to inhibition by PKA-dependent phosphorylation and that PKA plays an integral role in their gating.

A wide variety of neurons generate prolonged (> 2 s) post-spike after-hyperpolarizations (slow AHPs) including enteric AH neurons (Nishi & North, 1973; Hirst et al. 1974; Wood & Mayer, 1979) and hippocampal pyramidal neurons (Zhang et al. 1994; Sah & Bekkers, 1996). These events are important in the overall postsynaptic integration of inputs and for controlling neuronal excitability and firing patterns. Slow AHPs have generally been attributed to increases in Ca²⁺-activated K⁺ conductances. In some cases, postspike after-hyperpolarizations may be generated following activation of the Na⁺-K⁺-ATPase by the accumulation of Na⁺ during tetanic spiking (Cherubini & Lanfumey, 1987; Parker et al. 1996). Unlike slow AHPs, however, such events are not associated with any decrease in the input resistance.

We have recently described and characterized in enteric AH neurons a K⁺ channel with properties that make it a strong candidate to be the K⁺ channel that underlies the slow AHP (K_sAHP channel). This includes a pharmacological resistance to apamin and to TEA and a dependence on submicromolar internal [Ca²⁺] for activation (Vogalis et al. 2002a). The channel has an intermediate unit conductance and we have therefore referred to it as an IK_Ca-like channel, although none of the Ca²⁺-activated K⁺ (K_Ca) channels that have been cloned thus far, including the IK₁ (or SK4) K_Ca channels, have properties that fully match those of the K_sAHP channels in AH neurons (Vergara et al. 1998; Bond et al. 1999). For example, IK_Ca-like channels in enteric AH neurons are about 100-fold less sensitive to charybdotoxin and clotrimazole (Vogalis et al. 2002c), drugs that block IK_Ca channels at tens of nanomolar concentrations (Ishii et al. 1997). Similarly, the whole-cell currents underlying the slow AHPs in hippocampal neurons require micromolar levels of clotrimazole for complete block (Shah et al. 2001).

Despite the lack of specific pharmacological inhibitors of K_sAHP channels, a hallmark that distinguishes the macroscopic currents generated by the opening of these channels (I_sAHP) in many types of neuron is that the current is potently inhibited by elevation of intracellular cAMP (Pedarzani & Storm, 1995; Erdemli et al. 1998; Lancaster & Batchelor, 2000). However, it is unclear whether this is the result of alterations in Ca²⁺ entry and/or Ca²⁺ release (Torres et al. 1996), both of which are involved in the generation of slow AHPs in central and enteric neurons (North, 1973; Sah, 1996; Vogalis et al. 2001), or whether cAMP directly, or through downstream enzymatic mediators, acts on the K_sAHP channels to decrease their open probability (P_o; Pedarzani & Storm 1993, 1995). Myenteric neurons are capable of generating robust cAMP signals in response to a range of agonists (Xia et al. 1991; Liu et al. 1999), and this is associated with suppression of the slow AHP and an increase in the excitability of AH neurons. This particular response is elicited by a range of neurotransmitters acting through G-protein-coupled receptors and inflammatory agents including substance P and histamine (see Furness et al. 1998), although the involvement of the cAMP-protein kinase A (PKA) pathway has not been determined.

In the present study we obtained cell-attached, whole-cell and excised-patch recordings of putative K_sAHP channels from AH neurons in intact myenteric ganglia, and subjected them to agents that are known to act on the cAMP-PKA pathway, to determine whether their activity is dependent on this important intracellular transduction pathway.

METHODS

Preparation of intact myenteric plexus for in situ patch clamping

We recorded from myenteric neurons in intact ganglia from the guinea-pig duodenum, as described previously (Kunze et al. 2000). In summary, guinea-pigs of either gender (inbred Hartley strain, colony of the Department of Anatomy and Cell Biology at the University of Melbourne) were killed humanely by cervical dislocation, followed by exsanguination, as approved by the Animal Experimentation Ethics Committee of the University of Melbourne. The abdominal cavity was opened and a segment of duodenum (3–4 cm distal to the pylorus) was removed and placed in pre-oxygenated Krebs solution containing nicardipine (1 μm). The segment was then cut open longitudinally and pinned out in a dissecting dish, mucosa uppermost. The mucosa, submucosa and circular muscle layer were then removed by sharp dissection with the aid of a binocular microscope. The resultant longitudinal muscle with the attached myenteric plexus (LMMP preparation) was transferred to a tissue chamber and pinned out with the plexus uppermost, and protease (0.01 %)-containing Krebs solution was perfused through the chamber for 20 min. The enzyme was then washed out with enzyme-free Krebs solution and the connective tissue was gently brushed aside from a chosen ganglion using the blunt end of a fire-polished patch pipette. This method of cleaning allows the outlines of the cell bodies of myenteric neurons to be visualized, so the tips of the patch pipettes can be positioned on the somatic regions of neurons.

The LMMP preparations were then perfused continuously with Krebs solution pre-heated to 35 °C to which nicardipine (1 μm) was added to block contractile activity in the adherent longitudinal muscle. Drugs were added to the Krebs solution and perfused through the recording chamber. Patch electrodes were drawn from borosilicate capillary glass tubing (GC150F-10, Harvard Apparatus, UK) to have resistances of 4–10 MΩ when filled with the standard pipette-filling solution of the following composition (mm): KCl 130, NaCl 10, MgCl₂ 1, CaCl₂ 0.5, Hepes 10, EGTA 1 and K₂ ATP 2. The pH was titrated to 7.3 with 4 m KOH, which resulted in the addition of approximately 11 mm K⁺ to the pipette solution, yielding a final [K⁺]_i of 145 mm. [Ca²⁺]_i was estimated to be about 85 nm using Maxchelator (http://www.stanford.edu/∼cpatton/maxc.html). While whole-cell recording from these neurons would be expected to alter the intracellular ionic milieu, we found that by using patch pipettes with such relatively high resistances, the slow AHP could be maintained for up to at least 30 min following patch rupture, once gigaseal (> 2 GΩ) formation had occurred in cell-attached mode. In the whole-cell recording configuration, capacitive currents were nulled by using the series resistance and capacitance subtraction circuitry on the amplifier, and the series resistance for whole-cell recordings measured < 20 MΩ. In addition, series resistance was compensated up to about 50–60 %.

Neurons that were patch clamped were identified as AH neurons by visual inspection of ganglia with the aid of high-power (× 600) microscopy, as those cells having large oval-shaped cell bodies > 20 μm in diameter. While the exact morphology or immunohistochemical profile of these patched neurons was not determined in the present study, when stimulated appropriately they were all able to generate slow AHPs or the underlying current, I_sAHP, when whole-cell recording was established. We have therefore preferred to refer to them as AH neurons, although they may encompass more than one functional type.

