University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom
Submitted 10 January 2003 ; accepted in final form 12 September 2003
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ABSTRACT |
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hypoxia; chemoreception; background K channels; ATP
In addition to characterizing the KB channels present in type 1 cells, we have also investigated mechanisms that might be responsible for modulating their activity. Over the years a number of different hypotheses have been advanced to explain oxygen sensing by the carotid body. Historically one of the most prominent is that oxygen sensing could be linked to mitochondrial metabolism (1). Inhibitors of electron transport and mitochondrial uncouplers are all potent chemostimulants (17) and inhibit K+ currents in these cells (8, 34). Moreover, the regulation of K+ channels by adenine nucleotide levels is a key feature of other metabolic sensors (2). This raises the possibility that cytosolic factors such as ATP, ADP, AMP, or Mg2+ may be involved in the regulation of KB channels. Another hypothesis is that oxygen sensing could occur through the modulation of cell redox couples such as NADH/NAD+. Redox modulation of several voltage-sensitive K+ channels has also been reported (25, 36). We have therefore investigated the regulation of KB channels by metabolism, adenine nucleotides, Mg2+, and NADH to gain some insights into possible mechanisms whereby KB channel activity might be modulated.
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METHODS |
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Electrophysiology. Experiments were performed using the cell-attached and inside-out configurations of the patch-clamp technique. Data were acquired at 1020 kHz and filtered at 25 kHz, with an Axopatch 200B amplifier, Digidata 1200, and pClamp software (version 6.0 or 7.0, Axon Instruments). Electrodes were made from Clarke CG150 borosilicate glass capillaries (Clarke Electromedical, Pangbourne, Reading, UK) coated with Sylgard 184 (Dow Corning). Electrodes were fire polished before use and had resistances between 5 and 15 M; seal resistances were >10 G
. In the cell-attached patch, the potential across the membrane (Vm) is the difference between the resting potential of the cell (Vr) and the potential applied to the inside of the pipette (Vp) i.e., Vm = Vr - Vp. Pipette potentials are reported for cell-attached data, and membrane potentials are reported for all other data. Current is reported as membrane current according to the convention that current flow into the cell is represented as negative, or inward, current (unless otherwise specified i.e., Fig. 3Aa).
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The bath was earthed via an Ag-AgCl pellet. Data are presented with correction for liquid junction and reference potential errors, which were measured to be between 0.3 and 10 mV for different combinations of pipette and bathing solutions.
Solutions. Cells were bathed in standard -buffered saline containing (mM): 117 NaCl, 4.5 KCl, 23 NaHCO3, 1.0 MgCl2, 2.5 CaCl2, and 11 glucose bubbled with 5% CO2 and 95% air; pH 7.47.45. For testing cyanide (CN-) and 2,4-dinitrophenol (DNP) in cell-attached patches, we bathed cells in a modified calcium-free high-potassium
-buffered saline containing (mM): 100 KCl, 17 NaCl, 23 NaHCO3, 3.5 MgCl2, and 11 glucose, which was bubbled with 5% CO2 and 95% air. This solution was used to stabilize membrane potential and prevent large changes in intracellular calcium concentration ([Ca2+]i), since both DNP and CN- depolarize type 1 cells and evoke large increases in [Ca2+]i (8).
Extracellular pipette solutions for cell-attached and inside-out patches contained (mM): 140 KCl, 4 MgCl2, 1 EGTA, 10 HEPES, and 10 tetraethylammonium (TEA)-Cl, pH 7.4 with KOH. The final pipette [K+] was 146 mM. To test the ion selectivity of the channel, NaCl, N-methyl-D-glucamine (NMDG), or K2SO4 was substituted for KCl in the pipette.
Inside-out intracellular bath solutions contained (mM): 130 KCl, 5 MgCl2, 10 EGTA, 10 HEPES, and 10 glucose. pH was adjusted to 7.2 with KOH. The final [K+]i was 152 mM.
