Background and tandem-pore potassium channels in magnocellular neurosecretory cells of the rat supraoptic nucleus

Abstract

Magnocellular neurosecretory cells (MNCs) were isolated from the supraoptic nucleus of rat hypothalamus, and properties of K⁺ channels that may regulate the resting membrane potential and the excitability of MNCs were studied. MNCs showed large transient outward currents, typical of vasopressin- and oxytocin-releasing neurons. K⁺ channels in MNCs were identified by recording K⁺ channels that were open at rest in cell-attached and inside-out patches in symmetrical 150 mm KCl. Eight different K⁺ channels were identified and could be distinguished unambiguously by their single-channel kinetics and voltage-dependent rectification. Two K⁺ channels could be considered functional correlates of TASK-1 and TASK-3, as judged by their single-channel kinetics and high sensitivity to pH_o. Three K⁺ channels showed properties similar to TREK-type tandem-pore K⁺ channels (TREK-1, TREK-2 and a novel TREK), as judged by their activation by membrane stretch, intracellular acidosis and arachidonic acid. One K⁺ channel was activated by application of pressure, arachidonic acid and alkaline pH_i, and showed single-channel kinetics indistinguishable from those of TRAAK. One K⁺ channel showed strong inward rectification and single-channel conductance similar to those of a classical inward rectifier, IRK3. Finally, a K⁺ channel whose cloned counterpart has not yet been identified was highly sensitive to extracellular pH near the physiological range similar to those of TASK channels, and was the most active among all K⁺ channels. Our results show that in MNCs at rest, eight different types of K⁺ channels can be found and six of them belong to the tandem-pore K⁺ channel family. Various physiological and pathophysiological conditions may modulate these K⁺ channels and regulate the excitability of MNCs.

Magnocellular neurosecretory cells (MNCs) in the hypothalamus release vasopressin and oxytocin into the circulation via axon terminals in the neurohypophysis in response to various physiological stimuli such as dehydration, haemorrhage and nipple suckling. Synaptic events as well as intrinsic membrane properties regulate the firing rate of MNCs that determines the amount of hormone released into the circulation. An important component of the intrinsic membrane property is the background (also referred to as ‘leak’ or ‘resting’) K⁺ conductance that sets the resting membrane potential at a value negative to the firing threshold in excitable cells. The membrane potential of MNCs at rest under physiological medium is approximately −60 to −65 mV (Oliet & Bourque, 1992; Schrader & Tasker, 1997; Stern & Armstrong, 1997), indicating that a resting K⁺ conductance is present. The background K⁺ conductance is involved in not only setting the resting membrane potential but also the modulation of neuronal firing rate in response to neurotransmitters. For example, the depolarizing effect of glutamate on MNCs has been reported to involve a reduction in the resting K⁺ current (Schrader & Tasker, 1997). Also, the generation of depolarizing after-potentials (DAPs) in MNCs following a repetitive firing has been suggested to involve a reduction in outward K⁺ current that has properties of a background K⁺ current (Li & Hatton, 1997). At present, the identity K⁺ channel or channels that give rise to the background conductance in MNCs is not known, and is the subject of this study.

Recent studies have shown that tandem-pore (2P) K⁺ channels behave as background K⁺ channels, as judged by their openings across the physiological range of membrane potentials, relative voltage independence and rapid activation with little or no inactivation following depolarization (Lesage & Lazdunski, 2000; Goldstein et al. 2001). In situ hybridization studies show that mRNA expression of 2P K⁺ channels is widespread in rat and human brain (Medhurst et al. 2001; Talley et al. 2001). In the rat supraoptic nucleus (SON), mRNAs of 2P K⁺ channels such as TASK-1 (TWIK-related acid-sensing K⁺ channel), TASK-3, TREK-1 (TWIK-related K⁺ channel), TREK-2 and TRAAK (TWIK-related arachidonic acid activated K⁺ channel) are expressed (Talley et al. 2001). More recently, TASK-2 immunoreactivity was detected in the rat SON (Gabriel et al. 2002). These studies suggest that several 2P K⁺ channels may be present and serve as part or all of the background K⁺ conductance in MNCs.

In this study, we attempted to identify potential background K⁺ channels in MNCs by biophysical and pharmacological investigation of K⁺ channels that are open at rest. In particular, the presence of K⁺ channels with properties similar to 2P K⁺ channels was examined. As each 2P K⁺ channel possesses unique single-channel properties, it is possible to compare the native and cloned K⁺ channels and determine which K⁺ channel is functionally expressed in MNCs. Our results show that the eight different K⁺ channels, six of which have properties similar to those of 2P K⁺ channels, may contribute to the background K⁺ current in MNCs. The relative contribution of each of the eight K⁺ channels to the resting K⁺ conductance is discussed.

Methods

Dissociation procedures

MNCs were isolated from the SON of adult rats using an enzymatic procedure similar to that described earlier (Oliet & Bourque, 1992; Foehring & Armstrong, 1996). All animals were used in accordance with the Guide for the Care and Use of Laboratory Animals (DHEW Publication no. NIH85–23). Male Sprague-Dawley rats (110–150 g) were decapitated using a small animal guillotine without anaesthesia and the brains were removed. A thin piece (< 1 mm thick) of SON was obtained using ultra-fine micro-dissecting scissors. The tissue pieces were incubated for 15 min at 35 °C in an oxygenated (100 % O₂/5 % CO₂) solution containing (mm): 125 NaCl, 3 KCl, 2 CaCl₂, 2 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃ and 20 d-glucose, with 1 mg ml⁻¹ protease (Sigma, catalogue no. P8811). The tissues were removed and washed three times in oxygenated low Ca²⁺-medium (mm): 140 sodium isethionate, 2 KCl, 0.1 CaCl₂, 4 MgCl₂, 15 Hepes, 23 mm d-glucose). Tissues were then gently triturated with fire-polished glass pipettes. The dispersed cells were plated onto glass coverslips pretreated with poly-l-lysine and used in electrophysiological recording for up to 4 h.