The composition of the Krebs solution was (mm): NaCl 118.1, KCl 4.8, NaHCO₃ 25, NaH₂PO₄ 1.0, MgSO₄ 1.2, glucose 11.1 and CaCl₂ 2.5. After bubbling with carbogen (95 % O₂–5 %CO₂) the pH of the Krebs solution was 7.35.

Inside-out patch recordings

Recordings from excised inside-out patches were obtained as described previously (Vogalis et al. 2002a), using solutions of identical composition at room temperature (21–24 °C) in order to maintain control of the pH of the Hepes-based solutions. Patch pipettes were drawn from borosilicate glass (Clark GF105-10) and their tips were fire-polished to have resistances of 10–25 MΩ when filled with low-potassium gluconate (5 mm) pipette solution (PS). The standard zero-Ca²⁺ high-potassium gluconate PS (0 Ca²⁺ high-K PS) consisted of (mm): potassium gluconate 145, MgCl₂ 1, Hepes 10 and EGTA 1; brought to pH 7.3 with KOH. The free [Ca²⁺] was estimated to be < 10 nm, assuming a 50 μm total Ca²⁺ was present in the de-ionized water. Varying amounts of CaCl₂ were added to this solution to raise the free [Ca²⁺] to higher levels. The composition of low-potassium gluconate PS was similar to the standard 0 Ca²⁺ high-K PS, except that 145 mm potassium gluconate was replaced with 140 mm sodium gluconate and only 5 mm potassium gluconate. For cell-attached recordings, the pipette filling solution consisted of (mm): KCl 145 and NaCl 10, along with MgCl₂ 1, Hepes 10 and the pH was adjusted to 7.3 with KOH. The free [Ca²⁺] in this solution was adjusted to about 50 nm with 1 mm EGTA and 0.4 mm added CaCl₂. Except where noted, the following blockers were added to the pipette solution: TEA (5 mm), apamin (0.5 μm), Cs⁺ (1–2 mm) and Cd²⁺ (0.1 mm). All the drugs were dissolved in water and stored as 1 m stock solutions (TEA, Cd²⁺, Cs⁺). Apamin was stored as 1 mm aliquots at −20 °C.

Current and voltage signals for all the experiments were recorded using an Axopatch 200A amplifier and pCLAMP 8 software (Clampex 8), onto the hard disk of a computer. Current recordings were analog low-pass filtered at 1–2 kHz (eight-pole Bessel filter, Krohn-Hite) and digitized at 5 kHz. Analysis of channel recordings (all-points histogram binning and Gaussian curve fits) were performed using a combination of Clampfit (to further digitally filter the recordings of 50 Hz line frequency-derived noise) and custom-written procedure files in Igor 4 (Wavemetrics). To obtain estimates of P_o and single-channel current levels, all-points histogram distributions were constructed from 20 s recordings representative of the various treatments and multiple Gaussian curves were superimposed on the histograms and their position, height and width were adjusted manually to obtain satisfactory fits. Goodness of fit was assessed by calculating the minimum of the summed squared differences between the value of the bin count and the corresponding value on the fitted curve. For other fits we used the curve-fitting routine in Igor 4.

Voltage and current signals were low-pass filtered at 1–5 kHz (eight-pole Bessel filter) and recorded onto computer using an Axopatch 200A amplifier and Clampex 8.1 (Axon Instruments, USA) software. Clampfit 8.1 was used to analyse the recordings and Igor 4 (Wavemetrics, USA) was used to plot the data. Averaged data are presented as means ±s.e.m., where n is the number of cells. The level of statistical significance of the difference between the means was set at P < 0.05, using Student's paired t test, unless otherwise stated. Pooled data are presented as means ±s.e.m. and n is equal to the number of patches or, in the whole-cell recordings, to the number of cells. Throughout the text, V_p denotes the pipette potential (cell-attached recordings) and V_m denotes the membrane potential.

Rp-cAMPS, cAMP and ATP γS (Sigma, Australia) were dissolved in water and stock solutions were aliquoted (50 μl) and stored at −20 °C until the day of use, when they were dissolved directly into the whole-cell pipette filling (internal) solution. ATP γS was added to whole-cell internal solution from which ATP was omitted. Forskolin and 1,9-dideoxyforskolin (Sigma, Australia) were dissolved in DMSO (10⁻²m), aliquoted and stored at −20 °C until the day of use. ATPK₂ and MgCl₂ were added to 0 Ca²⁺ high-K PS and the pH was adjusted to 7.3 on the day of use. Aliquots (5–10 μl) of the catalytic subunit of PKA (PKA_cat; 2500 units, Promega, Australia) were added to 0 Ca²⁺ high-K PS (500 μl) for pipetting into the recording chamber as boli (50–200 μl). The inhibitory peptide PKI_5–24 (Promega) was added to the whole-cell internal solution. Alkaline phosphatase (calf intestinal, Promega) was added to the 0 Ca²⁺ high-K PS bathing inside-out patches. All other drugs were purchased from Sigma.

Immunolocalization of R_IIβ subunits in myenteric neurons

Preparation of full-thickness sheets (2 cm × 2 cm) of duodenum for immunohistochemistry was performed as described previously (Vogalis et al. 2002b). Labelling for the human IIβ regulatory subunit of PKA (R_IIβ) was performed by incubating tissue with a mouse monoclonal antibody raised against that particular subunit (BD Biosciences; no. P54720-050) diluted to 1:1000. Small pieces of tissue fixed in Zamboni's solution, were pre-incubated (10 % NHS and 1 % Triton X-100 in PBS for 30 min) and then washed in PBS (30 min) prior to the addition of the primary antibodies. The tissue was stored at 4 °C for 36 h in a humid box, then washed for 30 min in PBS prior to addition of the secondary antibody, anti-mouse IgG coupled to fluorescein isothiocyanate (FITC; Jackson Immunoresearch Laboratories, PA, USA) at a final concentration of 1:200. The tissue was incubated in the secondary antibody solution for 90 min in a humid box at room temperature and then washed for 10 min in PBS before being mounted in phosphate-buffered glycerol (pH 8.6). The specificity of the R_IIβ antibody was confirmed by performing Western blot analysis on myenteric ganglia lysates, which revealed labelling of a 53 kDa peptide corresponding to the molecular weight of the R_IIβ subunit (D. Poole, personal communication). Preparations were analysed by confocal microscopy on a Biorad MRC1024 confocal scanning laser system installed on a Zeiss Axioplan 2 microscope. Stained neurons were visualized using 488 nm excitation filter and 522/535 nm emission filter for FITC. Images (512 × 512 pixels, nominal optical thickness, 0.5 μm) were stored on computer and processed using Corel PhotoPaint and Corel Draw software programs.