The effect of high intracellular Ca2+ was tested on inside-out patches with an intracellular solution containing (mM): 130 KCl, 3.61 MgCl2, 8.27 CaCl2, 10 EGTA, 10 HEPES, and 10 glucose, pH 7.2 with KOH. This solution had a free [Ca2+]i of 1.0 µM, compared with 0.03 nM in control. Free [Mg2+]i was maintained at 3.3 mM. The effect of reducing intracellular Mg2+ was tested on inside-out patches with an intracellular solution containing (mM): 130 KCl, 10 EGTA, 10 HEPES, and 10 glucose, pH 7.2 with KOH. (The free intracellular levels of Mg2+ and Ca2+ in this solution were estimated to be
0.8 µM and 0.02 nM, respectively, based on the levels of Ca and Mg present as impurities in KCl.) Values for free intracellular divalent cation concentrations were calculated with published dissociation constants (29) and in-house software (P. Griffiths, University Laboratory of Physiology, Oxford).
TEA and 4-aminopyridine (4-AP) were added directly to the saline solutions, and the pH was adjusted as appropriate. Sodium cyanide and DNP were added directly to the saline solutions on the day of the experiment, and solutions were replaced every 12 h. K2ATP, K2ADP, and AMP were added to the intracellular solution directly on the day of the experiment, and the pH was adjusted as needed. In experiments with 2 and 5 mM K2ATP, intracellular Mg2+ was increased to 7 and 10 mM, respectively, so that free intracellular Mg2+ was maintained at 3 mM (MgATP was
1.95 and 4.9 mM, respectively). After addition of nucleotides and adjustment of the pH of the intracellular solution, the [K+]i increased to a maximum of 174 mM with 5 mM ATP. Solutions were superfused at
2 ml/min through a recording chamber with a volume of
80 µl. Experiments were performed at 2933°C.
Data analysis. Single channel recordings were analyzed with pClamp 6.0 software (Axon) or Spike3 (Cambridge Electronic Design). The value of channel amplitude was initially determined by eye using the amplitude of the longest open events. A Gaussian distribution was then fitted to level 1 open events to give an estimate of the single channel amplitude. Open events were defined using the 50% threshold criteria with events of 150%, 250%, etc. of threshold counted as multiple channel openings. Channel activity is reported as the open probability times the number of channels in a patch (NPopen). Kinetics and channel activity were also determined from the pClamp events list. For burst analysis, the minimum value of the closed duration that separates bursts of openings, the interburst interval, was determined using pClamp software, with bin width set to manual, test interburst interval of 0.1 ms, start value of 0.1 ms, and stop value of 2.0 ms. To verify the result, we also used the following equation (40), modified from Colquhoun and Sakmann (10)
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Sigmaplot 5.0 (SSPS, Chicago, IL) was used to calculate linear and nonlinear regressions of plotted data. Statistical significance of results was assessed by the Student's t-test.
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RESULTS |
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The current-voltage relationship of the main conductance state was linear, between -120 and 30 mV pipette potential, and had a mean slope conductance of 15.6 ± 0.04 pS (n = 12) and a reversal potential of -74 ± 2 mV (n = 10). Slight rectification was evident at pipette potentials more positive to 80 mV (approximately -150 mV membrane potential) and at pipette potentials more negative to -120 mV (50 mV membrane potential) (Fig. 1). These channels were active immediately and did not inactivate or run down over time.
Substitution of NaCl for KCl, in the pipette, led to a rightward (positive) shift in the single channel current-voltage relationship and the appearance of marked rectification at low extracellular K+ concentration ([K]e) (Fig. 1B). Reversal potentials were estimated by linear interpolation of 38 points nearest the x-axis. A plot of reversal pipette potential vs. log [K]e had a linear slope of -55 ± 2 mV/decade (Fig. 1C). (On converting pipette potential to membrane potential the slope value changes sign to 55 ± 2 mV/decade.) This is very close to that expected for a purely K+-selective channel (slope of 60 mV/decade at 30°C).
To confirm that the channels were indeed K+ selective, we also performed experiments in which the reversal potential was determined at different concentrations of extracellular (pipette) chloride and sodium. Reduction of extracellular chloride from 158 to 30 mM (by substitution of KCl with extracellular K2SO4) had no significant effect on the reversal potential or on the slope conductance measured between -120 and 40 mV pipette potential (-74 ± 2 mV and 16 ± 1 pS in 158 mM Cl-, n = 10; compared with -68 ± 3 mV and 15 ± 1 pS in 30 mM Cl-, n = 6). Indeed, the single channel current-voltage relationships obtained in low and normal chloride were superimposable (Fig. 2A). Similarly, replacing 70 mM Na+ with 70 mM NMDG (pipette K+ = 70 mM) had no significant effect on the reversal potential or the current-voltage relationship (reversal potential = -49 ± 8 mV with NMDG, n = 4; compared with -56 ± 2 mV with Na+, n = 10). Thus substantive changes in pipette chloride or sodium failed to have any affect on single channel current-voltage relationships, confirming that these channels were primarily K+ selective.