Immunocytochemistry

Vasopressin (VP)- or oxytocin (OT)-containing neurons were visualized by immunocytochemistry with primary antibody against VP-neurophysin (VP-NP; antibody PS-41) or OT-NP (antibody PS-36) generously supplied by Dr Harold Gainer (NIH). The two antibodies do not cross react (Ben-Barak et al. 1985). Isolated MNCs were fixed in 5 % acrolein and treated with 0.01 m sodium metaperiodate and 1 % sodium borohydride to remove aldehyde residues. Fixed cells were then incubated in 5 % normal donkey serum in 0.1 m phosphate-buffered saline (PBS) containing 0.4 % Triton X-100 to block non-specific staining. They were then incubated with a primary antiserum against VP-NP or OT-NP for 48 h at 4 °C. Following extensive washing in 0.05 m PBS containing 0.25 % Triton X-100, fixed cells were incubated for 90 min with biotinylated antibody (horse anti-mouse IgG). The cells were then washed and incubated for 90 min with Vectastain ABC reagent (Vector Labs), and then washed again. Further incubation with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma) activated with 0.03 % H₂O₂ for 5–7 min formed the reaction product. After air-drying, the stained neurons were identified using an inverted light microscope equipped with interference-contrast optics.

Transfection in COS-7 cells

Rat TASK-1, TASK-3 and TREK-2 were cloned previously (Kim et al. 1999, 2000; Bang et al. 2000). The coding DNA sequence of TASK-2 was cloned by RT-PCR using first strand cDNA prepared from rat liver total RNA. The coding regions of rat TASK-1, TASK-2, TASK-3 and TREK-2 were subcloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). COS-7 cells were seeded at a density of 2 × 10⁵ cells per 35 mm dish, 24 h prior to transfection in 10 % bovine serum in Dulbecco's modified Eagle's medium (DMEM). COS-7 cells were co-transfected with a plasmid (pcDNA3.1) containing a 2P K⁺ channel DNA and a plasmid (pcDNA3.1) containing green fluorescent protein (GFP) using LipofectAMINE and OPTI-MEM I reduced serum medium (Life Technologies, Rockville, MD, USA). Green fluorescence from cells expressing GFP was detected with the aid of a Nikon microscope equipped with a mercury lamp light source. Cells were used 2–3 days after transfection.

Electrophysiological studies

Electrophysiological recording was performed using a patch clamp amplifier (Axopatch 200, Axon Instruments, Union City, CA, USA). All recordings were performed at room temperature (≈24 °C). Single-channel currents were digitized with a digital data recorder (VR10, Instrutech, Great Neck, NY, USA), and stored on videotape. The recorded signal was filtered at 5 kHz using an 8-pole Bessel filter (-3 dB; Frequency Devices, Haverhill, MA, USA) and transferred to a computer (Dell) using the Digidata 1200 interface (Axon Instruments) at a sampling rate of 20 kHz. Threshold detection of channel openings was set at 50 %. Whole-cell currents were recorded after cancelling the capacitive transients. Whole-cell and single-channel currents were analysed with the pClamp program (version 7). For single-channel analysis, the filter dead time was 100 µs (0.3/cutoff frequency) such that events shorter than 50 µs in duration were missed. Data were analysed to obtain a duration histogram, an amplitude histogram and relative channel activity (relative NP_o) using Fetchan and pSTAT programs of the pClamp software. N is the number of channels in the patch, and P_o is the probability of a channel being open. Mean amplitudes were obtained from amplitude histograms at each membrane potential and used to plot current-voltage relationships. NP_o was determined from ≈1 min of current recording in patches that contained 1–3 channels. Single-channel current tracings shown in figures were filtered at 2 kHz. In experiments using cell-attached and excised patches, pipette and bath solutions contained (mm): 150 KCl, 1 MgCl₂, 5 EGTA and 10 Hepes (pH 7.3). The pH was adjusted using HCl or KOH to desired values. In whole-cell recordings, bath solution contained (mm): 135 NaCl, 3 KCl, 0.5 CaCl₂, 1 MgCl₂ and 10 Hepes. The pH was adjusted to 7.3 using NaOH. Free fatty acids were dissolved by sonicating for 5 min (Heat Systems-Ultrasonics, Inc. W-380, Farmingdale, NY, USA) in the bath recording solution at a desired concentration. All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA). For statistics, Student's t test and analysis of variance were used with P < 0.05 as the criterion for significance. Data are represented as means ± s.d.

Results

MNCs from the SON

Figure 1A shows photomicrographs of isolated MNCs each showing a large soma with dendrites that were truncated during the isolation procedure. Each isolated MNC that we used in this study was not identified as either a VP- or OT-containing neuron. However, immunocytochemistry using antibodies to VP-NP and OT-NP performed on several (n = 5) typical isolated cell preparations showed positive immunostaining for both VP and OT neurons. Figure 1A shows a photomicrograph of immunostained neurons for VP-NP. Of 362 neurons examined, 44 % showed positive immunostaining for either VP-NP or OT-NP. Of the immunostained cells, 71 % were positive for VP-NP and 29 % were positive for OT-NP. MNCs that immunostain positively with vasopressin and oxytocin antibodies have been reported to be of certain size such that their cross-sectional areas are greater than 160 µm² and they exhibit large transient outward currents (Bourque, 1988; Cobbett et al. 1989; Fisher et al. 1998). Therefore, cells with cross-sectional areas greater than 160 µm² were chosen for this study. The majority of cells chosen for study had cross-sectional areas greater than 200 µm². When neurons with areas greater than 160 µm² (n = 219) were counted, 76 and 47 % of cells were immunostained positively for VP-NP and OT-NP, respectively. This suggests, as reported previously (Glasgow et al. 1999), that as many as 20 % of SON neurons express both genes. Therefore, the neurons that we chose for recording can be considered to be either VP- and/or OT-containing neurons.

Figure 1. Activation of transient outward and sustained K⁺ currents by depolarization in magnocellular neurosecretory cells.

A, photomicrographs of magnocellular neurosecretory cells (MNCs) isolated from the supraoptic nucleus (SON, first two pictures). Note the presence of short dendrites. The lengths and widths of the ellipsoidal cells were estimated using a calibrated Klarman reticle (KR-2078P) inserted into the eyepiece of the microscope. The areas of MNCs were then calculated assuming an ellipsoidal shape (r₁r₂π). The scale bar represents 16 µm in length for the first two pictures. The third picture shows immunocytochemical identification of VP-containing neurons. In this field, all three MNCs showed immunoreactivity against VP-NP. The scale bar represents 30 µm in length. B, cell membrane potential was held at −80 mV, then hyperpolarized to −120 mV for 1 s and then stepped to −10 mV for 300 ms before stepping back to −80 mV. Application of 4-aminopyridine (4-AP) reduced the transient outward current. 4-AP-sensitive current (I_TO) is shown on the right. C, in another MNC, the same voltage protocol was used to elicit the I_TO. In this cell the sustained outward current was large. Application of 10 mm TEA reduced the sustained current. TEA-sensitive K⁺ current is shown on the right.

