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Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Acknowledgements

The patch-clamp technique was used to record from intact ganglia of the guinea-pig duodenum in order to characterize the K+ channels that underlie the slow afterhyperpolarization (slow AHP) of myenteric neurons. Cell-attached patch recordings from slow AHP-generating (AH) neurons revealed an increased open probability (Po) of TEA-resistant K+ channels following action potentials. The Po increased from < 0.06 before action potentials to 0.33 in the 2 s following action potential firing. The ensemble averaged current had a similar time course to the current underlying the slow AHP. TEA- and apamin-resistant Ca2+-activated K+ (KCa) channels were present in inside-out patches excised from AH neurons. The Po of these channels increased from < 0.03 to approximately 0.5 upon increasing cytoplasmic [Ca2+] from < 10 nm to either 500 nm or 10 μm. Po was insensitive to changes in transpatch potential. The unitary conductance of these TEA- and apamin-resistant KCa channels measured approximately 60 pS under symmetric K+ concentrations between −60 mV and +60 mV, but decreased outside this range. Under asymmetrical [K+], the open channel current showed outward rectification and had a limiting slope conductance of about 40 pS. Activation of the TEA- and apamin-resistant KCa channels by internal Ca2+ in excised patches was not reversed by washing out the Ca2+-containing solution and replacing it with nominally Ca2+-free physiological solution. Kinetic analysis of the single channel recordings of the TEA- and apamin-resistant KCa channels was consistent with their having a single open state of about 2 ms (open dwell time distribution was fitted with a single exponential) and at least two closed states (two exponential functions were required to adequately fit the closed dwell time distribution). The Ca2+ dependence of the activation of TEA- and apamin-resistant KCa channels resides in the long-lived closed state which decreased from > 100 ms in the absence of Ca2+ to about 7 ms in the presence of submicromolar cytoplasmic Ca2+. The Ca2+-insensitive closed dwell time had a time constant of about 1 ms. We propose that these small-to-intermediate conductance TEA- and apamin-resistant Ca2+-activated K+ channels are the channels that are primarily responsible for the slow AHP in myenteric AH neurons.

Slowly developing post-spike afterhyperpolarizations (slow AHPs) are a feature of many types of neuron. These include vagal primary afferent neurons (Cordoba-Rodriguez et al. 1999), sensory neurons with cell bodies in dorsal root ganglia (Gold et al. 1996), sympathetic neurons with cell bodies in prevertebral ganglia (Jobling et al. 1993), central neurons such as hippocampal pyramidal neurons (Sah & Clements, 1999; Bekkers, 2000) and neurons with cell bodies in the myenteric plexus in the wall of the intestine (Hirst et al. 1974; Wood & Mayer, 1978). The slow AHP limits the duration of firing of neurons and may confer a bursting pattern of activity. This is because the underlying conductance develops slowly, over hundreds of milliseconds, permitting cells to fire briefly before action potential firing is curtailed by hyperpolarization of the membrane potential and the short-circuiting of local currents.

Myenteric neurons that generate slow AHPs (AH neurons) serve as the primary afferent neurons in the enteric neuronal circuits (Furness et al. 1998). AH neurons are activated by mechanical distortion of their processes (Kunze et al. 1998) or by application of chemical stimuli to the mucosa to which they send processes (Bertrand et al. 1998). As well as limiting the firing rate of these neurons, the slow AHP gates the trans-somatic passage of action potentials (APs) (Wood, 1994). The Ca2+-activated K+ conductance (gK-Ca) that underlies the slow AHP is triggered by Ca2+ entry during the action potential (North, 1973; Hirst et al. 1985a). However, the immediate sources of Ca2+ for activation of this gK-Ca are the internal Ca2+ stores from which Ca2+ is released through ryanodine- and caffeine-sensitive Ca2+ channels (Hillsley et al. 2000; Vogalis et al. 2001). Some of the properties of the slow AHP suggest that the KAHP channels are different from the two major types of Ca2+-activated K+ (KCa) channels, i.e. the large-conductance BK channels and the small-conductance SK channels. BK channels respond quickly, within tens of milliseconds, to changes in cytoplasmic [Ca2+] (Vergara et al. 1998), and SK channels open and close with fast kinetics in response to Ca2+ transients (Hirschberg et al. 1998, 1999). Recent studies in myenteric AH neurons from the guinea-pig small intestine have shown that the Ca2+ transient measured in the soma decays at a faster rate than the slow AHP (Hillsley et al. 2000), suggesting that the KAHP channels may be gated differently by intracellular Ca2+ from BK- or SK-type channels. Moreover, neither low concentrations of TEA (2–5 mm) nor apamin, which block BK and SK channels, respectively, significantly affect the magnitude of the slow AHP in AH neurons (Furness et al. 1998).

In a recent study in which we performed noise analysis of the whole-cell IAHP in AH myenteric neurons, the chord conductance of the KAHP channels at −55 mV in physiological (asymmetric) K+ concentrations was estimated to be about 10 pS (Vogalis et al. 2001). This value is similar to the conductance of slow AHP channels in other neurons (Sah, 1995; Cordoba-Rodriguez et al. 1999). Channel openings of small-to-intermediate conductance channels were also recorded in cell-attached patches following action potential firing. In the present study, we have performed patch-clamp recordings in excised and cell-attached patches from AH neurons to determine the properties of these non-BK and non-SK type K+ channels. Our results suggest that these channels have unique properties that are consistent with their involvement in the slow AHP.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Acknowledgements