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Inside-out patches. Continuous recordings of channel activity from patches in the cell-attached configuration through patch excision to the inside-out configuration revealed a rapid rundown in channel activity (Fig 3A). For these studies cells were superfused with the standard inside-out recording medium (see METHODS) after seal formation and for at least 1015 s before patch excision to obtain continuous recordings of channel activity before, during, and after patch excision. Within the limits of resolution of the timing of patch excision (approximately ± 1-s), rundown appeared to begin immediately and was complete within 40 s of patch excision with channel activity having fallen to 10% of the cell-attached level (Fig. 3Ab). Despite this rundown single channel, events were clearly and routinely observed in inside-out patches with 146 mM [K+]e in the pipette solution and 152 mM [K+]i (Fig. 3B) and had a single channel amplitude at -76 mV of 1.15 ± 0.06 pA (n = 7).
The current-voltage relationship of these channels was determined in the inside-out patch with 146, 100, and 70 mM [K+]e in the pipette. With 146 mM [K+]e and 152 mM [K+]i, the current-voltage relationship was linear, in the range 40 to -100 mV, with a slope conductance of 14.2 ± 0.9 pS (n = 6) and a reversal potential of 8 ± 2 mV. Weak rectification was again evident in inside-out patches, as with cell-attached patches. This rectification did not decrease over time, suggesting it is not due to the presence of diffusible polyamines (26). As in cell-attached patches, the reversal potential was shifted toward more negative potentials as [K+]e was decreased consistent with a K+-selective channel (Fig. 3, C and D). Reversal potentials were measured by linear interpolation and a plot of reversal potential against log [K+]e could be fitted with a linear slope of 60 ± 5 mV (Fig. 3C).
We again performed a number of recordings in which extracellular chloride and sodium were reduced to confirm the K+ selectivity of the channels observed in excised patches. As in cell-attached patches, reduction of extracellular chloride from 158 to 30 mM had no significant effect on reversal potential (8 ± 2 mV in 158 mM Cl-, n = 6; compared with 4 ± 2 mV in 30 mM Cl-, n = 6). Similarly, reduction in extracellular Na+ from 70 mM to 0 (NMDG substituted) had no effect on single channel conductance or reversal potential (-17 ± 3 mV in 0 Na+, n = 5; compared with -10 ± 3 mV in 70 mM Na+, n = 5; Fig. 2B).
Thus aside from a lower level of activity, the single channels observed in excised inside-out patches appeared to be similar to those observed in cell-attached patches. The strong biophysical similarities between the channel activity seen in the cell-attached patch and that seen in the inside-out patch suggest that the channels observed under these different recording conditions are one and the same. In view of the high K+ selectivity of these channels and the fact that they are open at all potentials, they will be termed KB channels.
Effects of TEA and 4-AP. TEA at 10 mM was present in the pipette solution in all above single channel recordings. Removal of TEA from the extracellular solution had no effect on single channel conductance or channel activity (NPopen = 0.30 ± 0.1, n = 7 -TEA; 0.27 ± 0.04, n = 29 +TEA; measured at 0 mV), nor did it visibly alter the channel kinetics of the inward currents (9) (Fig. 4A), confirming that, like the whole cell background K+ current (4), these background channels were insensitive to extracellular TEA. In cerebellar granule neurons, one of the channels thought to be a major contributor to background K+ currents, the type 4 channel, whilst also being insensitive to extracellular TEA, is strongly inhibited by intracellular TEA (19). Application of 20 mM TEA to the intracellular surface of inside-out patches (in the presence of 10 mM extracellular TEA), however, had no effect on channel activity (107 ± 21% and 96 ± 5% of control for inward and outward currents, respectively; n = 3) or single channel amplitude (91 ± 7% and 104 ± 7% of control for inward and outward currents, respectively; n = 3). The KB channels of type 1 cells are therefore clearly different from the type 4 channel of cerebellar granule neurons.