In all six cells examined, clearly identifiable transient outward currents, with peak amplitudes ranging from 2–4 nA, were elicited by applying voltage steps from −120 to −10 mV (Fig. 1B). As the experiments were carried out in medium mimicking a normal physiological solution, the observed current includes Na⁺, Ca²⁺ and K⁺ currents. 4-Aminopyridine (4-AP; 10 mm), a well-known blocker of transient outward current (I_A or I_TO), abolished the appearance of I_TO, leaving a sustained outward current. The 4-AP-sensitive current, representing the I_TO, is shown as the subtracted current. Tetraethylammonium (TEA; 10 mm) did not reduce the peak I_TO and increased the apparent peak-to-steady state ratio by reducing the sustained current, as reported previously (Bourque, 1988). An example of this effect of TEA is shown in a MNC that possessed a high level of sustained current (Fig. 1C). In our studies, TEA (10 mm) decreased the sustained current by 58 ± 11 % (n = 4), indicating that the sustained current was a non-inactivating K⁺ current, probably consisting of delayed rectifying and background K⁺ currents (Bourque, 1988; Cobbett et al. 1989). In this study, we attempted to identify potential background K⁺ channels that might give rise to the resting K⁺ conductance in MNCs to understand the nature of this important current that regulates the resting membrane potential and cell excitability.

K⁺ channels present in cell-attached patches of MNCs

To identify putative background K⁺ channels in MNCs, we recorded single ion channel currents in cell-attached patches with pipette and bath solutions containing 150 mm KCl. In 680 cell-attached patches formed from MNCs, we recorded eight different types of channels that were K⁺ selective, as judged by their expected shifts in reversal potential after a change in [K⁺]_o, as described in the sections below. Figure 2A shows expanded current tracings of eight different K⁺ channels recorded at −40 mV in MNCs. The abbreviations to designate the K⁺ channels are on the left of each current tracing. Some K⁺ channels were more active than others, but all eight of them were found to be open at rest in many patches (Fig. 2A). As described below, each of the eight K⁺ channels could be clearly distinguished from each other based primarily on their single-channel kinetics, and also from different pharmacological and modulatory effects. By forming cell-attached patches from one MNC several times, we were able to determine that as many as six different K⁺ channels could be present in a single MNC. The K⁺ channels shown in Fig. 2A represent those that are present in the cell soma only as we have not recorded from dendrites.

Figure 2. Single-channel currents recorded from cell-attached patches in MNCs.

A, eight types of K⁺ channel currents were recorded from MNCs at −40 mV. Bath and pipette solutions contained 150 mm KCl. Each channel type was determined to be K⁺ selective by measurement of reversal potential shifts after changing bath or pipette [KCl] and by lack of current in Na⁺-containing solution. AS, acid/alkali sensing; AA, arachidonic acid; TRAAK, TWIK-related AA-activated K⁺ channel; IRK, inwardly rectifying K⁺ channel. B, the bar graph shows the percentage of patches showing each type of K⁺ channel. C, the bar graph shows the estimated relative total current for each type of K⁺ channel, calculated from the equation I =NP_oi, where N is the number of K⁺ channels of a particular type in the patch, P_o is the open probability determined from 10–12 patches and i is the single-channel amplitude at +40 mV.

The bar graph in Fig. 2B shows the percentage of cell-attached patches in which each of the eight K⁺ channels were observed in MNCs. Each bar corresponds to the channel tracing on its left. It is clear that one of them (third current tracing from the top) is the most abundant while seven others are found in much less abundance and distributed relatively evenly. The relative current produced by each K⁺ channel type was estimated by determining the total channel activity (NP_oi) for that channel. In this calculation, N is the total number of K⁺ channels of a particular type observed (from Fig. 2B), P_o is the open probability of the K⁺ channel averaged from 10–12 recordings and i is the amplitude of the K⁺ channel current at +40 mV. This is an estimation calculated at one membrane potential using high [KCl] and clearly does not reflect the K⁺ channel current under physiological conditions. Nevertheless, Fig. 2C illustrates that, under the ionic conditions and membrane potential used, the most abundantly found K⁺ channel would provide the major fraction (72 % of total) of the total K⁺ conductance in the soma of MNCs. The other seven K⁺ channels together provide ≈30 % of the K⁺ current under these ionic conditions. Below, we describe the properties of each of the eight K⁺ channels and compare them to those of the cloned 2P K⁺ channel that have been described to encode various background K⁺ channels.

Two native K⁺ channels (i_K.AS1 and i_K.AS2) similar to TASK-1 and TASK-3

Two of the eight K⁺ channels that we observed in MNCs possessed characteristics similar to those of TASK-1 and TASK-3, which are members of the 2P K⁺ channel family (Duprat et al. 1997; Kim et al. 1999, 2000; Rajan et al. 2000). In Fig. 3, expanded current tracings of native K⁺ channels (that we have designated i_K.AS1 and i_K.AS2; where AS stands for acid/alkali sensing), and cloned TASK-1 and TASK-3 expressed in COS-7 cells are compared. Current-voltage relationships show that the two native K⁺ channels have single-channel conductances (14 ± 1 and 27 ± 1 pS at −60 mV) similar to those of TASK-1 and TASK-3 (14 ± 1 and 28 ± 1 pS at −60 mV). In symmetrical 150 mm KCl, both i_K.AS1 and i_K.AS2 in MNCs exhibited weak inward rectification, similar to those of TASK-1 and TASK-3 (Fig. 3). Mean open times measured at 60 mV were 0.7 ± 0.1 and 1.0 ± 0.1 ms for i_K.AS1 and i_K.AS2, respectively. This is not statistically different from the mean open times of TASK-1 (0.8 ± 0.1 ms) and TASK-3 (1.1 ± 0.1 ms) measured from COS-7 cells (P < 0.05; n = 4). The reversal potentials of i_K.AS1 and i_K.AS2 shifted by 33 ± 3 and 34 ± 3 mV (n = 3) when bath KCl concentration was changed from 150 mm to 35 mm in inside-out patches. A shift of 37 mV is expected from the Nernst equation for a K⁺-selective ion channel. In inside-out patches, both i_K.AS1 and i_K.AS2 were clearly present when KCl in the bath was replaced with potassium glutamate (-60 to +60 mV), but neither was observed when KCl was replaced with NaCl, indicating that they are not non-selective cation channels.