Preparation of longitudinal muscle-myenteric plexus

All experimental procedures were approved by the Animal Experimentation Ethics Committee at the University of Melbourne. Guinea-pigs (200–250 g) were killed by cervical dislocation, and exsanguinated. Segments (2 cm in length) of duodenum were removed and placed in pre-oxygenated Krebs solution containing nicardipine (1 μm). Each segment was dissected longitudinally along the mesenteric border and pinned out flat in a dissecting dish, mucosa uppermost, and both the mucosa and underlying circular muscle layer were removed by sharp dissection under a binocular microscope. The Krebs solution was exchanged frequently during dissection. The myenteric plexus and the adherent underlying longitudinal muscle layer (LMMP) were then pinned out in a tissue chamber positioned on the stage of an inverted microscope. Images of myenteric ganglia were projected onto a video monitor, which allowed neurons to be identified by size, shape and position. The LMMP preparation was perfused continuously (3–5 ml min−1) with pre-oxygenated and pre-heated (to 35–37 °C) Krebs solution. A low concentration of protease (0.01 %, type XIV, Sigma, USA) dissolved in Krebs solution was then superfused over the preparation for 10–20 min (Kunze et al. 2000). Following enzymatic treatment, individual ganglia were selected under the microscope for cleaning, which consisted of running the blunt end of a fire-polished glass pipette back and forth over the surface of the ganglion with the aid of a micromanipulator. After a sufficient period of cleaning (up to 60 min) to remove the surface connective tissue, blood vessels and satellite cells, neurons could be easily identified within myenteric ganglia.

Patch-clamp recording from myenteric neurons

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 either pipette solution. For cell-attached patch recordings, the LMMP preparations were perfused continuously with Krebs solution at 35–37 °C and patch-pipettes were filled with 0 Ca2+ high-KCl physiological solution (PS; composition given below). Similarly, excised outside-out patches were bathed in pre-heated Krebs solution and pipettes were filled with high-K+ PS. For inside-out patch recordings, however, LMMP preparations were perfused (0.5 ml min−1) with 0 Ca2+ high potassium gluconate PS at room temperature (22–24 °C). Although under these depolarizing conditions it was not possible to identify AH neurons electrophysiologically, such neurons were recognizable by their size and shape. Test solutions with varying concentrations of Ca2+ were perfused directly into the tissue chamber (500 μl in volume) at room temperature at approximately 0.5 ml min−1. To minimize electrical noise, the depth of the pipette in the solution was kept to a minimum.

Currents were recorded, using an Axopatch 200A amplifier and pCLAMP 8 software (Clampex 8), onto the hard disk of a computer. Current recordings were analog filtered at 2 kHz at the amplifier (-4 dB Bessel filter) and digitized at 5 kHz. Analysis (all-points histogram binning and Gaussian curve fits) was 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 open probabilities and single channel current levels, Gaussian curves were superimposed on the all-points histogram distributions, 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 corresponding value on the fitted curve. For other fits, we used the curve fitting routine in Igor 4.

Composition of solutions and channel blockers

The composition of the Krebs solution was as follows (mm): NaCl, 118.1; KCl, 4.8; NaHCO3, 25; NaH2PO4, 1.0; MgSO4, 1.2; glucose, 11.1; and CaCl2, 2.5. The Krebs solution was maintained at room temperature (22 °C) during dissection, but was heated to 35–37 °C during recording. Nicardipine (1 mm) was added to the Krebs solution to block spontaneous muscle contraction.

The standard 0 Ca2+ high potassium gluconate PS (0 Ca2+ PS) consisted of (mm): potassium gluconate, 145; MgCl2, 1; Hepes, 10; and EGTA, 1; pH 7.3 with KOH. The free [Ca2+] was estimated to be < 10 nm, assuming 50 μm total Ca2+ was present in the deionized water. Varying amounts of CaCl2 were added to this solution to raise the free [Ca2+] to higher levels. Calculations of free [Ca2+] were performed using Sliders (http://www.stanford.edu/~cpatton/maxc.html). Low potassium gluconate PS, used for recording K+ channel currents under asymmetric (physiological) K+ concentrations, was similar to the standard 0 Ca2+ high potassium gluconate PS except 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 145 mm KCl and 10 mm NaCl, along with 1 mm MgCl2, 10 mm Hepes, pH 7.3. 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 this solution: TEA, 5 mm; apamin, 0.5 μm; Cs+, 1–2 mm; and Cd2+, 0.1 mm. These concentrations of TEA and apamin almost fully block current flow through BK channels (Vergara et al. 1998) and SK channels (Grunnet et al. 2001). Cs+ and Cd2+ were added to block hyperpolarization-activated current (IH) and voltage-gated Ca2+ channels. 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. The pooled data are presented as means ±s.e.m. and n is the number of neurons. Student's unpaired t test was used to determine statistically significant differences. Throughout the text, Vp denotes the pipette potential (cell-attached recordings) and Vh denotes the holding potential (excised patches).

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Acknowledgements

Cell-attached patch recordings of AHP channels

Cell-attached patch recordings from the somatic region of AH neurons (identified from the inflection on the action current) in intact ganglia revealed an increase in the open probability (Po) of a population of channels in the patch following stimulation of neuronal action potentials (APs) (Fig. 1Ab). Such channel openings were much less frequent when the neuron was quiescent for > 20 s (Fig. 1Aa). The Po of these channels was estimated by integrating Gaussian curves fitted to all-points histogram plots constructed from 2 s recordings following AP firing (Fig. 1B). This type of analysis indicated that Po increased about fivefold from 0.06 ± 0.03 to 0.33 ± 0.05 (n = 9) following one to three APs. The amplitude of the corresponding unitary current (estimated from the differences between peaks of adjacent Gaussian curves at a transmembrane potential of approximately 30 mV more positive to the resting membrane potential) averaged 0.72 ± 0.05 and 0.77 ± 0.06 pA before and after AP firing, respectively. A typical unitary current (i)-voltage (V) relationship (i-V) of these channels recorded from a cell-attached patch is shown in Fig. 1Bc. The unitary slope conductances of these channels had values of between 20 and 40 pS. Confirmation that the cell from which these recordings were obtained was an AH neuron was obtained after patch rupture and stimulation of a slow AHP under current clamp (Fig. 1C).