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The effect of 5 mM 4-AP was also tested with 10 mM extracellular TEA present. In cell-attached patches, extracellular, pipette, 4-AP had no significant effect on single channel conductance at negative membrane potentials (16.7 ± 0.6 pS with 4-AP, n = 5; 16 ± 1 pS in control, n = 10, Fig. 4B), reversal potential (-61 ± 6 mV pipette potential, n = 5), or channel activity (measured at 0 mV pipette potential, i.e., resting membrane potential, NPopen = 0.21 ± 0.06, n = 9 with 4-AP; 0.27 ± 0.04, n = 29 control). In the absence of extracellular 4-AP, however, outward channel openings were less well defined than inward channel openings (often of variable size), and there was more apparent channel activity and baseline noise than for inward currents. By contrast, in the presence of extracellular 4-AP (in addition to TEA), outward currents were more clearly resolved (Fig. 4C), and inward rectification was significantly reduced (P < 0.05, n = 5, Fig. 4B). Moreover, under these conditions channel activity was independent of voltage from -140 to 40 mV (9). Thus the apparent voltage sensitivity of channel activity and rectification seen in the absence of extracellular 4-AP is probably due to the presence of other, contaminating, voltage-gated 4-AP-sensitive K+ channels in the same patch.
Kinetics of the KB channel. It is clear from visual inspection that the KB channel has very rapid single channel kinetics, consisting of short bursts of rapid openings (Fig. 5). Kinetic parameters were measured from cell-attached recordings acquired at 20 kHz and filtered at 5 kHz. In these, as well as all other patch clamp recordings, both whole cell and inside out, we failed to obtain any recordings in which there were no double, or higher, conductance state openings (see DISCUSSION). Consequently measurements were taken from those recordings in which large sections of trace were of one main single channel level, and multiple openings (or higher conductance states) were rare.
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Open time histograms were best fitted with a single exponential and were corrected for sampling and bin promotional errors with pClamp6 software (Fig. 5Ba). At 70 mV pipette potential in cell-attached patches, the mean open time constant, o, was 0.28 ± 0.02 ms (n = 15). Mean open times were independent of pipette potential between 0 and 100 mV [
o = 0.27 ± 0.04 ms (n = 5) at 0 mV and 0.29 ± 0.02 ms (n = 10) at 100 mV].
Closed time histograms were best fitted with a double exponential, consistent with the channel displaying bursting kinetics (Fig. 5Bb). Due to relatively short lengths of recording and the possible presence of multiple channel levels, the measured duration of the long closed state was very variable and could be anywhere between 2 and 30 ms. The variability in long closed time may, in part, be a consequence of varying numbers of channels in the patch. It is also possible, however, that modulation of channel activity could involve changes in the stability of the long closed state. The mean short closed time was 0.11 ± 0.08 ms at 70 mV pipette potential (binned logarithmically without correction for sampling promotional errors). Values for both the mean open time and the mean short closed time are longer than the true values since the time constants are close in value to the filter dead time (0.04 ms) (11). Burst durations were measured to be 1.7 ± 0.3 ms (n = 12) at 70 mV pipette potential. Burst durations were also independent of pipette potential between 0 and 100 mV [1.8 ± 0.4 ms (n = 4) at 0 mV, and 1.3 ± 0.1 ms (n = 8) at 100 mV]. The burst duration should be fairly close to the true value since the errors in open time are primarily due to missed gaps within a burst (11).
We have previously reported that KB channel activity is reversibly inhibited by hypoxia in cell-attached patches (9, 41). We have therefore also analyzed the effect of hypoxia on channel kinetics. In cell-attached patches, hypoxia significantly increased the long closed time (166 ± 26% of control, P < 0.05, n = 9) without affecting mean open time (103 ± 6%), mean short closed time (85 ± 11%), or mean burst duration (99 ± 7%).
Regulation of KB channels: effects of divalent cations on the KB channel. Because [Ca2+]i was buffered to very low levels in our standard intracellular media (10 mM EGTA, no added Ca2+), below that normally found at rest in these cells (100 nM, Ref. 5), it was important to establish whether the KB channel was sensitive to changes in [Ca2+]i and whether the fall in [Ca2+]i upon patch excision might account for channel rundown. In inside-out patches, increasing intracellular Ca2+ from 0.03 nM to 1 µM had no significant effect on channel activity or single channel amplitude (Fig. 6, A and B); single channel amplitude at -100 mV was 1.30 ± 0.07 pA in control and 1.35 ± 0.08 pA with 1 µM Ca2+. Outward current channel activity in the presence of 1 µM Ca2+ was 86 ± 15% of that in control (n = 4). These results confirm that the KB channel is not directly Ca2+ activated. Thus the rundown in channel activity upon patch excision is unlikely to be due simply to the reduction in [Ca2+]i.