RESULTS

Modulation of I_sAHP by agents targeting the cAMP/PKA pathway

The diterpene, forskolin, is a potent activator of adenylyl cyclase and increases the intracellular level of cAMP. Under whole-cell recording conditions we found that forskolin (1 μm) blocked the slow AHP that was evoked using triple-pulse stimulation (three, 10 ms depolarizing pulses, 250–400 pA, 50 Hz; Fig. 1A). Although overall forskolin did not significantly affect the resting membrane potential (RMP) of AH neurons (control, −60.0 ± 5.6 mV; forskolin, −55.5 ± 3.9 mV, n = 6), in four of the neurons on which it was tested, the input resistance (R_in) was increased (P = 0.07) from 306 ± 96 to 412 ± 107 MΩ and the neurons tended to develop spontaneous firing in the presence of forskolin (Fig. 1A(ii)). Forskolin did not significantly affect the duration of action potentials at half-peak (AP_half-dur; control, 2.24 ± 0.13 ms; forskolin, 2.34 ± 0.32 ms, n = 4) and did not affect the inflection on the repolarizing phase, although the amplitude of the action potential was reduced from 108 ± 6 to 99 ± 10 mV (Fig. 1A(i), inset). The current underlying the slow AHP (I_sAHP) which was stimulated by step-depolarizing the neuron to +50 mV for 50 ms from a holding potential of −65 mV and was then recorded at −55 mV, was also significantly decreased by more than 91 % (Fig. 1B(i)) from a mean of 298 ± 93 to 26 ± 36 pA (n = 6) (P < 0.05). This suppression occurred within 5 min of wash-in (Fig. 1B(ii)) and was partially reversible, with the I_sAHP recovering to about 50 % of its maximum following 20–30 min of wash-out of forskolin in two neurons tested.

Figure 1. Suppression of the slow after-hyperpolarization (AHP) and the current underlying the slow AHP (I_sAHP) by forskolin.

A(i), whole-cell current-clamp recording from an enteric AH neuron bathed in normal Krebs solution, which generated a slow AHP following action potential firing triggered by triple-pulse stimulation (at the dot; three intracellular current pulses, 250 pA, 10 ms in duration, at 10 ms intervals). A(ii), same neuron similarly stimulated, in the presence of forskolin 1 μm. Forskolin had no effect on the action potential configuration (inset in A(i)). The dotted line represents the resting membrane potential (RMP). B(i), whole-cell voltage-clamp recordings of the I_sAHP (lower traces) in the absence and in the presence of forskolin (traces are superimposed). The upper trace shows the voltage protocol used to trigger an I_sAHP, which consisted of a 50 ms step depolarization to +50 mV from a holding potential of −65 mV, and the I_sAHP was recorded at −55 mV for 25 s. The dotted line in current recordings represents 0 pA in this and in other traces. B(ii), time course of the decrease in the peak I_sAHP as forskolin (1 μm) was washed into the bath. Currents were stimulated every 45 s. C(i), cumulative suppression of I_sAHP by increasing concentrations of forskolin. C(ii) concentration-percentage inhibition relationship between forskolin and the I_sAHP. Data points represent means ±s.e.m., from four AH neurons and are fitted with the Hill equation of the form:

where y represents the percentage inhibition of the I_sAHP relative to its amplitude in the absence of forskolin, and n_H is the Hill coefficient. The fit yielded an IC₅₀ of 8 nm and a Hill coefficient of 0.97. The inset shows the effect of increasing concentrations of forskolin on the means ±s.e.m. of the holding current at −65 mV, in the same neurons.

Although we used forskolin at 1 μm to block the slow AHPs rapidly, we found that this concentration was supramaximal. We found that forskolin at 10 nm produced about 50 % inhibition of the I_sAHP (Fig. 1C(i)). Mean data obtained from four AH neurons revealed that the percentage inhibition of the I_sAHP by forskolin could be fitted with the Hill equation, which yielded an IC₅₀ of 8 nm and a Hill coefficient of 0.97 (Fig. 1C(ii)). In contrast, there was no significant change in holding current in these neurons at the IC₅₀ (Fig. 1C(ii), inset). In two other neurons tested, application of 1,9-dideoxyforskolin (1 μm) caused a < 20 % reduction of the I_sAHP, which was subsequently blocked by equimolar application of forskolin.

In another series of recordings, cAMP (50–100 μm) was introduced directly into AH neurons through the patch pipette. We found that within 10 min of gaining whole-cell access, the I_sAHP had decreased significantly from 266 ± 32 to 127 ± 13 pA (n = 5) and continued to decrease with time such that by 30 min it was < 20 % of its peak (Fig. 2A). As with forskolin treatment, internal cAMP had no effect on the RMP, which averaged −55.4 ± 2.3 mV at break-in and −54.0 ± 5.8 mV 10 min later (n = 5; Fig. 2B). Likewise, R_in was unaffected (288 ± 91 MΩ at break-in vs. 219 ± 31 MΩ, 10 min post break-in, n = 5). AP_half-dur averaged 2.63 ± 0.15 ms immediately following whole-cell access and 2.31 ± 0.17 ms 10 min later (n = 5; Fig. 2B(ii) inset).

Figure 2. Decrease in the I_sAHP in an AH neuron perfused internally with cAMP.

A(i), whole-cell currents (superimposed) showing the decrease in the magnitude of the I_sAHP with time following whole-cell break-in. cAMP (50 μm) was added directly to the internal pipette-filling solution and allowed to diffuse into the neuron after gaining whole-cell access. A(ii), time course of the decrease of the I_sAHP following whole-cell access. B, slow AHPs triggered by triple-pulse stimulation (at the dot) were recorded within 1 min of patch rupture (i) and approximately 30 min later (ii). Note that the RMP was slightly hyperpolarized by internal perfusion of cAMP and that an after-depolarization (ADP) was generated following perfusion, which produced a burst of action potentials (arrow) that preceded the slow AHP. The ADP triggered a prolonged poststimulus burst of five action potentials to increase Ca²⁺ entry and overcome the cAMP-mediated inhibition of the current (see Vogalis et al. 2002b). The action potential configuration was unaffected by cAMP and action potentials retained their Ca²⁺-current-mediated ‘humps’ (inset).