Figure 3. Electrophysiological properties of two native K⁺ channels (i_K.AS1 and i_K.AS2) in MNCs.

A, single-channel openings of i_K.AS1 in a MNC and TASK-1 expressed in a COS-7 cell. Current-voltage relationships of i_K.AS1 (open circles) and TASK-1 (filled circles) are shown (n = 3). In outside-out patches, changing the pH_o from 7.3 to 6.3 and 5.8 produced marked decreases in channel activity of both i_K.AS1 and TASK-1. B, single-channel openings of i_K.AS2 in a MNC and TASK-3 expressed in a COS-7 cell. Current-voltage relationships of both currents are shown (n = 3). In outside-out patches, changing the pH_o from 7.3 to 6.3 and 5.8 produced marked decreases in channel activity of both i_K.AS2 and TASK-3. Each point and bar is the mean ± s.d. of 3 determinations. The values obtained at pH 6.3 and 5.8 were significantly different from that at pH 7.3 (P < 0.05). However, no difference was observed between i_K.AS and TASK at each pH value (P > 0.05), as determined by analysis of variance.

Agents that have either no effect (TEA, Cs⁺) or inhibit TASK channels (quinidine, arachidonic acid (AA)) were found to have nearly identical effects on the two native K⁺ channels. Thus, 1 mm TEA and 1 mm CsCl applied to the cytoplasmic side of inside-out patches had no significant effect (P > 0.05; n = 4), whereas 10 µm AA caused 67 ± 12 and 56 ± 6 % inhibition of i_K.AS2 and i_K.AS2 in MNCs, respectively (n = 4). A distinguishing property of TASK-1 and TASK-3 is that they are highly sensitive to changes in extracellular pH (Duprat et al. 1997; Kim et al. 1999; Talley et al. 2000). In outside-out patches, the two native K⁺ channels were also inhibited markedly when the pH_o was changed from 7.3 to 6.3 and 5.8 (Fig. 3). Although data from only three patches were obtained due to difficulties in getting outside-out patches containing only TASK-1 or TASK-3, the results strongly suggest that the TASK-1 and TASK-3 encode the two native K⁺ channels in MNCs shown in Fig. 3. The results also show that i_K.AS1 and i_K.AS2 are active at rest and probably contribute to the resting K⁺ conductance in the cell soma of MNCs.

Another native pH_o-sensing K⁺ channel (i_K.AS3)

A K⁺ channel that was observed most frequently showed interesting single-channel behaviour (Fig. 4A). In nearly all patches that contained this channel, the open probability was relatively high (> 0.4) at potentials positive to the reversal potential, and gradually decreased as the membrane potential was held at more negative values. The channels tended to open in long bursts at depolarized potentials compared to those at hyperpolarized potentials, producing the higher P_o at depolarized potentials (Fig. 4C). Open-time duration histograms could each be fitted with a single-exponential function from which the channel mean open times were determined to be greater at depolarized potentials (3.6 ± 0.3 ms; −40 mV) than at hyperpolarized potentials (0.8 ± 0.1 ms; −40 mV). The single-channel current-voltage relationship was nearly linear with a slope conductance of 35 ± 2 pS (Fig. 4B). As expected for a K⁺-selective channel, the reversal potential shifted −34 ± 2 mV when KCl in the bath solution was changed from 150 mm to 35 mm in outside-out patches (n = 4).

Figure 4. A K⁺ channel (i_K.AS3) with high open probability and sensitive to extracellular pH in MNCs.

A, single-channel currents in a cell-attached patch at different membrane potentials. Duration and amplitude histograms at +40 and −40 mV are shown. B, current-voltage relationship is relatively linear with a conductance of 35 ± 1 pS. C, plot showing the voltage dependence of channel activity. D, current tracings show the effect of changes in pH_o on channel activity in outside-out patches. E, plot of channel activity as a function of pH_o and pH_i. Inside-out patches were used to study the effect of changes in pH_i. Each point is the mean ± s.d. of 6 determinations. * Significantly different from the corresponding control value (P < 0.05) as determined by analysis of variance.

To further characterize this K⁺ channel, we studied its sensitivity to various pharmacological agents, as well as pH. Unlike TASK-1 and TASK-3, this K⁺ channel was inhibited by 86 ± 7 % when 1 mm TEA was applied to the intracellular side of the membrane (n = 5). TEA showed no significant effect from the extracellular side of the membrane. Ba²⁺ (1 mm) and Cs⁺ (1 mm) produced 91 ± 9 and 43 ± 6 % inhibition, respectively, of i_K.AS3 from the extracellular side (n = 5). Another interesting feature of this K⁺ channel was that 20 µm AA produced an activation from the cytoplasmic side of the membrane (2.4 ± 0.9-fold at −60 mV; n = 5), in contrast to the marked inhibition of i_K.AS1–2 and TASK channels by AA.

Similar to i_K.AS1 and i_K.AS2, i_K.AS3 showed a high sensitivity to changes in pH_o. Figure 4D shows expanded current tracings obtained from an outside-out patch at different pH_o values. The major effect of pH_o was on the frequency of opening. The relative current plotted as a function of pH_o shows the marked inhibitory effect of acid medium and a strong stimulatory effect of alkaline medium. Similar changes in intracellular pH with pH_o kept constant at 7.3 produced a much smaller effect on channel activity, as shown by the slope of the two lines in Fig. 4E. Therefore, the extracellular effect of acid or alkali was clearly not via a change in intracellular pH, since one pH_o unit change is known to produce only a small change (< 0.1 pH unit) in pH_i in a typical cell (Deitmer & Schneider, 1998). Our literature search so far has failed to identify the K⁺ channel gene that encodes this acid- and alkali-sensing K⁺ channel. The relatively high density and high P_o of i_K.AS3 suggests that this K⁺ channel is likely to be responsible for a large fraction of background K⁺ conductance in MNCs.