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The effect of reducing intracellular Mg2+ on channel activity was also tested. Neither the single channel current-voltage relationship nor channel activity was affected by a reduction in [Mg2+]i from 3.3 mM to 0.8 µM (Fig. 6D; in low [Mg2+]i channel activity was 104 ± 10% and 97 ± 5% of control for inward and outward currents, respectively; n = 5). In contrast, reducing extracellular Mg2+ from 3.7 mM to 0.9 µM (nominally Mg2+ free) significantly increased single channel conductance to inward currents from 15.6 ± 0.4 pS (n = 12) to 28 ± 3 pS (n = 4; P < 0.05; Fig. 6C).
Regulation of KB channels: effect of intracellular nucleotides. Rundown in ion channel activity following patch excision has been observed for many different ion channels and is often associated with the loss of cytosolic ATP. We therefore tested the effects of 2 and 5 mM ATP on inside-out patches at least 1 min following patch excision (i.e., after rundown). In the majority of patches studied, ATP induced a clear, and reversible, increase in single channel activity of approximately fourfold in the presence of 5 mM ATP (Fig. 7, A and C); NPopen = 0.04 ± 0.01 control and 0.14 ± 0.03 ATP (n = 18, P < 0.005). At 5 mM, ATP had no significant effect on mean open times, albeit measured from data obtained with a 2-kHz bandwidth, but significantly decreased the long closed time to 17 ± 3% of control (n = 7, P < 0.05). We also investigated the effects of ADP and AMP on channel activity. At a high physiological concentration of 100 µM, ADP had no discernable effect on single channel activity in the excised patch; NPopen = 0.05 ± 0.02 control and 0.07 ± 0.03 plus ADP (n = 5, Fig. 7C). At much higher concentrations, however, ADP mimicked the effects of ATP; i.e., 2 mM ADP reversibly increased channel activity in the majority of patches by, on average, twofold (Fig. 7, B and C); NPopen = 0.05 ± 0.01 control and 0.10 ± 0.03 in the presence of ADP (n = 13, P < 0.01, see Fig. 7). Application of 1 mM intracellular AMP had no significant effect on channel activity (Fig. 7C); mean NPopen = 0.03 ± 0.004 control and 0.02 ± 0.004 plus AMP (n = 6).
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The possibility that the channel might also be regulated by a change in the redox state of the cell was tested on inside-out patches by application of 2 mM intracellular NADH in the presence of 2 mM ATP. There was no significant effect of NADH on channel activity (control = 0.09 ± 0.05, NADH = 0.08 ± 0.04, wash = 0.05 ± 0.02; n = 3).
Regulation of KB channels: metabolic regulation of the channel. Given the sensitivity of KB channel activity to ATP in excised patches, an obvious question is whether changes in metabolism could play a role in the regulation of channel activity in vivo. We therefore examined the effects of two classical inhibitors of energy metabolism on KB channel activity in cell-attached patches. For these experiments cells were bathed in a calcium-free high-K+ Tyrode solution to depolarize and stabilize membrane potential and prevent large changes in [Ca2+]i.
Application of 2 mM ClCN-, an electron transport inhibitor, rapidly and reversibly inhibited channel activity in cell-attached patches by 56 ± 10% (n = 8, P < 0.05; Fig. 8). The mitochondrial uncoupler DNP (250 µM) also caused a rapid and reversible inhibition of channel activity in cell-attached patches by 57 ± 10% from that in control (n = 8, P < 0.05; Fig. 8). DNP had no effect on mean open time or mean short closed time but significantly increased the mean long closed time by 370 ± 68% (kinetics determined from data obtained with a 2-kHz filter). In contrast, when applied to the intracellular side of inside-out patches, neither DNP nor CN- had any effect on channel activity (Fig. 8).