There is evidence that cAMP is generated basally in myenteric neurons (Xia et al. 1997; Liu et al. 1999), suggesting that PKA is also basally active. To investigate whether this pathway was active at rest and modulated the activity of K_sAHP channels, we tested the actions of Rp-cAMPS, a PKA inhibitor that binds to the regulatory subunit of the holo-enzyme and blocks dissociation of the active catalytic subunits. Internal perfusion of Rp-cAMPS (500 μm) into AH neurons led to a significant hyperpolarization of the RMP within 5 min of gaining whole-cell access, from a mean of −57.5 ± 2.3 mV at break-in to −78.0 ± 2.6 mV (n = 8; Fig. 3B(i), (ii)). This hyperpolarization was paralleled by a decrease in R_in from 264 ± 42 to 104 ± 19 MΩ (n = 8) and was accompanied under voltage clamp by the development of a net outward current at the holding potential (−65 mV), which increased from −33 ± 19 to 202 ± 48 pA (n = 9; Fig. 3A(ii), arrow). The development of this outward current coincided with a net increase in the absolute I_sAHP current (current recorded at −55 mV) from an average of 434 ± 85 pA within 1 min of break-in to 533 ± 85 pA (n = 8), 5 min later. This enhanced I_sAHP had a slower rate of deactivation, as measured by fitting a mono-exponential function to the decay phase of the I_sAHP. The time constant of deactivation averaged 22.6 ± 5.9 s (n = 4; Fig. 3A(i), (ii)) compared with 6.0 ± 0.9 s (n = 7) for currents recorded from the same neurons shortly after break-in. These data suggest that the cAMP-PKA pathway is involved in the deactivation of the K_sAHP channels and that inhibition of this pathway leads to their persistent opening. As shown in Fig. 3A and B, clotrimazole (10 μm) substantially decreased both the I_sAHP and the standing outward current that developed in the AH neuron filled with Rp-cAMPS, and partially restored the RMP and the R_in (Fig. 3B(iii)). In five AH neurons filled with Rp-cAMPS, bath application of forskolin (1 μm) was still able to decrease the peak I_sAHP significantly by about 70 % from 435 ± 96 to 135 ± 50 pA.

Figure 3. Enhancement of the I_sAHP by internal perfusion of Rp-cAMPS.

A, the whole-cell I_sAHP was recorded from an AH neuron that was perfused internally with the standard pipette-filling solution to which was added Rp-cAMPS (500 μm) to inhibit the cAMP-dependent protein kinase (PKA). Shown are currents recorded about 1 min after break-in (i), about 7 min later (ii) and following the addition of clotrimazole (10 μm) to the bathing solution. The stimulus protocol is shown in the upper traces in each panel. Note the increase in the holding current at −65 mV (arrow in current trace (ii)) following perfusion of Rp-cAMPS. B, current-clamp recordings from the same AH neuron at approximately the same times as those in A. At each time point are shown superimposed the responses to hyperpolarizing current steps of fixed magnitude (100 pA) and the suprathreshold responses in response to depolarizing current steps under the three conditions. Note the large decrease in input resistance that developed with perfusion of Rp-cAMPS into the cell and the decrease in the amplitude of the evoked action potential (ii), indicating electrotonic spread from an extrasomatic region. The increased conductance was inhibited by clotrimazole, which partially restored the action potential amplitude (iii).

Effect of internal inhibitory peptide (PKI) and PKA on I_sAHP

To test further the possibility that K_sAHP channels are regulated by basal kinase activity, we introduced ATP γS (1 mm) into AH neurons, with no added ATP present in the internal solution, in an attempt to induce irreversible phosphorylation of the K_sAHP channels. This led to an almost complete block of the I_sAHP within 5–10 min of patch rupture (Fig. 4A) and substantial suppression of the slow AHP (Fig. 4B). In the three neurons tested, the I_sAHP (i.e. the peak current at −55 mV following the stimulus pulse to +50 mV) was decreased from 348 ± 172 to −14 ± 45 pA. Although the RMP was depolarized from −54.0 ± 4.8 to −44.8 ± 7.5 mV, there was no change in the action potential configuration (Fig. 4B(ii), inset).

Figure 4. Suppression of the slow AHP and the I_sAHP by perfusion of ATP γS into an AH neuron.

A(i), superimposed are currents recorded just after whole-cell break-in (1 min) and the current remaining after about 7 min of whole-cell perfusion of a pipette solution containing ATP γS (1 mm) but no added ATP. A(ii), time course of the decrease in I_sAHP showing that there was an immediate suppression of the current following patch rupture. B, current-clamp recordings of the slow AHPs immediately after gaining whole-cell access (i) and the response to triple-pulse current injection about 8 min later (ii). The inset shows superimposed action potentials recorded at break-in and following perfusion of ATP γS into the cell, revealing minimal diminution in the action potential duration.

We also microperfused PKA_cat into AH neurons. In eight AH neurons tested, internal application of PKA_cat (50–100 units ml⁻¹) significantly decreased the I_sAHP from an average of 502 ± 65 pA within 1 min of whole-cell access, to 245 ± 51 pA 5–10 min later (Fig. 5A and B). In contrast, in five representative AH neurons that were not filled with PKA_cat during this series of experiments, the I_sAHP averaged 499 ± 106 pA within 1–2 min of patch rupture, and 396 ± 57 pA 10 min later, a difference that was not statistically significant. The standing current at the holding potential (−65 mV) was not altered significantly with intracellularly perfused PKA_cat (Fig. 5A(ii)). Likewise, in current-clamp mode, neither the RMP was unaltered by internal perfusion of PKA_cat (−61.2 ± 3.0 mV at break-in, vs.−63.0 ± 2.5 mV 5–10 min later) nor was the AP_half-dur (2.30 ± 0.47 vs. 2.24 ± 0.56 ms, n = 4). As shown in Fig. 5B, inhibition of the I_sAHP by PKA_cat was associated with suppression of the accommodation in firing which is normally observed with depolarizing currents, and is due to the activation of the slow AHP (Fig. 5B(i), arrow).

Figure 5. Inhibition of the slow AHP and the I_sAHP by perfusion of exogenous PKA into the cell.

A(i), the I_sAHP was evoked in response to the standard stimulus protocol (upper trace) immediately after whole-cell break-in (< 1 min) from an AH neuron that was perfused internally with standard pipette solution containing 100 units ml⁻¹ of the catalytic subunit of PKA (PKA_cat). Superimposed is the I_sAHP recorded from the same neuron about 5 min later (5 min). A(ii), averaged data show the decrease in amplitude of the I_sAHP at break-in (open column) and the amplitude of the current at 5–10 min later (filled column), showing a significant reduction of about 50 %. Also shown is the magnitude of the holding current (I_{–65 mV}) recorded in the same cells, which was not significantly different at the two corresponding time-points. B, current-clamp recordings from the same neuron showing the response to a suprathreshold depolarizing current step immediately after whole-cell break-in (i), and about 6 min later (ii). Note the suppression of the slow AHP (the onset of which is indicated by the arrow in (i)) and increased excitability of neuron following perfusion of PKA_cat (ii).