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Figure 1. Afterhyperpolarization (AHP) channels in a cell-attached patch recording from an electrophysiologically identified AH neuron

Aa, the pipette potential was held at −60 mV and then stepped to −50 mV. Few channel openings were observed in the patch in the absence of action potential firing: c, closed state; o1, one channel open. b, stepping the pipette potential (Vp) from −60 mV to +60 mV for 110 ms evoked a single action current (arrow) in the neuron and this was followed by the opening of up to three channels at VP=−50 mV. Ba and b, all-points histograms of the corresponding traces shown in A. Note the increase in Po following AP firing. Unitary current amplitude was 0.37. c, i-V relationship of KAHP channels at three values of Vp. A linear regression line through the data points gave a slope conductance of approximately 40 pS. C, whole-cell current clamp recording from the same neuron which generated a slow AHP following a three-pulse stimulus train. The bath contained Krebs solution and the pipette was filled with 145 mm KCl, containing 50 nm Ca2+.

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The ensemble-averaged current constructed from the openings of these channels resembled the macroscopic whole-cell slow AHP current (IAHP) in time course (Fig. 2). As shown in one cell-attached patch in Fig. 2A, there were usually few channel openings when the neuron was not firing (the asterisk in Fig. 2Aa marks a single channel opening) at a Vp of +20 mV. However, immediately following stimulation of an AP with a 100 ms pre-pulse to VP=+60 mV, the frequency of openings of small-to-intermediate conductance channels increased markedly. These channels were resistant to TEA (5 mm) and to apamin (0.5 μm), both of which were present in the pipette solution. Consecutive traces depicting channel openings are shown in Fig. 2Ab-g, with up to four channels being open simultaneously. Digital summation and averaging of these traces yielded an ensemble averaged current trace that peaked within the stimulus pre-pulse (100 ms), but had decayed by only about 50 % after 2 s (Fig. 2Ah), consistent with the development of the slow AHP in the neuron. The unitary current amplitude derived from the all-points histogram constructed from the traces containing active channels was 0.67 pA (Fig. 2B). In the whole-cell configuration, the same AH neuron generated a typical slow AHP (Fig. 2C). To investigate the properties of these KAHP channels more closely, and to be able to control the voltage across the membrane more accurately, further recordings were obtained from excised patches.

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Figure 2. Cell-attached patch recording from an AH neuron with BK and SK channels blocked with TEA (5 mm) and apamin (0.5 μm)

Aa, in the absence of APs, few channel openings were evident (the asterisk marks the opening of one channel). VP=−20 mV. Traces b-g were recorded following 110 ms step depolarizations of the patch (VP=+60 mV) to trigger an AP in the neuron (not shown). Following AP firing, there is an increase in channel activity in the patch. In traces d and e, channel openings continued past the end of the recording. Up to four TEA- and apamin-resistant channels are seen to open simultaneously. h, ensemble current averaged from 10 traces containing channel openings. The patch current during the step depolarization has been removed for clarity. Note slow decay of current. B, all-points histogram constructed from traces containing channel openings, revealing three equidistant peaks and fitted with Gaussian curves (closed state on left and two open states; the third open state is within the baseline). Unitary current was 0.67 pA. C, whole-cell recording after patch rupture shows prominent slow AHP following three evoked action potentials (filled bar) recorded under current clamp.

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Ca2+-activated K+ channels in outside-out patches

To establish that it was possible to isolate these TEA-resistant K+ channels in excised patches, we obtained outside-out patches from AH neurons. This was done by withdrawing the pipette from the soma, following whole-cell access. Patch pipettes were filled with potassium gluconate PS, containing 500 nm free Ca2+, in order to activate KCa channels resident in the patch, from the cytoplasmic side. The outsides of patches were bathed in normal Krebs solution containing nicardipine (1 μm) to inhibit smooth muscle contraction. As shown by a typical recording in Fig. 3A, ramp depolarization of an outside-out patch from −100 mV to +50 mV (from a holding potential, Vh, of 0 mV) revealed the presence of two types of Ca2+-activated K+ (KCa) channels. The larger conductance channels (there were three in this patch) opened with an increasingly higher probability at potentials positive to about 0 mV, while channels of a smaller conductance were active throughout the ramp depolarization (Fig. 3A). Following the addition of TEA (5 mm) to the bathing solution, the openings of the larger conductance channels were blocked, but the openings of the smaller conductance channels were unaffected (Fig. 3B). The current through the smaller conductance channels decreased as the ramp potential approached the Nernst potential for K+ (EK; −90 mV), indicating that these channels were selective for K+. The addition of Ba2+ (1 mm) to the bathing solution inhibited the opening of these smaller conductance K+ channels, being more effective at potentials negative to 0 mV (Fig. 3C). These observations suggest that TEA-sensitive and voltage-dependent large-conductance KCa (BK channels) may be co-localized with smaller-conductance TEA-insensitive KCa channels that appear to possess little or no voltage dependence in their Po at submicromolar levels of cytoplasmic Ca2+. To investigate the Ca2+ and voltage dependence of these putative KAHP channels more closely, we performed single channel recordings from inside-out patches.