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DISCUSSION |
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Comparison of background channels with the oxygen-sensitive KB current. The properties of the KB channel described here correspond closely with those of the oxygen-sensitive whole cell background, or leak, K+ current recorded by Buckler (4) in neonatal rat carotid body type 1 cells. Both the KB channel and the whole cell K+ current are insensitive to 10 mM TEA and 5 mM 4-AP (at negative potentials) and show an almost linear current-voltage relationship in symmetrical K+ concentrations in the presence of these drugs (9). The KB channel and the whole cell K+ current are both inhibited by hypoxia to a similar extent (a reduction of 35 ± 5% compared with 40 ± 6% in the whole cell; see Ref. 9), and both currents are inhibited by 250 µM DNP, a mitochondrial uncoupler, to a similar extent (a reduction of 57% for the KB channel vs. 56% for the whole cell K+ current; Ref. 8). Finally, both the KB channel and the whole cell K+ current is activated by the general anesthetic halothane and inhibited by the local anesthetic bupivacaine (9). These observations support the conclusion that the channels described here are primarily responsible for the oxygen-sensitive background K+ conductance of carotid body type 1 cells (4).
Single channel properties/kinetics. The KB channel in type 1 cells has a unitary conductance for the main open state of 15 pS as measured for inward currents over a range of negative membrane potentials in a symmetrical K+ gradient. Whilst this was the predominant form of channel activity seen, visual inspection of all patches showing such channels (>300) also revealed the presence of brief openings to higher conductance levels in addition to the 15-pS level. In none of our recordings, however, did we see similar higher conductance channel openings in isolation (i.e., without the lower 15-pS openings). The higher conductance levels observed could therefore represent the opening of more than one channel simultaneously or different conductance states of the same channel. Although some patches inevitably contained multiple channels, variable conductance levels were also present in patches with very low overall channel activity. This observation suggests that higher conductance levels are unlikely to be solely due to coincident openings of more than one channel and may therefore represent multiple conductance states for the same channel. Thus in contrast to the study of Han et al. (19) on cerebellar neurons, we were able to identify only one major channel type that could be associated with the TASK-like background K+ current in type 1 cells.
Studies on channel gating revealed that the KB channel opened in short bursts with a mean duration of 2 ms comprising a single brief open state (
0.3 ms for the main conductance level) and even briefer closings (
0.1 ms). These bursts were interspersed with a highly variable long closed time. These measurements suggest a minimal kinetic model of one open state and two closed states. Channel modulation by hypoxia appeared to be mediated primarily through changes in the stability/duration of the long closed state with no detectable effect on conductance,
o, short closed time constant, or burst duration.
Identity of the KB channels. As previously described (9), there is compelling evidence to suggest that this oxygen-sensitive background K+ current/channel is a TASK-like member of the two-P domain K+ channel family. Here we have further shown that unitary outward currents at positive potentials (>50 mV) were unaffected by reducing [Mg2+]i, supporting the notion that this channel is not a weakly rectifying inwardly rectifying K+ channel (30) and that increasing [Ca2+]i had no effect on channel activity, confirming that it is not a Ca2+-activated K+ channel.
The biophysical properties of the KB channel show a number of similarities with those of the cloned channel TASK-1: i.e., 1) TASK-1 has a main single channel conductance of 1316 pS for inward currents and what may be larger subconductance states (23, 27), 2) weak inward rectification of single channel conductance at positive potentials that is insensitive to intracellular magnesium, 3) brief open times of 0.70.3 ms, and 4) at least two closed states (23, 27). There are, however, also some differences between the properties of the KB channels observed in type 1 cells and those of TASK-1. One possibly minor point of difference is the much briefer short closed times of the KB channel (0.11 ms) compared with that reported for TASK-1 (5 ms, Ref. 27). These kinetic parameters may, however, be subject to both intracellular modulation and methodological error in their determination due to limiting bandwidth of single channel recordings. A more striking observation, however, was that removal of extracellular magnesium (in 0 Ca2+) doubled unitary conductance for inward currents (see Fig. 6D). Similar observations have also been reported for TASK-3, i.e., that removal of extracellular divalent cations increases the conductance to inward current (35), whereas whole cell TASK-1 currents appear to be unaffected by the removal of extracellular divalent cations (27, 28, J. Cochoran and K. J., Buckler unpublished observations). Although TASK-1 and TASK-3 share a number of common features, which makes any distinction between them difficult, one key difference is in the single channel conductance. TASK-3 is reported to have a single channel conductance for inward currents of 27 pS in 2 mM external Mg2+ (0 Ca2+) or 1 mM Mg2+ and 1 mM Ca2+ (22, 35), 36 pS in 1 mM Mg2+ (19), and 100 pS in 0 Mg2+ (35); these values are clearly much higher than those obtained for the KB channel (15 pS in 4 mM Mg2+ and 28 pS in 0 Mg2+ and 0 Ca2+). It would therefore seem unlikely that the KB channel is a simple TASK-3 homomer.