In a separate series of experiments, we introduced into AH neurons the inhibitory peptide PKI_5–24, which acts as a pseudosubstrate for endogenous PKA, thereby reducing its activity on physiological targets. We found that internal perfusion of PKI (100–200 μm) did not significantly affect the magnitude of the I_sAHP, which averaged 652 ± 125 pA at break-in and 542 ± 88 pA, 5–10 min later (n = 6); in four non-PKI-filled neurons, the current decreased from an average of 512 ± 183 to 484 ± 74 pA over the same time period. In all the PKI-filled neurons, an outward current of variable amplitude developed soon after patch rupture at the holding potential (−65 mV), averaging 180 ± 77 pA. This standing outward current was associated with a comparatively negative RMP (−68.6 ± 4.7 mV) and a lower than normal R_in (143 ± 51 MΩ). A recording from one such PKI-filled neuron is shown in Fig. 6, which illustrates that forskolin (1 μm) failed to fully block the I_sAHP, reducing it by about 60 % (Fig. 6A), and inhibited the slow AHP by < 30 % (Fig. 6B) in current-clamp mode. This indicates that the actions of forskolin are mediated in part through cAMP-mediated activation of PKA.

Figure 6. Response of an AH neuron filled with the inhibitory peptide of PKA (PKI) to application of forskolin.

A(i), superimposed I_sAHP recorded from an AH neuron that was perfused internally with PKI_5–24 (100 μm), a pseudosubstrate of endogenous PKA (control). The degree of inhibition of the I_sAHP by forskolin (1 μm) was reduced (cf. Fig. 1B). A(ii), time course of the inhibition of the I_sAHP by forskolin. Note the run-up in the amplitude of the I_sAHP with perfusion of the PKI into the neuron. B(i), slow AHP recorded immediately following gaining whole-cell access, triggered by triple-pulse stimulation. (ii) The slow AHP following bath application of forskolin was only partially inhibited (cf. Fig. 1A). Inset shows that forskolin under these conditions had no effect on the action potential configuration.

Current recordings from membrane patches

We confirmed in cell-attached recordings that action potential firing in AH neurons is followed by the opening of channels in the membrane (Fig. 7A), which increase their P_o from < 0.01 to 0.3–0.5 within the initial 2–3 s following action potential firing. The ensemble-averaged current from such recordings deactivated with a time constant of the order of 2–3 s (Fig. 7B(ii)) and the channels had slope conductances of about 30 pS under these quasi-symmetrical transmembrane [K⁺] conditions (Fig. 7B(i)). In contrast, activation of these IK_Ca-like channels in cell-attached patches from AH neurons that had been treated with forskolin was substantially diminished or absent following action potential firing (Fig. 7C), consistent with these channels being responsible for the macroscopic I_sAHP in AH neurons.

Figure 7. Cell-attached patch recordings from AH neurons.

A, a whole-cell patch pipette filled with standard pipette-filling solution was sealed onto an AH neuron. The channel activity in the patch was monitored at a pipette potential (V_p) of −30 mV. Following stimulation of an action potential in the neuron (arrow), there was an increase in channel activity over the ensuing 5–10 s. c, closed channel level; o1–o3, open channel levels. B(i), single-channel current amplitude (estimated from all-points histogram plots) plotted as a function of V_p for the patch depicted in A. The linear regression line through the data points yields a unit conductance of 30 pS. B(ii), the ensemble averaged current constructed from 10 traces recorded at V_p = −30 mV illustrating the time course of deactivation (time constant of a single exponential was 2.3 s approximated the whole-cell I_sAHP. C, cell-attached patch recording from a different AH neuron that was bathed in forskolin (1 μm). Under these conditions few channel openings could be recorded following action potential firing in the neuron (arrow). When channels were activated, their conductance was similar to the conductance of channels that opened under normal conditions.

In a previous study, we showed that these IK_Ca-like channels are activated by submicromolar Ca²⁺ applied to the cytoplasmic face of membrane patches excised from AH neurons (Vogalis et al. 2002a) and reported that they failed to deactivate when Ca²⁺ was washed out for more than 1 h. This suggests that under cell-free conditions, a cytoplasmic ‘factor’ was absent, and this absence prevented the IK_Ca-like channels from returning to their ‘resting’ low P_o. One possible explanation for this mode switch is that IK_Ca-like channels are regulated directly by PKA. To investigate this possibility, we tested the actions of exogenous PKA_cat (50–200 units ml⁻¹) applied to inside-out patches from AH neurons containing IK_Ca-like channels. Currents were recorded at 0 mV under asymmetric trans-patch [K⁺]. Under these conditions, the NP_o (where N = 1–5 channels) of IK_Ca-like channels averaged 0.012 ± 0.006 (n = 17) when the cytoplasmic face of the patch was bathed in 0 Ca²⁺ high-K PS. The NP_o increased to 2.89 ± 0.772 (n = 17) following wash-in of Ca²⁺-containing (1–2 μm) high-K PS and remained elevated at 2.58 ± 0.73 (n = 17) following wash-out of Ca²⁺. In seven of these patches containing more than three channels, the P_o (per channel) averaged 0.56 ± 0.08, 0.51 ± 0.07 and 0.54 ± 0.07 at −30 mV, 0 mV and at + 30 mV (V_m), respectively, indicating that the IK_Ca-like channels were not voltage dependent.

In twelve of these inside-out patches, removal of Ca²⁺ by wash-in of 0 Ca²⁺ high-K PS to which ATP (3 mm) and Mg²⁺ (2–3 mm) had been added, failed to reduce the NP_o of IK_Ca-like channels in patches, which averaged 2.79 ± 1.21. This indicates that these channels were not sensitive to internal ATP. We then added 30–150 μl of 0 Ca²⁺ high-K PS solution that contained PKA_cat (50–200 units ml⁻¹) and ATP and/or Mg²⁺ directly to the bath and monitored the channel activity over the next 5–10 min (Fig. 8A(i)). In 9 of 12 patches, this led to a clear decrease in the NP_o of IK_Ca-like channels, while in the other three, no such change was evident. Overall the NP_o was decreased to 1.16 ± 0.30 (n = 12) in 12 patches, or a decrease to 64 ± 17 % of the control NP_o, while in the 9 patches in which PKA_cat inhibited channel activity, the NP_o decreased to 39 ± 13 % of control values.

Figure 8. Recordings from an inside-out patch containing several IK_Ca-like channels, showing the inhibitory effect of PKA_cat.