A native K⁺ channel similar to TRAAK

Another type of channel that was found in cell-attached patches of isolated MNCs is shown in Fig. 5A. The P_o varied greatly among patches and tended to increase slightly after patch excision. Unique characteristics of this K⁺ channel are that the open-time duration is very short (0.3–0.5 ms) and the single-channel conductance is relatively large (105 ± 8 pS at −40 mV; n = 3) at potentials negative to reversal potential. In outside-out patches, a change in bath [KCl] from 150 mm to 35 mm shifted the reversal potential from 0 mV to −34 ± 3 mV (n = 3), close to the −37 mV shift calculated from the Nernst equation. This channel was not observed when K⁺ in the bath was replaced with Na⁺, showing that it is a K⁺ channel, not a non-selective cation channel. The current-voltage relationship was mildly inwardly rectifying in symmetrical 150 mm KCl. This K⁺ channel was not significantly blocked by 0.1 mm Ba²⁺, 1 mm Cs⁺ or 1 mm TEA (P > 0.05; n = 4 each) applied to the external solution. These single-channel characteristics are reminiscent of TRAAK, a 2P K⁺ channel cloned from brain (Maingret et al. 1999a; Kim et al. 2001). The single-channel conductance of TRAAK expressed in COS-7 cells was 110 ± 7 pS at −40 mV and the mean open time was 0.3 ± 0.1 ms (Fig. 5). TRAAK produced less than 5 % changes in response to 0.1 mm Ba²⁺, 1 mm Cs⁺ or 1 mm TEA (P > 0.05; n = 4 each).

Figure 5. A native K⁺ channel with properties similar to TRAAK.

A, single-channel currents in a cell-attached patch of a MNC at different membrane potentials are shown along with amplitude and duration histograms (+40 mV) and the current-voltage relationship. B, single-channel currents in a cell-attached patch of a COS-7 cell expressing TRAAK at different membrane potentials are shown along with amplitude and duration histograms (+40 mV) and the current-voltage relationship. C, the effect of applying negative pressure, changing intracellular pH and applying AA in inside-out patches of MNCs are shown. The bar graph on the right shows the summary of these results. Each bar is the mean ± s.d. of 4–5 determinations. [KCl] in pipette and bath solutions was 150 mm. * Significantly different from the corresponding control value (P < 0.05).

To obtain additional evidence that the K⁺ channel recorded in MNCs may be the native functional correlate of TRAAK, the effects of three manoeuvres (membrane stretch, alkali and free fatty acid) that are known to activate TRAAK were studied in MNCs. As shown in Fig. 5C, application of negative pressure to the pipette interior produced a clear and reversible increase in channel activity. In some cell-attached patches where channel openings were not present, application of small negative pressure was enough to elicit activation of this K⁺ channel. Perfusion of the cytoplasmic side of inside-out patches with solution at different pH levels showed that alkali but not acid conditions caused activation of the native K⁺ channel in MNCs, as also observed with TRAAK (Kim et al. 2001). AA (10 µm) applied to the cytoplasmic side of the membrane also activated the K⁺ channel in MNCs. Linoleic and oleic acids (10 µm), which are also unsaturated free fatty acids, produced similar activation of the K⁺ channel. These results suggest that the effects of free fatty acids are likely to be directly on the membrane. A summary of these experiments is shown in the bar graph (Fig. 5). Recently, TRAAK mRNA has been detected in the SON of rat brain (Talley et al. 2001). We have also confirmed the expression of TRAAK mRNA by RT-PCR using first strand cDNA prepared from total RNA of SON (data not shown). Thus, these results provide strong evidence that the native K⁺ channel present in MNCs is very likely to be encoded by TRAAK. This is the first description of the functional expression of a native, TRAAK-like K⁺ channel in the mammalian system.

Two native K⁺ channels (i_K.AA1 and i_K.AA2) similar to TREK-1 and TREK-2

TREK-1 and TREK-2 are 2P K⁺ channels whose single-channel kinetics and modulatory properties have been well-characterized in previous studies (Maingret et al. 1999b; Bang et al. 2000). In cell-attached and inside-out patches of MNCs, we identified two K⁺ channels with single-channel properties nearly identical to those of TREK-1 and TREK-2. Figure 6 shows the single-channel currents of native K⁺ channels recorded from MNCs and those of TREK-1 and TREK-2 expressed in COS-7 cells. The single-channel conductance of the two native channels that we designate here as i_K.AA1 and i_K.AA2 (AA-activated) were 120 ± 8 and 66 ± 5 pS, respectively, at +40 mV, values that are similar to those of TREK-1 (116 ± 8 pS) and TREK-2 (62 ± 4 pS). The current-voltage relationships and the associated rectification, mean open times and the intrinsically noisy open state of i_K.AA1 and i_K.AA2 were also very similar to those of TREK-1 and TREK-2 (Fig. 6). As with TREK, i_K.AA1 and i_K.AA2 were activated by application of pressure to the membrane and free fatty acids, as illustrated by two examples in Fig. 6A and B. In contrast to TRAAK, only acid conditions are known to activate both TREK-1 and TREK-2. Both i_K.AA1 and i_K.AA2 in MNCs were activated by acidic conditions (see bar graphs). Like TREK-1 and TREK-2, i_K.AA1 and i_K.AA2 in MNCs were not significantly (P > 0.05, n = 4) blocked by 1 mm TEA and 1 mm Cs⁺.

Figure 6. K⁺ channels in MNCs (i_K.AA1 and i_K.AA2) with properties similar to TREK-1 and TREK-2.

A, single-channel currents (i_K.AA1) in cell-attached patches of a MNC and a COS-7 cell expressing TREK-1 are shown at 3 membrane potentials. The current-voltage relationships of i_K.AA1 and TREK-1 are almost identical. In inside-out patches of MNCs, the effect of applying negative pressure, acidic pH and AA are shown in the bar graph. A current tracing illustrating an example of the effect of applying negative pressure to an inside-out patch of a MNC is shown. B, single-channel currents (i_K.AA2) in cell-attached patches of a MNC and a COS-7 cell expressing TREK-2 are shown at 3 membrane potentials. Inwardly rectifying current-voltage relationships of both channels are shown. In inside-out patches of MNCs, the effects of applying negative pressure, acidic pH and AA are shown in the bar graph. A current tracing illustrating an example of the effect of applying AA to an inside-out patch of a MNC is shown. All points are means ± s.d. of 3 values and bars are means ± s.d. of 5 values. [KCl] in pipette and bath solutions was 150 mm. * Significantly different from the corresponding control value (P < 0.05).