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Figure 3. Outside-out patch recording from AH neurons showing two populations of KCa channels

The bath was perfused with Krebs solution and the patch pipette was filled with 145 mm KCl containing 500 nm Ca2+. A, the outside-out patch was ramp-depolarized from −100 mV to +50 mV over 10 s (upper trace). Note activation of BK-type channels (three channels open simultaneously) at potentials positive to approximately +20 mV in the current recording (lower trace). B, ramp current elicited after TEA (5 mm) was added to the Krebs solution. Openings of BK-type channels were abolished, but openings of the smaller conductance channels persisted. C, following the addition of Ba2+ (1 mm) to the TEA-containing Krebs solution, the opening of the smaller conductance channels was markedly inhibited at potentials negative to 0 mV.

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KCa channels in inside-out excised patches

Inside-out patches were excised from AH neurons that were bathed in high-K+ (145 mm potassium gluconate) PS which contained 1 mm EGTA and no added Ca2+ (0 Ca2+ PS; free [Ca2+] was estimated to be < 10 nm assuming the presence of 50 μm of contaminating Ca2+). Chloride was replaced with gluconate in the PS to eliminate possible contamination by currents through Cl channels. In addition, these recordings were performed at room temperature (22 °C) to help maintain the buffering of Ca2+ with EGTA constant. Initial excision of membrane patches from neuronal cell bodies was carried out in 0 Ca2+ PS in order to minimize run-up in the activity of the putative KAHP channels (see below). On most occasions, excision of patches directly into 0 Ca2+ PS resulted in the formation of membrane vesicles. These were ruptured by exposing the tip of the patch pipette in air several times, resulting in a near-doubling in the amplitude of the recorded unitary currents and in more rapid current jumps.

The first series of experiments was carried out with the patch pipettes filled with 0 Ca2+ low-K+ PS (5 mm potassium gluconate and 140 mm sodium gluconate), thus favouring net outward current flow through K+ channels at potentials positive to EK (-90 mV). Also present in the pipette solution were TEA (5 mm) to block BK channels, apamin (0.5 μm) to block SK channels, Cs+ (1–2 mm) to block hyperpolarization-activated cation (IH) channels, and Cd2+ (0.1 mm) to block any co-localized voltage-gated Ca2+ channels and non-selective cation channels. Under these conditions, with 0 Ca2+ high-K+ PS perfused through the bath (i.e. on the cytoplasmic face of the patch), there was little or no channel activity detectable at a Vh of 0 mV (Fig. 4A and Ba). Upon wash-in of 500 nm Ca2+-containing high-K+ PS (> 500 nm free Ca2+), the frequency of unitary outward currents increased over time, consistent with increased openings of K+-selective channels. The time course for activation of these channels was variable from patch to patch, but on average, a period of 5–10 min of continuous perfusion of Ca2+ PS (approximately 5–10 bath volumes) was necessary before channel activity had reached steady state (Fig. 4Ab and Bb). In nine such inside-out patches tested, the increase in activity of these TEA- and apamin-resistant KCa channels could not be reversed after washing out of the Ca2+-containing PS and washing in of 0 Ca2+ PS (Fig. 4Ab and Bc). This activity persisted for the life of the patch or experiment, in one patch persisting for 80 min of continuous perfusion of 0 Ca2+ PS. In four inside-out patches, we confirmed that these Ca2+-stimulated, persistently active KCa channels were indeed K+ selective by blocking currents with 0 Ca2+ PS in which the potassium gluconate had been substituted with equimolar sodium gluconate (Fig. 4Bd). Subsequent re-perfusion of 0 Ca2+ PS containing potassium gluconate through the bath reinstated the previous level of channel activity.

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Figure 4. Activation by cytoplasmic Ca2+ of TEA- and apamin-resistant KCa channels in an inside-out patch

Aa, two TEA- and apamin-resistant KCa channels were stimulated to open following wash-in of 500 nm Ca2+-containing high-K+ PS. The patch was held at 0 mV under asymmetric K+ concentrations at room temperature (5 mm potassium gluconate in pipette; 145 mm potassium gluconate in bath). The pipette solution contained 5 mm TEA and 0.5 μm apamin. b, time course of activation of KCa channels by Ca2+ expressed as NPo (where N is number of channels), showing persistent activity after wash-out of 500 nm Ca2+ PS. The dotted lines represent gaps in continuous recordings where recordings at other potentials were executed. B, activity of KCa channels before (a) and in the presence of (b) Ca2+ PS. c, activity of KCa channels persisted following 5 min wash-out of Ca2+ PS. d, wash-in of 0 Ca2+ PS containing 145 mm sodium gluconate blocked current through KCa channels.

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In contrast to TEA- and apamin-resistant KCa channels, the activation of BK-type channels in inside-out patches (TEA was omitted from the pipette solution in these recordings) by Ca2+-containing high-K+ PS was rapid, requiring less than 30 s for the increase in activity to reach steady state (Fig. 5A). Moreover, the increased activity was reversible after 0 Ca2+ PS was washed back into the bath (Fig. 5A). As shown in Fig. 5B, in the same inside-out patch containing a single BK channel, increasing cytoplasmic [Ca2+] shifted the voltage dependence of activation of BK channels to more negative potentials. When cytoplasmic [Ca2+] was less than 10 nm (0 Ca2+ PS), BK channels failed to open even at potentials as positive as +50 mV (Fig. 5Ba). However, when cytoplasmic [Ca2+] was increased to 500 nm, BK channels began to open at potentials positive to 20 mV (Fig. 5Bb); 10 μm cytoplasmic [Ca2+] caused BK channels to open at potentials as negative as −70 mV (Fig. 5Bc). Similar results were obtained in two other inside-out patches containing BK channels. These recordings illustrating the prompt activation and deactivation of BK channels in response to changes in cytoplasmic [Ca2+] suggest that the failure of the TEA- and apamin-resistant putative KAHP channels to deactivate following wash-out of Ca2+-containing PS is not an artefact of our perfusion system (i.e. that Ca2+ fails to wash out of the bath), and indicates that the persistent activity is an inherent property of these KCa channels in inside-out patches.