A(i), continuous recording from the patch that had been previously exposed to a Ca²⁺-containing high-K solution that was then washed out with 0 Ca²⁺ high-K solution (0 Ca²⁺). The recording shows the effect of bolus application of PKA_cat (50 units ml⁻¹; gap in trace) on the activity of IK-like channels (openings are upward). A(ii), decrease in the patch-current average over 1 s intervals with the application of PKA_cat. B, portions of the recordings shown on an expanded time base ((i) and (ii)) and the corresponding all-points histogram plots ((iii) and (iv)) showing that more than five channels were open simultaneously in the patch before application of PKA_cat.

In patches containing multiple (< 6) IK_Ca-like channels, bolus application of PKA_cat reduced the number of channels that were open simultaneously, leading to a reduction in the average patch current (Fig. 8A(ii) and B) and to a decrease in the NP_o (Fig. 8B(ii), (iv)). We did not observe any substantial decrease in the NP_o of IK_Ca-like channels in two inside-out patches when we applied PKA_cat that had been left overnight at room temperature and which had presumably denatured.

A notable example of the inhibitory effect of PKA_cat on an inside-out patch containing a single IK_Ca-like channel is shown in Fig. 9A(i). In this patch, as in others, following application of PKA_cat and the subsequent reduction in the P_o of the IK_Ca-like channel, there was an apparent increase in the P_o of a channel of a larger conductance, approximately double that of the IK_Ca-like channel (Fig. 9B(ii)). The identity of this channel was not determined. In two of the inside-out patches, the suppression of IK_Ca-like channel activity following bolus application of PKA_cat partially recovered following wash in of 0 Ca²⁺ high-K PS, while in three other patches application of Ca²⁺-containing solution restored the NP_o. These data are summarized in Fig. 9C, in which NP_o has been normalized in each patch to the value following the first wash-out of Ca²⁺ solution.

Figure 9. Single-channel recording from an inside-out patch from an AH neuron.

A(i), the patch contained a single IK_Ca-like channel (openings are upward) that was activated by previous exposure to 2 μm Ca²⁺-containing high-K PS, which was subsequently washed out with 0 Ca²⁺ high-K PS. The recording was obtained in the presence of 0 Ca²⁺ high-K PS containing 3 mm ATP-Mg²⁺. A bolus application of this solution containing PKA_cat (200 units ml⁻¹; noise in trace at bar) caused the activity of the IK_Ca-like channel to switch off and unmasked openings of a larger conductance channel. A(ii), all-points histogram distribution constructed from a 20 s section of the recording prior to the application of PKA_cat, and fitted with two Gaussian functions representing the closed state (centred about 0 mV) and the open state. The open probability (P_o) was estimated to be 0.705. A(iii), all-points histogram dominated by a closed-state current level following the application of PKA_cat. B, expanded portions of the recording shown in A(i), showing characteristic flickering activity of the IK_Ca-like channel before the addition of PKA_cat (i), and absence of opening following application of PKA_cat (ii); note the opening of a larger conductance channel. C, mean data of IK_Ca-like channel activity recorded in excised patches under different conditions. The open probability (NP_o) of IK_Ca-like channels recorded at a membrane potential (V_m) = 0 mV was normalized to the absolute value for channel activity recorded following the first wash-out of Ca²⁺-containing high-K PS (WO Ca²⁺). * Significant difference (P < 0.05; unpaired t test) compared to WO Ca²⁺. The numbers on or above columns refer to the number of patches.

Our results on excised patches indicate that phosphorylation of IK_Ca-like channels by PKA may be involved in the return of these channels to a resting mode of low P_o, following their activation by Ca²⁺. This gating scheme suggests that dephosphorylation is then involved in the activation of the IK_Ca-like channels. As shown in Fig. 10 in one of two inside-out patches, application of alkaline phosphatase (20 units ml⁻¹) to an inside-out patch that was bathed in 0 Ca²⁺ high-K PS resulted in a significant increase in the P_o of IK_Ca-like channels (Fig. 10B), suggesting that they are subject to regulation by phosphatases.

Figure 10. Effect of alkaline phosphatase on IK_Ca-like channel activity in an inside-out patch.

A, continuous recording from an inside-out patch under similar conditions to the recordings depicted in Fig. 8, with the cytoplasmic face of the patch exposed to 0 Ca²⁺ high-K PS. Perfusion of 0 Ca²⁺ high-K PS containing 20 units ml⁻¹ of alkaline phosphatase from bovine intestine caused the IK_Ca-like channels to increase their activity. B, expanded portions of the trace shown in A (corresponding to labels a and b), showing sparse openings (o, open channel current level; c, closed channel current) of IK_Ca-like channels prior to exposure to alkaline phosphatase (i), and continuous activity following wash in of the enzyme (ii). The all-points histograms to the right of each trace show the increase in the P_o of the channel with a unit current of about 1.2 pA at V_m = 0 mV.

Immunolocalization of PKA R_IIβ subunits

PKA is composed of two catalytic and two regulatory subunits, with the latter acting to compartmentalize the distribution of the holo-enzyme. To determine whether we could detect the presence of PKA in enteric neurons, we used a mouse monoclonal antibody raised against an epitope on the human IIβ regulatory subunit of PKA (R_IIβ), which is highly expressed in the brain. As illustrated in the fluorescence and the confocal micrographs in Fig. 11A and B, enteric neurons with the morphological characteristics of AH neurons exhibited intense staining of the cytoplasm and their proximal processes (arrows in Fig. 11A and B), but not the nucleus. This was confirmed in three preparations from different animals. These data indicate that PKA is expressed strongly in the cytoplasm of enteric AH neurons.

Figure 11. Immunochemical localisation of the regulatory subunit of PKA (R_IIβ) in myenteric AH neurons.

A, fluorescence micrograph showing myenteric neuron labelled with the monoclonal antibody (raised against R_IIβ) in the cytoplasm of the soma (arrow) and in the proximal processes. B, confocal micrograph showing labelling of the cytoplasm of an AH neuron (arrow) and the processes, but no localization within the nucleus.

DISCUSSION

Slow AHPs in enteric neurons and cAMP actions

Neurons that generate slow AHPs are found throughout the nervous system and the slow AHP is especially prominent in enteric AH neurons. The results of functional and structural studies have indicated that these neurons act as intrinsic afferent neurons that participate in the generation of intrinsic neural reflexes in the intestine (Furness et al. 1998). By controlling the excitability of AH neurons, slow AHPs are functionally important in grading the amount of afferent information that is transmitted by AH neurons to second-order enteric neurons (i.e. inter- and motoneurons) and may enable AH neurons to encode different types of afferent signals. Recently we identified a K⁺ channel that underlies the slow AHP in AH neurons (Vogalis et al. 2002a). This channel, which we termed an IK_Ca-like channel, had an intermediate unit conductance and was activated by submicromolar levels of Ca²⁺. We also reported that the channel had the unusual property of failing to deactivate following wash-out of Ca²⁺ from the cytoplasmic face of excised membrane patches (Vogalis et al. 2002a). In the present study, we provide evidence that cAMP-dependent PKA inhibits the P_o of these channels and that this action may be involved in channel deactivation.