In many cell-attached patches, the presence of i_K.AA1 and i_K.AA2 was not obvious as they were in the closed state. However, opening of these channels could be observed following application of one of the three activators (pressure, acid or free fatty acid). In 22 % of cell-attached patches, a low basal activity of either i_K.AA1 or i_K.AA2 was present without any intervention. It was possible that i_K.AA1 and i_K.AA2 may have been active at rest because some cell swelling occurred. In a near-physiological bath solution (310 mosmol l⁻¹) that did not cause cell swelling, opening of i_K.AA1 and i_K.AA2 could still be observed using pipettes that contained 150 mm KCl without applied negative pressure.

A novel TREK-like K⁺ channel (i_K.AA3)

In addition to i_K.AA1 (TREK-1-like) and i_K.AA2 (TREK-2-like) channels, we were able to identify another putative member of the TREK-like K⁺ channel family in MNCs. The biophysical characteristics of i_K.AA3 were very similar to those of i_K.AA1 and i_K.AA2 with respect to single-channel openings, such as noisy open state and openings in bursts (Fig. 7A). As shown in the current-voltage relationships of the three K⁺ channels, the single-channel conductance of i_K.AA3 was different from those of i_K.AA1 and i_K.AA2. The conductance of i_K.AA3 at negative potentials (inward current) was lowest and the conductance at positive potentials was slightly higher than that of i_K.AA2 but lower than that of i_K.AA1 (Fig. 7B). Other features of i_K.AA3 that are very similar to those of TREK channels include activation by application of negative pressure to the membrane, acidic conditions and AA (Fig. 7C). Although not shown, other unsaturated free fatty acids (linoleic, oleic and linolenic acids) also caused reversible activation of i_K.AA3. K⁺ channel blockers were also generally ineffective in inhibiting this K⁺ current, similar to the effects observed with the other two TREK-like K⁺ channels. Thus, 1 mm TEA and 1 mm Cs⁺ applied either to the extracellular or intracellular sides of the membrane had no effect on i_K.AA3 (P > 0.05; n = 5) and 1 mm Ba²⁺ produced only a small inhibition (22 ± 4 %; n = 5) when applied to the cytoplasmic side of the membrane. Although the gene that encodes i_K.AA3 has not yet been reported, the characteristics of this K⁺ channel suggest strongly that it is likely to be a member of the TREK family of 2P K⁺ channels. Of the three i_K.AA1–3, i_K.AA3 was the most frequently observed channel in MNCs. As with i_K.AA1 and i_K.AA2, i_K.AA3 was also in the quiescent state in many, but not all, cell-attached patches (72 %). A search in GenBank for a DNA sequence similar to those of TREK-1, TREK-2 or TRAAK failed to yield any novel TREK-like K⁺ channel gene.

Figure 7. A novel K⁺ channel (i_K.AA3) sensitive to pressure, acid and AA.

A, single-channel currents in a cell-attached patch of a MNC are shown at different membrane potentials in 150 mm KCl. B, amplitude and duration histograms of channel openings at −40 mV are shown. The current-voltage relationship of i_K.AA3 is compared with those of i_K.AA1 and i_K.AA2. C, examples of current tracings showing the effects of negative pressure, acidic and alkaline pH, and AA are illustrated. A summary of these results is shown in the bar graph. Each bar represents the mean ± s.d. of 5 values. * Significantly different from the corresponding control value (P < 0.05).

A native K⁺ channel similar to a classical inward rectifier K⁺ channel

One of the eight K⁺ channels recorded in MNCs showed characteristics typical of a classical inward rectifier K⁺ channels (Kir2 family). Thus, the single-channel currents showed long openings, and strong inward rectification (Fig. 8A). The single-channel conductance for the inward current was 14 ± 4 pS, a value that is similar to that of IRK3 (Kir2.3) which has a conductance of 13 pS and has been shown to be expressed in the brain (Morishige et al. 1994). An earlier study showed no expression of IRK3 mRNA in the SON by in situ hybridization (Karschin et al. 1996). Therefore, the inward rectifier K⁺ channel found in MNCs of the SON may be a new member of the IRK family. We have not observed K⁺ channels similar to IRK1 and IRK2 that have single-channel conductances of 22 and 34 pS, respectively (Isomoto et al. 1997). The probability of observing the IRK3-like K⁺ channel in MNCs was lowest among all eight K⁺ channels, suggesting that the IRK3-like K⁺ channel contributes very little to the overall background K⁺ conductance in the cell soma of MNCs.

Figure 8. A strongly inwardly rectifying (IRK-like), Ca²⁺-activated and ATP-sensitive K⁺ channel in MNCs.

A, current tracings show single-channel currents from a cell-attached patch of a MNC. The pipette and bath [KCl] were 150 mm. The current-voltage relationship shows the strong inward rectification of this channel. B, opening of a large conductance Ca²⁺-activated K⁺ channel in an inside-out patch is shown. The pipette and bath [KCl] were 150 mm. Intracellular [Ca²⁺] was ≈1 µm. C, opening of an ATP-sensitive K⁺ channel in an inside-out patch is shown. The pipette and bath [KCl] were 150 mm. The scales for the current tracings were drawn to be identical for all three types of K⁺ channel to show the marked differences in the single-channel amplitudes and opening characteristics.

Other K⁺ channels

During the course of our study, we observed the opening of large conductance Ca²⁺-activated (K_Ca) K⁺ channels and ATP-sensitive (K_ATP) K⁺ channels in inside-out patches, with single-channel conductances of 188 ± 10 and 68 ± 5 pS at −60 mV, respectively (n = 4 each; Fig. 8B and C). These two types of K⁺ channels were not open at rest under physiological solution in cell-attached patches. The large conductance, 188 pS K_Ca channels could be activated by addition of 1–10 µm Ca²⁺ to the cytoplasmic side of the membrane, as expected (not shown). The presence of this K_Ca channel in MNC cell bodies has been reported in a previous work (Dopico et al. 1999).

K_ATP channels with a single-channel conductance of 68 pS (-60 mV) were activated upon forming inside-out patches, presumably due to depletion of intracellular ATP, and closed upon addition of 2 mm ATP (not shown). The single-channel conductance of the K_ATP channel in MNCs was nearly identical to that of the cardiac K_ATP channel (70 ± 2 pS, −60 mV). Interestingly, 3–4 channels were usually present in patches that contained K_ATP channels (14 out of 270 patches), suggesting that K_ATP channels may be clustered on the membrane surface at a low density. As these K⁺ channels are not part of the background K⁺ conductance, we did not study them further.