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Figure 5. Rapid activation and deactivation of BK-type channels by Ca2+-PS in another inside-out patch

A, the patch which contained a single BK-type channel was held at 0 mV under asymmetrical transmembrane K+ concentrations. Upon wash-in of Ca2+-containing (10 μm) high-K+ PS, there was a rapid increase in the Po of the BK channel; activity quickly reversed following wash-in of 0 Ca2+ PS. B, recordings from the same patch in response to ramp depolarization in the presence of 0 Ca2+ PS in the bath (a) and after wash-in of 500 nm Ca2+(b). PS ramp depolarization activated BK channels at potentials positive to about +20 mV. c, in the presence of 10 μm Ca2+-containing PS in the bath, the ramp depolarization activated BK channels at potentials positive to about −80 mV. TEA was omitted from the pipette solution.

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Lack of voltage dependence of TEA- and apamin-resistant KCa channels

Ramp depolarization of inside-out patches from −100 mV to +50 mV over 10 s failed to reveal any apparent voltage dependence in the opening of TEA- and apamin-resistant KCa channels at a cytoplasmic [Ca2+] of 500 nm (Fig. 6B). In the presence of 0 Ca2+ PS, ramp depolarization of the patches triggered the opening of only a few channels, usually at positive potentials (Fig. 6A). To investigate more closely whether these KCa channels possessed any intrinsic voltage dependence at maintained membrane potentials, we determined their Po at several levels of Vh by integrating Gaussian curves fitted to all-points histogram plots of 20–30 s recordings (Fig. 7). As illustrated for one such patch in Fig. 7A, there was little apparent change in channel activity over a 60 mV voltage range in the presence of 500 nm cytoplasmic Ca2+. This lack of voltage dependence is reflected in the all-points histogram plots and fitted curves (Fig. 7B), which show that these channels spent proportionally the same amount of time in the closed state as they did in the open state at all three membrane potentials. Close inspection of the recordings indicated that there were possibly two channels present in the patch, although simultaneous openings of both channels were rare (the asterisk marks simultaneous opening in Fig. 7Ab).

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Figure 6. The effect of ramp depolarization of an inside-out patch containing TEA- and apamin-resistant KCa channels

A, ramp depolarization from −100 to +50 mV over 10 s failed to activate channel openings except at very positive potentials (cytoplasmic [Ca2+] < 10 nm). B, following equilibration in 500 nm Ca2+-containing PS on the cytoplasmic side of the patch, ramp depolarization of the patch activated TEA- and apamin-resistant KCa channels throughout the ramp. Insets in each panel show expanded portions of the ramp currents around 0 mV (continuous line indicates closed channel level).

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Figure 7. Lack of voltage dependence of opening of TEA- and apamin-resistant KCa channels

A, inside-out patch containing an estimated two channels which were activated with 500 nm Ca2+ high-K+ PS. Steady-state activity is shown at three potentials. The asterisk in trace b indicates the possible simultaneous opening of two channels. o, open state; c, closed state. B, all-points histograms of 20 s recordings of channel activity at the three potentials, fitted with superimposed Gaussian curves. Note that the curves representing the open state distribution are of roughly equal relative area. C, Po-V plot constructed from the mean data, from recordings obtained under asymmetrical K+ concentrations. Data points have been fitted with a Boltzmann function, of the form: Po=Po,max/[1 + exp(V0.5/Vslope)]+Po,basal, where Vslope= 40 mV, V0.5 is −20 mV, Po,max was 0.56 and Po,basal was 0.37. D, i-V relationship of TEA- and apamin-resistant KCa channels under asymmetric K+ concentrations. Data points have been fitted with a form of the GHK current equation, showing open channel outward current rectification. From the fitted curve, the limiting slope conductance at positive potentials is approximately 40 pS and the chord conductance at −50 mV is 12 pS.

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The pooled data of Po plotted as a function of patch potential are shown in Fig. 7C. This Po-V curve indicates that the opening of TEA- and apamin-resistant KCa channels at submicromolar levels of internal Ca2+ has only a very weak dependence on membrane potential at best, or none at all. There is an increase in Po by less than 15 % at positive potentials from a non-deactivating level of approximately 0.4 (Fig. 7C). The slope factor of a Boltzmann curve fitted to the voltage-sensitive component of the Po-V plot was +40 mV (see also Fig. 8C).

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Figure 8. Ca2+-dependent activity of TEA- and apamin-resistant KCa channels under symmetrical K+ concentrations

A, wash-in of 5 μm Ca2+-containing PS on an inside-out patch held at −60 mV and containing a single TEA- and apamin-resistant KCa channel, resulted in an increase in channel activity. B, all-points histogram plots of channel activity recorded in 0 Ca2+ PS and in the presence of 5 μm Ca2+, at −60 mV, fitted with superimposed Gaussian curves. Note basal activity of KCa channels (left graph). C, Po-V curve constructed from the mean data collected from three to eight patches exposed to 2–5 μm cytoplasmic Ca2+. Data points have been fitted with a straight line indicating no voltage dependence. D, i-V plot of the mean unitary current ±s.e.m., fitted with a linear regression line between −60 and +60 mV, yielding a unitary conductance of 62 pS.