Enteric neurons are capable of generating strong cAMP signals when stimulated with the appropriate agonists or with forskolin (Zafirov et al. 1985; Palmer et al. 1987). This is associated with a slow EPSP-like depolarization of the resting potential and concomitant inhibition of the slow AHP (Nemeth et al. 1986). In the present study we have confirmed that forskolin inhibits the slow AHP and the underlying current, I_sAHP, but like Baidan et al. (1995), we found that forskolin had little or no effect on the Ca²⁺-current-mediated plateau, or ‘hump’, of the action potential that is largely responsible for the relatively long duration of action potentials in AH neurons (Vogalis et al. 2001; Rugiero et al. 2002). This indicates that inhibition of the slow AHP by forskolin is not due to inhibition of N-type Ca²⁺ channels, but is mediated by actions on sites downstream of Ca²⁺ entry. However, other agents such as adenosine may suppress the slow AHP by inhibiting Ca²⁺ entry (Baidan et al. 1995).

In addition to inhibiting the slow AHP and the underlying current, forskolin also increased the input resistance of neurons by about 30 %. This was associated with spontaneous firing. Internal perfusion of cAMP produced a similar effect to forskolin, suggesting that the mechanism of action of forskolin is largely due to the stimulation of adenylyl cyclase (Zafirov et al. 1985; Liu et al. 1999). This effect is consistent with the results of earlier studies using ‘sharp’ microelectrodes, in which forskolin was shown to the elicit a depolarization of AH and/or type 2 neurons that was associated with an increase in input resistance (Nemeth et al. 1986). It should be noted, however, that the increase in input resistance that was elicited by forskolin in those studies, and also by cAMP injection (Zafirov et al. 1985), in which AH neurons were impaled with microelectrodes, was much greater than we recorded using patch-clamp pipettes. We believe that this discrepancy is due largely to the different magnitudes of resting conductance that are obtained using the two methods of recording and to differences in the resting potentials. With ‘sharp’ microelectrodes, resting values of input resistance typically range between 10 and 100 MΩ, and resting potentials range between −60 and −75 mV (e.g. see Hirst et al. 1985; Nemeth et al. 1986; North & Tokimasa, 1987), while whole-cell patch-clamp recordings from these neurons yield values of input resistance that are threefold higher (∼ 300 MΩ) and resting potentials that are between −50 and −60 mV (Vogalis et al. 2001; Rugiero et al. 2002). This suggests that in sharp microelectrode recordings, there is a K⁺ conductance that is basally active, and this conductance is absent or minimally activated when whole-cell patch-clamp recordings are obtained. Therefore, inhibition of this resting conductance by forskolin and cAMP will produce a relatively larger change in input resistance in microelectrode-impaled AH neurons than in patch-clamped neurons. Consistent with this interpretation is the fact that the input resistance of some AH neurons that are treated with forskolin is increased to roughly similar values that are recorded using patch electrodes (i.e. about 300 MΩ; Nemeth et al. 1986).

According to the studies of North & Tokimasa (1987), about 15 % of the resting conductance is due to a Ca²⁺-dependent K⁺ component, whereas agonists such as substance P and acetylcholine reduce the resting conductance by a factor of two or three. On the other hand, forskolin and cAMP decreased the resting conductance of impaled neurons severalfold (Zafirov et al. 1985; Nemeth et al. 1986), suggesting that the cAMP pathway inhibits a resting Ca²⁺-dependent K⁺ conductance plus another resting K⁺ conductance. Therefore, when agonists are applied, the decrease in resting conductance exceeds the value that is attributed to the Ca²⁺-dependent component.

Although the increased leakage of Ca²⁺ into the cytoplasm in impaled neurons may artificially inflate values of resting conductance, the converse may also hold, that whole-cell recordings in which cytoplasmic [Ca²⁺] was buffered to ∼−70 nm with EGTA may underestimate the resting conductance. Yet another difference between our recordings and those made using microelectrodes were the neuronal preparations. In microelectrode recordings, the glial cell layer covering myenteric ganglia is largely intact, whereas in our recordings, glial cells are mechanically removed. It is possible that glial cells may release agents that maintain the resting conductance of AH neurons high and that the removal of glial cells may deprive AH neurons of a tonic paracrine inhibitory input, thereby decreasing their resting conductance.

Internal perfusion of cAMP has been shown to suppress the I_sAHP in other types of neuron, including hippocampal neurons (Erdemli et al. 1998), in which cAMP is thought to act by stimulating PKA (Pedarzani & Storm, 1993; Torres et al. 1995). The widespread actions of PKA on intracellular targets, and the fact that the I_sAHP is generated by a multicomponent pathway, suggests that several sites of action are involved in the inhibition of slow AHPs by cAMP-PKA. In hippocampal neurons, suppression of the I_sAHP by stimulation of 5HT₄ receptors and subsequent activation of the cAMP-PKA pathway is mediated by inhibition of Ca²⁺-induced Ca²⁺ release (Torres et al. 1996). However, in neurons from the same brain region, the magnitude of the Ca²⁺ transients in the cytoplasm that were recorded simultaneously with the I_sAHP was unchanged following stimulation with β-adrenoceptors (Sah & Clements, 1999), ruling out Ca²⁺ entry and Ca²⁺-induced Ca²⁺ release as targets of cAMP-PKA.

In another study on hippocampal neurons, cAMP analogues were shown to inhibit an outward current that was attributable to the persistent activation of the same conductance responsible for the I_sAHP (Lancaster & Batchelor, 2000), despite the presence of high intracellular concentrations of Ca²⁺ chelators, which clamped intracellular Ca²⁺ to extremely low levels. The results of that particular study indicated that the actions of cAMP on the persistently active outward current were not mediated through alterations in intracellular Ca²⁺ levels. The results of our present study support the notion that inhibition of slow AHPs by cAMP-PKA is not mediated by alterations in intracellular Ca²⁺ levels, because we have shown that in excised patches PKA directly inhibits the P_o of IK_Ca-like channels, which in AH neurons are likely to be the K_sAHP channels.