Discussion

The electrical properties of MNCs are critical for their role in vasopressin and oxytocin release that occurs under various physiological conditions. The pattern and frequency of firing in MNCs that determine the quantity of hormones released into circulation depend on time- and voltage-dependent activities of various ionic currents. Many types of K⁺ currents are expressed in MNCs, including transient outward K⁺ current (I_TO), Ca²⁺-activated K⁺ current I_(K,Ca), delayed rectifier K⁺ current (I_K), hyperpolarization-activated inward current (I_H), and background (or leak) K⁺ current (Renaud & Bourque, 1991; Schrader & Tasker, 1997; Armstrong & Stern, 1998; Ghamari-Langroudi & Bourque, 2000). A sustained outward K⁺ current that is activated by depolarization has also been described in OT-releasing MNCs (Stern & Armstrong, 1997). Of all the K⁺ currents, the background K⁺ current is the least characterized.

The resting membrane potential of MNCs is approximately −65 mV (Oliet & Bourque, 1992; Stern & Armstrong, 1996). This is probably due to the presence in MNCs of an active cation channel that has been shown to be mechanosensitive and senses the changes in plasma osmolality (Oliet & Bourque, 1994; Bourque & Oliet, 1997; Bourque & Chakfe, 2000). We have also identified in ≈30 % of cell-attached patches a non-selective cation channel that was active at rest. Without these cation channels, we would expect the resting membrane potential to be closer to −90 mV, as measured in cerebellar granule neurons, for example (Watkins & Mathie, 1996). Therefore, both cation and K⁺ conductances regulate the resting membrane potential in MNCs and may be the target of modulation by various physiological factors. Prior to this study, it was not known whether the background K⁺ current reflects a single population of K⁺ channels or several different types of K⁺ channels. Therefore, the objective of our study was to clearly identify and characterize individual K⁺ channels that may serve as background K⁺ channels in MNCs. Additionally, we wished to determine whether they belong to the family of recently described 2P K⁺ channels that are reported to behave like background K⁺ currents (Lesage & Lazdunski, 2000; Goldstein et al. 2001; Patel & Honore, 2001).

Although we have identified eight K⁺ channels in resting MNCs, it is not clear which of these truly serve as background K⁺ channels in MNCs for the following reasons. We measured K⁺ channel currents only from the cell soma and not from dendrites. MNCs in situ have long dendrites that provide a large proportion of the total membrane of the neuron, and K⁺ channels are also likely to be expressed in dendrites. K⁺ channel activities were determined at high [KCl] and at room temperature. Therefore, our K⁺ channel measurements will not accurately reflect the true activities under physiological conditions at 37 °C. Despite these limitations, our study provides important and new information on the properties and molecular identity of the K⁺ channels found in MNCs of the SON. The preparations of MNCs that we have used in this study contain both VP- and OT-containing cells. Therefore, whether specific differences exist in the types of K⁺ channel expression in the two groups of cells are not known. As both VP- and OT-containing cells have similar resting membrane potentials (Stern & Armstrong, 1996), it seems reasonable to speculate that similar K⁺ channels are present in the two types of neurons.

TASK-1 (i_K.AS1), TASK-3 (i_K.AS2) and another pH_o-sensing background K⁺ channel (i_K.AS3)

We found three K⁺ channels that are highly sensitive to extracellular pH in MNCs. Based on the single-channel kinetics, pH_o sensitivity and some pharmacology, we believe that TASK-1 and TASK-3 encode two of these K⁺ channels. The gene that encodes the third K⁺ channel (I_K.AS3) that is also pH_o-sensitive has not yet been identified. Therefore, whether this novel K⁺ channel belongs to the 2P K⁺ channel family is uncertain. Both TASK-1 and TASK-3 are active at rest in MNCs. Due to their low P_o, however, it is expected that they would play a relatively minor role as a background K⁺ conductance in MNCs. In high [KCl] medium, TASK-1 and TASK-3 would contribute ≈5 % of the K⁺ conductance at rest, if we assume that all eight K⁺ channels are present in one MNC. By contrast, the P_o and density of i_K.AS3 is relatively high compared to all other background K⁺ channels we report here, and we estimate that i_K.AS3 contributes ≈70 % of the resting K⁺ conductance. Interestingly, i_K.AS3 is nearly completely inhibited by intracellularly applied TEA, whereas it is insensitive to extracellular TEA. Both TASK-1 and TASK-3 are insensitive to TEA from both sides of the membrane. Therefore, we suspect that i_K.AS3 may not belong to the TASK family of K⁺ channels.

The physiological significance of the three acid-sensing K⁺ channels is not evident at this time. Recently, TASK-like K⁺ channels have been identified in arterial chemoreceptor cells (Buckler et al. 2000) and respiration-sensitive cells in the brain stem such as serotonergic neurons of the caudal raphe (Ballantyne & Scheid, 2000; Washburn et al. 2002). In these cells, TASK-like K⁺ channels have been suggested to be involved either directly or indirectly in the regulation of respiration, as any changes in plasma CO₂ content that occur during ventilation would be detected by the corresponding change in pH. Whether changes in plasma pH actually alter the firing frequency of MNCs and hormone release is currently not known. Local changes in extracellular pH may occur during neuronal activity and influence K⁺ channel activity in neurons (Chesler, 1990; Rose & Deitmer, 1995; Newman, 1996; Ransom, 2000). Neuronal activity is associated with K⁺ release into the extracellular space. K⁺-induced depolarization of glial cells nearby leads to an increased Na⁺-$\text{HC}{\text{O}}_3^{\text{ -}}$ co-transport activity, producing acidification of the extracellular medium. The fall in pH_o produced by glial cells, in concert with other neuronal activity-evoked changes in pH_o, is hypothesized to modulate neuronal excitability via effects on pH_o-sensitive molecules in the plasma membrane. Our study suggests that some of the K⁺ channels (i_K.AS1–3) described in this study may act as pH_o sensors that regulate neuronal activity.

TASK channels are particularly sensitive to inhibition by agonists whose receptors are coupled to Gq/11 protein (Millar et al. 2000; Talley et al. 2000; Czirjak et al. 2001). Therefore, it is quite plausible that certain neurotransmitters (such as noradrenaline (norepinephrine)) and neuropeptides (such as angiotensin II and substance P) that act via Gq/11 partly regulate hormone release via effects on these K⁺ channels. Whether i_K.AS3 is also sensitive to receptor agonists needs to be examined, particularly as this K⁺ channel may provide the major background K⁺ conductance. In our recent study of 2P K⁺ channels in cerebellar granule neurons, we found that a channel possessing properties very similar to i_K.AS3 was also the main background K⁺ channel (Han et al. 2002). Therefore, further study of i_K.AS3 would be important to better understand how this channel is regulated in MNCs and how this affects the firing rate.