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The corresponding i-V relationship of the unitary currents through TEA- and apamin-resistant KCa channels, recorded under asymmetrical K+ concentrations, was reasonably well fitted with the Goldman-Hodgkin-Katz (GHK) current equation (Hille, 1992) (Fig. 7D). From this fit, the limiting slope conductance at positive potentials was estimated to be about 40 pS, whereas the chord conductance at −50 mV was estimated to be about 12 pS.

The Po of TEA- and apamin-resistant KCa channels, at a Vh of 0 mV, in the presence of 500 nm cytoplasmic Ca2+ (0.48 ± 0.04, n = 23) was not statistically different from the mean Po of these channels in patches that were exposed to 10 μm internal Ca2+ (0.51 ± 0.06, n = 7; P > 0.05). The corresponding values of Po in 0 Ca2+ PS for the two series of experiments were 0.021 ± 0.008 (n = 22) and 0.035 ± 0.02 (n = 7), respectively. These data indicate that TEA- and apamin-resistant KCa channels have a high sensitivity to cytoplasmic Ca2+ whereby maximal activation is attained at submicromolar Ca2+ levels. Although the mean Po of these KCa channels in 500 nm internal Ca2+ was similar to the mean Po in 10 μm Ca2+ (at Vh= 0 mV) under steady-state conditions, we noted that the onset of the increase in activity was faster when PS containing higher levels of internal Ca2+ (2–10 μm) was perfused through the bath at the same rate (compare Fig. 4A with Fig. 8A).

The weak or lack of voltage dependence of the TEA- and apamin-resistant KCa channels was also evident when recordings were performed under symmetrical K+ concentrations. As illustrated in Fig. 8A, perfusion of high-K+ PS containing 5 μm Ca2+ on to the inside surface of an inside-out patch activated these channels at physiological potentials (-60 mV). The corresponding all-points histograms indicate that these KCa channels have a low Po in nominally 0 Ca2+ PS at −60 mV, and this increases to about 0.4 when channels are exposed to high-K+ PS containing 5 μm Ca2+ (Fig. 8Ba and b). The pooled values of Po derived from recordings such as these are plotted as a function of Vh in Fig. 8C, which shows that the Po-V relationship in symmetrical [K+] is essentially voltage independent and averaged just under 0.4. The corresponding i-V relationship constructed from the pooled data was approximately linear between −60 and +60 mV, but rectified outside this voltage range (Fig. 8D). The slope conductance at potentials between −60 and +60 mV was 62 pS, while at potentials positive to +60 mV and negative to −60 mV, the chord conductance of channels decreased to approximately 40 pS.

Open and close dwell times of TEA- and apamin-resistant KCa channels

The activity of TEA- and apamin-resistant KCa channels in myenteric AH neurons was characterized by rapid flicker-type openings and closings. In order to resolve single channel openings, traces were further digitally filtered at 1 kHz (8-pole Bessel filter in Clampfit 8) before being subjected to idealized fitting with Fetchan (pCLAMP 8). The mean open and closed dwell times were determined by idealized fitting of selected portions of these recordings (20–30 s) that contained either one channel open at any given time, or a single channel in the patch, using the 50 % threshold criterion for detection of channel transitions. As illustrated in Fig. 9Aa and b, channel activity was stimulated in an inside-out patch by increasing cytoplasmic Ca2+ from < 10 nm to 500 nm. The amplitude histogram of the idealized channel transitions to the open state gave a unitary current of 1.05 pA when fitted with a single Gaussian curve (Fig. 9Ac, o), while transitions to the closed level were centred around 0 pA (Fig. 9Ac, c). The Po of idealized transitions to the open state for the channels in this patch was 0.39 (assuming two channels were present in the patch). Both values of unitary current and Po at 0 mV are similar to the values derived from all-points histograms constructed from the raw data (see Fig. 7C).

image

Figure 9. Kinetic analysis of TEA- and apamin-resistant KCa channel activity recorded under asymmetrical K+ concentrations

Aa, activity in a typical inside-out patch at 0 mV in 0 Ca2+ PS; b, steady-state activity following wash-in of high-Ca2+ PS on the cytoplasmic surface. The trace was digitally filtered at 1 kHz and contains two channels, but simultaneous openings were rare (c). Amplitude histogram plot of the idealized transitions to the open state (o) and to the closed state (c), each fitted with a Gaussian curve. The single channel current was 1.07 and the Po was 0.39. B, dwell time distributions showing that the open dwell time was fitted with a single exponential curve with a time constant of 1.25 ms (a), while the closed dwell time was best fitted with the sum of two exponential functions having time constants of 1.44 and 7.89 ms (b).

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The distribution of open dwell time was well fitted with a single exponential function (Fig. 9Ba). The time constant of this exponential averaged 1.72 ± 0.34 ms (n = 16) at 0 mV. However, curve fitting of the closed dwell time distribution required the sum of at least two exponential functions (Fig. 7Bb), of which the time constants averaged 0.97 ± 0.11 ms (n = 16) and 7.33 ± 2.36 ms (n = 16), in the presence of 500 nm cytoplasmic Ca2+. The relative proportion of these two dwell times was 0.61 and 0.39, respectively. In six patches in which channel activity was analysed in 0 Ca2+ PS, the mean open dwell time (i.e. time constant of the fitted exponetial) averaged 1.8 ± 0.17 ms, while the two closed dwell times averaged 1.0 ± 0.11 ms and 116.5 ± 45 ms, with 64 % of closings occurring to the short-lived closed state and 36 % to the long-lived closed state. These estimates of the open and closed dwell times indicate that neither the open state nor the short-lived closed state is influenced by cytoplasmic Ca2+, suggesting that the rate constants governing transitions between these two states are not Ca2+ dependent. The increase in Po of these putative KAHP channels is largely attributable to a decrease in the time spent in the long-lived closed state.