Suppression of the slow AHP by cAMP-dependent PKA

In the present study we have shown that a neuronal isoform of the PKA regulatory subunit (R_IIβ; Chin et al. 2002) is expressed strongly in myenteric neurons that have morphological characteristics of AH neurons. We found strong expression in the somatic regions and on proximal processes, but less so on the finer processes and none in the nuclei of these neurons. It is generally accepted that binding of cAMP to the regulatory sites on the holo-enzyme leads to the liberation of the two active catalytic subunits of PKA that proceed to phosphorylate serine and threonine residues on target proteins, while the regulatory subunits act to localize the enzyme to specific subcellular compartments. Kinase activity is terminated when both types of subunits re-associate (Chin et al. 2002). We found that internal perfusion of Rp-cAMPS, an analogue of cAMP that is resistant to hydrolysis by phosphodiesterases and that binds to the regulatory site of PKA but prevents dissociation of the active subunits, led to a significant increase in the magnitude of the I_sAHP and a slowing of its rate of deactivation. Rp-cAMPS also elicited a persistent outward current and hyperpolarization of the resting potential of AH neurons by up to 20 mV, as was also reported to occur in hippocampal neurons (Pedarzani et al. 1998). AH neurons were also hyperpolarized when internally perfused with PKI_5–24 to inhibit PKA, suggesting that PKA is active under basal conditions and maintains a low P_o of K_sAHP channels. Consistent with this idea of basally active PKA, we found that internal perfusion of ATP γS led to an almost complete suppression of the I_sAHP, as has been reported to occur in hippocampal neurons (Pedarzani et al. 1998).

As expected, internal perfusion of PKA_cat resulted in a decrease in the magnitude of I_sAHP by about 50 % within 10 min of patch rupture, which is consistent with PKA inhibiting the P_o of K_sAHP channels. On the other hand, PKI, a peptide inhibitor that binds to PKA_cat, did not consistently increase the I_sAHP over the same time period, as one would expect if it is assumed that PKA was inhibiting the activation of K_sAHP channels. However, the average magnitude of the I_sAHP recorded from AH neurons that were internally perfused with PKI was about twofold larger immediately following patch rupture than in AH neurons that were not filled with the inhibitory peptide, despite having similar whole-cell capacitance values (30–40 pF). This suggests that inhibition of PKA by PKI occurs quickly following patch rupture, leading to an immediate enhancement of the I_sAHP and to the accumulation of active (i.e. open) K_sAHP channels that produce a persistent outward current at resting potentials. This conclusion is supported by the finding that PKI-filled neurons had resting potentials that were about 10 mV more negative than normal and a correspondingly lower input resistance, which we hypothesize, as with the actions of Rp-cAMPS, is due to the removal of PKA-mediated inhibition of K_sAHP channels.

Actions of PKA_cat on IK_Ca-like channels in excised patches

The failure of IK_Ca-like channels in patches to switch off following wash-out of Ca²⁺ (Vogalis et al. 2002a) is unusual for a Ca²⁺-activated K⁺ channel, because the three major types of K_Ca channels (i.e. the BK_Ca, SK_Ca and IK_Ca channels) are all activated by cytoplasmic Ca²⁺ in a reversible manner (Vergara et al. 1998). The persistent activity of IK_Ca-like channels in excised patches suggests that Ca²⁺ induces switching of the channels from a mode in which they are largely inactive (i.e. a low P_o mode), to one in which they are highly active (a high P_o mode). The present results on excised patches suggest that the inhibitory actions of cAMP-PKA on the P_o of IK_Ca-like channels involve the stabilization of the low P_o mode. According to this hypothesis, the failure of IK_Ca-like channels to switch back to a low P_o mode following their activation by Ca²⁺ in excised patches may be due to the absence of appropriate cytoplasmic factors. One of these factors may be PKA, because when PKA_cat was added to these patches, the persistent opening of K_sAHP channels by Ca²⁺ was partially reversed.

In contrast to the IK_Ca-like channels in AH neurons, the intermediate-conductance Ca²⁺-activated K⁺ (IK) channels in epithelial cells have been shown to be activated by the cAMP-PKA pathway (Gerlach et al. 2000) and by an unidentified kinase (von Hahn et al. 2001). This suggests that the IK_Ca-like channels that we have identified and characterized are different from the IK channels expressed in epithelial cells. The effect of exogenous PKA_cat on the IK_Ca-like channels from AH neurons, however, is reminiscent of the effect of this kinase on SK channels found in molluscan sensory neurons (Shuster et al. 1985).

Phosphorylation-dependent gating of IK_Ca-like channels

The present results on AH neurons are in general agreement with the proposed regulation of the I_sAHP in hippocampal neurons by competing phosphorylation and dephosphorylation (Pedarzani et al. 1998), with PKA being the principal kinase. According to this scheme, any increased drive of the cAMP-PKA pathway will decrease the slow AHP and increase AH neuronal excitability by making K_sAHP channels less likely to open. The slow AHP in AH neurons is inhibited by a range of amines and peptides (Furness et al. 1998), but not all of these are known to act through the cAMP-PKA cascade. It is possible, however, that other kinases may converge on PKA. For example, activation of 5-HT receptors in AH neurons initially leads to the activation of protein kinase C, which in turn activates the cAMP-PKA pathway by activating adenylyl cyclase (Pan et al. 1997). It is also possible that the IK_Ca-like channels may be subject to direct phosphorylation by other kinases.

The proposal that IK_Ca-like channels are ‘mode-gated’ by PKA-mediated phosphorylation is novel and will require further testing. The K_sAHP channels of enteric AH neurons have yet to be identified at the molecular level and this hypothesis cannot be tested using mutagenesis of potential domains that are targeted by PKA. It is also unclear at present what role dephosphorylation plays in the activation of these channels by Ca²⁺. We showed that it is possible to activate IK_Ca-like channels in excised patches by a broad-spectrum phosphatase, but we have yet to determine the nature of the endogenous phosphatase, or whether dephosphorylated (activated) IK_Ca-like channels can be turned off by PKA. Enzymatic regulation of the K_sAHP channels may also explain the reported strong temperature dependence of the I_sAHP in AH neurons (Hirst et al. 1985; North & Tokimasa, 1987) and elsewhere (Sah, 1996), which have Q₁₀ values in excess of 3.

Functional implications of phosphorylation-dependent gating of K_sAHP (IK_Ca-like) channels

An advantage of phosphorylation of IK_Ca-channels as a mechanism for regulating their activity is that phosphorylation would render these channels resistant to changes in intracellular parameters such as pH, ionic strength and membrane potential. However, it would also enable other enzymes to control their activity by acting on the terminal kinase, which we propose to be PKA, and enable these channels to participate in various forms of functional plasticity. In summary, we have demonstrated that the cAMP-PKA pathway is likely to play an important role in the regulation of the gating of K_sAHP channels in AH neurons of the intestine. Our results indicate that PKA inhibits the opening of these channels by stabilizing a mode of low P_o and that this mechanism may be responsible for resetting these channels following their activation by Ca²⁺ and for maintaining a low resting P_o.