An earlier study reported that TASK-2 immunoreactivity was present in the SON (Gabriel et al. 2002). To determine whether any of the K⁺ channels might be encoded by TASK-2, we expressed TASK-2 in COS-7 cells and studied its single-channel characteristics. TASK-2 exhibited distinct channel kinetics and conductance that were different from all other known 2P K⁺ channels. So far, we have not observed a K⁺ channel exhibiting behaviours similar to TASK-2 in MNCs. This may be due to the extremely low expression of TASK-2 in MNCs. This is consistent with the recent report that expression of TASK-2 mRNA in the CNS is low and shows an unremarkable pattern of localization (Talley et al. 2001).

TRAAK

TRAAK is a member of the 2P K⁺ channel family that is activated by membrane stretch, arachidonic acid and intracellular alkaline conditions (Maingret et al. 1999a; Kim et al. 2001). Although its mRNA is expressed in many areas of the brain, a native K⁺ channel with properties identical to the cloned TRAAK has not been reported previously. In MNCs, we were able to identify for the first time a native K⁺ channel with biophysical and other modulatory characteristics nearly identical to those of cloned TRAAK expressed in mammalian cells. In MNCs, the TRAAK-like K⁺ channel showed low activity in cell-attached patches, and its density was also low (see Fig. 1B). Therefore, TRAAK probably does not contribute much to the background K⁺ conductance at rest under normal physiological conditions, but may become functionally important, as would TREK channels, under conditions of chronic hyposmolality or hyponatraemia resulting from a syndrome of inappropriate hypersecretion of antidiuretic hormone.

TREK (i_K.AA)

In earlier studies using dissociated neurons from different regions of rat brain, we identified three types of native K⁺ channels that were activated by arachidonic acid (and other unsaturated free fatty acids), membrane stretch and acidic pH_i conditions (Kim et al. 1995). Two of them (i_K.AA1–2) can now be considered functional correlates of TREK-1 and TREK-2, based on their electrophysiological and pharmacological properties (Kim et al. 1995; Maingret et al. 1999a,b; Bang et al. 2000). The putative gene that codes for third K⁺ channel that has the lowest single-channel conductance (45 pS at +60 mV; Kim et al. 1995), has not yet been identified. More often, however, we recorded another type of K⁺ channel (that we designate i_K.AA3) that showed kinetic properties very similar to i_K.AA1–2 and exhibited similar behaviour with respect to responses to applied pressure, pH and fatty acids. Co-transfection of TREK-1/TREK-2, TREK-1/TRAAK and TREK-2/TRAAK failed to yield channels with properties of i_K.AA3. This suggests that i_K.AA3 is probably a functional correlate of a novel member of the TREK family of K⁺ channels.

It is somewhat surprising that a cell or a group of cells need three K⁺ channels (i_K.AA1–3) that behave similarly. Apart from small but distinct differences in single-channel conductances, we were unable to distinguish the native K⁺ channels (i_K.AA1–3) from each other by any other tests performed in this study. The relative densities and open probabilities of the three i_K.AA were also similar, suggesting that all three contribute equally, albeit to a minor extent, to the background K⁺ conductance. In isolated MNCs, it has been shown that cell swelling that occurs in response to a decrease in osmolality of the medium inhibits a cation conductance (Oliet & Bourque, 1993). This would lead to hyperpolarization and a decrease in the frequency of firing, thereby reducing VP release and helping to restore plasma osmolality. The same investigators have shown that changes in osmolality (± 8–30 mmol kg⁻¹) affect the conductance of a whole-cell current with a reversal potential of −41 mV but do not cause a shift in reversal potential of whole-cell current in MNC. The lack of a shift in the reversal potential would suggest that a K⁺ current is neither activated nor inhibited by small changes in osmolality. This suggests that i_K.AA is not affected by cell swelling that occurs within the physiological range of plasma osmolality. Perhaps i_K.AA comes into play when other factors such as changes in intracellular pH and fatty acid content are involved. Clearly, the role of these mechanosensitive background K⁺ channels in hormone release needs to be studied further.

Physiological significance

The physiological regulation of VP and OT release from the nerve terminals of MNCs is highly complex, and involves many neurotransmitters and peptide hormones (Chakfe & Bourque, 2000; Sladek, 2000; Sladek & Kapoor, 2001). Such complexity in signalling pathways that converge on the firing frequency and pattern of MNCs is further complicated by the presence of many background K⁺ channels that may also be targets of the same neurotransmitters and peptides. Inhibition of these K⁺ channels via receptor ligands would bring the resting membrane potential closer to the firing threshold, and facilitate the occurrence of neuronal discharges in response to other depolarizing stimuli in MNCs. Some of the effects produced by neurotransmitters and peptides may involve K⁺ channels reported here, as TASK and TREK channels have been reported to be inhibited by ligands that act on G protein-coupled receptors (Lesage et al. 2000; Talley & Bayliss, 2002). In a previous study, glutamate has been reported to modulate several K⁺ currents, including a leak K⁺ current (Schrader & Tasker, 1997). Cell swelling and other chronic physiological or pathophysiological conditions may inhibit hormone release by activation of TREKs and shifting the resting membrane potential away from the firing threshold. Caesium (Cs⁺) has been reported to block depolarizing after-potentials and phasic firing in rat supraoptic neurons (Ghamari-Langroudi & Bourque, 1998). It was suggested that the effect of Cs⁺ is due to inhibition of hyperpolarization-activated inward current (I_H) and inhibition of a background K⁺ current such as TASK (Ghamari-Langroudi & Bourque, 2001). Our finding of a Cs⁺-sensitive K⁺ channel (i_K.AS3) suggests that i_K.AS3 may be involved in Cs⁺-induced effects and in controlling the resting membrane potential. The high sensitivity of several K⁺ channels to pH_o described here is also intriguing. Although it is not known whether changes in blood pH alter VP or OT release, the presence of highly pH_o-sensitive K⁺ channels in MNCs suggests that there may exist certain physiological processes whereby protons may be used as a signalling molecule. These potentially important mechanisms need to be studied further to better understand the role of each background K⁺ channel present in MNCs.