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Acknowledgements

A novel KCa channel in myenteric AH neurons

We have demonstrated in the present study that myenteric AH neurons express an intermediate conductance KCa channel with novel properties. This 40-pS channel is insensitive to low concentrations of TEA, is resistant to external apamin, displays little if any voltage dependence, has a flickery opening and closing behaviour, and is activated by submicromolar levels of cytoplasmic Ca2+ from which it fails to readily deactivate in a cell-free environment. The resting cytoplasmic [Ca2+] in AH neurons loaded with membrane-permeant, Ca2+-sensitive dye has been estimated to be as low as 90 nm (Tatsumi et al. 1988). This suggests that, based on our cell-attached recordings, 90 nm of cytoplasmic Ca2+ would maintain the Po of the 40-pS KCa channels at about 0.05, which increases to 0.33 in the 2 s following action potential firing. Although cytoplasmic [Ca2+] may increase up to 700 nm following maximal stimulation of AH neurons (Hillsley et al. 2000), our data from inside-out patches suggest that these 40-pS KCa channels may attain a maximal Po with 500 nm cytoplasmic Ca2+, which makes them more sensitive to Ca2+ than are SK channels (Kohler et al. 1996).

A distinguishing feature of these 40-pS KCa channels was their inability in inside-out patches to deactivate once they had been exposed to submicromolar cytoplasmic Ca2+, even following prolonged wash-out of the Ca2+-containing solution. This persistent activity contrasted markedly with the behaviour of BK channels in response to a transient increase in cytoplasmic [Ca2+]. Typically, BK channels activated within tens of seconds of an increase in cytoplasmic [Ca2+] and then promptly deactivated upon wash-out of Ca2+. This response is consistent with the known Ca2+ dependence of neuronal BK channels and their participation in the repolarization phase of the action potential (Franciolini et al. 2001).

Evidence that the 40-pS KCa channel is the KAHP channel

The properties of the 40-pS KCa channels are consistent with these channels being primarily responsible for the Ca2+-activated K+ current that underlies the slow AHP (IAHP). The open probability of 40-pS KCa channels increased after the action potential and the ensemble averaged current caused by the opening of these channels followed the same time course as the slow AHP. Moreover, calculation of the total current flowing through 40-pS channels after the action potential was sufficient to account for IAHP (see below). Like the IAHP (Hirst et al. 1985b; North & Tokimasa, 1987), the 40-pS channels are resistant to block by external TEA (5–10 mm). They are also resistant to apamin, as is the IAHP (Tack & Wood, 1992; Kunze et al. 1994). Other parallels include the finding that these channels are voltage insensitive, as is the IAHP (Morita et al. 1982; Hirst et al. 1985b; North & Tokimasa, 1987; Vogalis et al. 2000) and that they are blocked by external Ba2+, which again parallels the properties of IAHP (North & Tokimasa, 1983; Hirst et al. 1985b). Other Ca2+-activated K+ channels in myenteric AH neurons are unlikely to be significantly involved in the slow AHP. The open probability of BK channels is highly voltage sensitive (Kunze et al. 2000) and SK channels are generally apamin sensitive (Grunnet et al. 2001). In addition, these channels are unlikely to be non-specific cation channels (i.e. IH channels) because they were recorded in the presence of external Cs+ (1–2 mm) which blocks IH channels (Galligan et al. 1990).

Conductance and kinetic properties of 40-pS KCa channels

Under symmetrical (physiological) concentrations of K+ across patches, the limiting slope conductance of TEA- and apamin-resistant KCa channels at potentials positive to +40 mV was estimated to be about 40 pS. However, the chord conductance of these channels at −50 mV, which was extrapolated from the line fitted to the data points according to the GHK current equation, yielded a value of about 12 pS (see Fig. 7D). This value is similar to the chord conductance of the KAHP channels that was determined using noise analysis of the whole-cell IAHP (about 10 pS; Vogalis et al. 2001). However, under symmetrical trans-membrane K+ concentrations, we found that the slope conductance of these KCa channels between −60 mV and +60 mV was higher (62 pS), but that it decreased at potentials negative to −60 mV and positive to +60 mV. Collectively, these unitary conductance values are larger than those reported for SK-type channels, but are similar to the conductance values reported for intermediate-conductance KCa channels (Joiner et al. 1998; Vergara et al. 1998). For convenience, we refer to these putative KAHP channels as 40-pS KCa channels.

Ca2+ dependence of 40-pS KCa channel

As with BK-type channels, activation and deactivation of SK-type channels by internal Ca2+ in cell-free patches has been shown to occur quickly, with time constants of the order of milliseconds (Hirschberg et al. 1998; Hirschberg et al. 1999). Therefore, it would seem unlikely that the slowly activating and non-deactivating 40-pS KCa channels that we have characterized in inside-out patches excised from AH neurons are structurally related to either SK channels, which are gated by Ca2+ through constitutively bound calmodulin (Xia et al. 1998), or to BK channels, which have a Ca2+ binding region formed by the c-terminal portions of the pore-forming subunits (Vergara et al. 1998). The relatively slow onset of activation of the 40-pS KCa channels by cytoplasmic Ca2+ and their failure to deactivate following wash-out of Ca2+ suggests that the conformational change(s) caused by Ca2+ may involve interaction with an endogenous protein, perhaps an enzyme. If a kinase is involved in deactivation, then it is possible that activation of these channels may be mediated by a Ca2+-dependent phosphatase, since in a cell-free environment, phosphorylation would be precluded by the absence of ATP. Furthermore, inhibition of KAHP channels by phosphorylation is suggested by the data of Palmer et al. (1986). Alternatively, it is possible that a cytoplasmic factor which maintains the channels in a conformation state of low Po dissociates from the channel with the increase in internal [Ca2+], which in excised patches, would leave the channels unable to deactivate.

Voltage-independence of 40-pS KCa channel

The open probability of the 40-pS KCa channels in AH neurons showed only a weak or non-existent dependence on membrane potential. In the presence of submicromolar levels of Ca2+, the Po of these channels averaged just under 0.5 in symmetrical and asymmetrical K+ concentrations. This lack of voltage dependence is similar to SK-type channels (Bond et al. 1999) and is consistent with the lack of voltage dependence of the IAHP in AH neurons (Hirst et al. 1985b). Recordings in inside-out patches performed under symmetrical K+ concentrations revealed that the 40-pS KCa channels displayed considerable rectification at positive and negative potentials, whereby the unitary conductance decreased at potential negative to −60 mV. Although we did not investigate the cause of this rectification in the present study, the presence of Mg2+ in our solutions may have contributed to this phenomenon, as has been demonstrated for inwardly rectifying K+ channels (Quayle et al. 1997).

KAHP channels in cell-attached patches

As reported in a previous study (Vogalis et al. 2001), stimulation of action potential firing in the soma of AH neurons was followed by the opening of intermediate conductance K+ channels in cell-attached patches. Because these channel openings occurred when the membrane potential of AH neurons would normally be experiencing time-dependent hyperpolarization (i.e. the slow AHP), the unitary conductance derived from such cell-attached recordings is subject to some error, since the driving force on K+ would be changing with time. A typical slow AHP triggered by a strong stimulus hyperpolarizes the membrane potential by about 10–15 mV at its peak. Despite the confounding influence of the unclamped slow AHP, it was still possible to distinguish approximately equidistant peaks in the all-points histograms constructed from these recordings, at physiological transmembrane potentials. The unitary conductance approximated in this way was between 20 and 40 pS.

The ensemble-averaged current generated by the opening of these 40-pS putative KAHP channels decayed slowly with an estimated time constant of over 2 s, which is faster than the time course of a typical IAHP recorded in the whole-cell configuration (Vogalis et al. 2001), but is similar to IAHPs recorded with intracellular microelectrodes (Hirst et al. 1985b). The discrepancy may be due to perturbation by whole-cell dialysing pipettes or intracellular microelectrodes of the intracellular Ca2+/enzyme milieu that may regulate the activity of KAHP channels.

Openings of these 40-pS KAHP channels in cell-attached patches occurred even though the outsides of these patches were exposed to high-K+ physiological solution containing nanomolar levels (< 10 nm) of Ca2+. This suggests that activation of KAHP channels is unlikely to depend on Ca2+ entry through co-localized N-type voltage-gated Ca2+ channels (Cloues et al. 1997; Marrion & Tavalin, 1998) because of the low [Ca2+] in the pipette solution and the presence of Cd2+. Activating Ca2+ most probably is derived from Ca2+ stores from which it is released following Ca2+ entry onto the inside of the membrane patch (Vogalis et al. 2001).

Role of KAHP channels in the slow AHP

As primary afferent neurons in the enteric nervous system, AH neurons respond to mechanical and chemical stimuli with bursts of action potentials (Furness et al. 1998). The slow AHP is largely responsible for the pronounced adaptation in firing of AH neurons. However, excitatory slow synaptic inputs from other myenteric AH neurons can modulate this firing pattern to produce long discharges of action potentials primarily by causing a diminution in the magnitude of the Ca2+-activated K+ conductance (gK-Ca) that underlies the slow AHP (Kunze & Furness, 1999). The 40-pS KCa channels that we have identified in the present study are likely to make a significant contribution to this gK-Ca. A rough estimate of the magnitude of this contribution can be made from the following measurements: (1) the Po of these channels following AP firing (equal to about 0.33); (2) the area of a typical inside-out patch used in the present study (approximately 1 μm2 for a 20 MΩ pipette); (3) the number of channels per patch (approximately two or three); and (4) the area of a cell body of a typical AH neuron (approximately 4000 μm2, assuming a whole-cell capacitance of 40 pF; Vogalis et al. 2001). From these values, we estimate that there would be 3200 of these putative KAHP channels open at the peak of the slow AHP, assuming maximal Po is attained. If the chord conductance is about 12 pS at −50 mV, then peak conductance would be equal to 25 nS. This is about one and a half times greater than the value derived from whole-cell recordings at the peak of the IAHP (Vogalis et al. 2001), suggesting that sufficient current can pass through the 40-pS channel population to account for the IAHP, but not all the available KAHP channels are activated during the slow AHP. Given their high sensitivity to cytoplasmic [Ca2+], these channels would also be partially active at rest and contribute to the resting gK-Ca in AH neurons (North & Tokimasa, 1987). This is further supported by the finding that AH neurons depolarize when Ca2+ stores are depleted with ryanodine and cytoplasmic [Ca2+] subsequently decreases (Hillsley et al. 2000).

Summary

The current underlying the slow AHP cannot be confidently attributed to any of the Ca2+-activated K+ channels that have been characterized in neuronal and non-neuronal tissues prior to this study (Vergara et al. 1998). The channel that we have characterized in the present study is likely to be responsible for the slow AHP in myenteric AH neurons. It behaves differently from both BK-type and SK-type KCa channels. This channel has a small to intermediate unitary conductance, and once activated by Ca2+ does not readily deactivate in a cell-free environment.

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  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Acknowledgements
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Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Acknowledgements

This work was supported by an NHMRC grant to F.V. and J.B.F. F.V. is the recipient of a CR Roper Fellowship from the University of Melbourne.