The observation that the properties of the KB channels described here do not precisely match those of either TASK-1 or TASK-3 raises a number of intriguing possibilities, e.g., 1) the background channel may be a heterodimer of TASK-1 and TASK-3. Such a heteromerization between these two channels has been suggested from coexpression studies in Xenopus oocytes (12). 2) The KB channel could be a TASK-1 or TASK-3 homomer whose properties have been subtly altered by association with another protein. 3) The channel we have observed is neither TASK-1 nor TASK-3 but another member of the tandem-p domain K+ channel family; given that the biophysical and pharmacological properties of this channel do not conform to those of any other tandem-p domain channel that has thus far been described, however, the only remaining possibility is that it is one of those channels that have not yet been successfully expressed, e.g., TASK-5 [which has a high degree of homology with TASK-3 (3, 21)].
Intracellular regulation of channel activity: a role for ATP? The abrupt rundown of activity of the KB channel following patch excision is presumably due to the rapid loss of some intracellular constituent. This suggests that the KB channel may be subject to a high degree of regulation by cytosolic factors. In this context it is interesting to note that rapid changes (increase) in KATP channel activity also occur upon patch excision, due to the loss of the metabolic second messenger ATP (2, 33). In studies utilizing excised patches, we observed that both ATP and very high levels of ADP reversibly increased channel activity, whereas lower, physiological, levels of ADP and AMP had no effect.
The activity of KB channels would therefore seem to be dependent on the presence of ATP, the loss of which may account for some of the rundown in channel activity seen upon patch excision. In this respect the KB channels studied here differ from both cloned TASK channels and endogenous TASK-like channels in cerebellar granule neurons that have been reported to be insensitive to cytosolic ATP (19, 23). Some endogenous TREK-like channels, however, have also been found to be activated by intracellular ATP, unlike heterologously expressed TREK-1 (16, 38). ATP sensitivity would therefore appear to be conferred on some tandem-p domain channels when expressed endogenously. This raises the question as to whether this ATP sensitivity results from interaction with a regulatory subunit.
We also noted that both CN- and the mitochondrial uncoupler DNP markedly reduced background channel activity in cell-attached patches (but not in excised patches). This observation suggests that changes in cytosolic ATP levels could play a role in modulating channel activity in the intact cell. [during metabolic inhibition the rise in ADP is expected to be relatively small compared with fall in ATP; for example, during CN- poisoning of the heart ATP falls from 8 to 4.8 mM but ADP rises only from 40 to 100 µM (24).]
The above observations inevitably lead to the question as to whether oxygen sensing itself could be mediated via changes in oxidative phosphorylation and cellular ATP levels. Of central importance to this issue is the question as to whether the oxygen sensitivity of mitochondrial metabolism is sufficient to account for the oxygen-dependent regulation of channel activity, and there is some evidence to suggest that this may be the case (13). At this stage, however, there is no direct proof that oxygen sensing by KB channels does indeed involve inhibition of mitochondrial ATP synthesis. Our observations do, however, permit a number of predictions to be made that should allow this hypothesis to be tested. For example, if this hypothesis were true, it should be demonstrable 1) that as hypoxia and mitochondrial inhibitors modulate the same channels then their effects on channel activity should be mutually exclusive; 2) that levels of hypoxia that inhibit channel function should also impair mitochondrial function sufficiently to lower cytosolic ATP levels; and 3) that appropriate, direct manipulation of adenine nucleotide levels should prevent changes in channel activity during hypoxia. These issues now need to be addressed.
Conclusions. In summary, this work gives further support to the idea that the oxygen-sensitive K+ channel in neonatal rat carotid body type 1 cells is a TASK-like background K+ channel. The biophysical properties of these channels do not, however, precisely conform to those of either TASK-1 or TASK-3, suggesting that the KB channels are not simple homomultimers of TASK-1 or TASK-3.
The activity of these KB channels is highly dependent on cytosolic factors including ATP and is also sensitive to the inhibition of oxidative phosphorylation. Changes in cellular energy metabolism could therefore play a key role in regulating the activity of these channels. The extent to which changes in metabolism might be involved in the oxygen-dependent regulation of KB channel activity, however, remains to be determined.
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ACKNOWLEDGMENTS |
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GRANTS
This work was funded by the British Heart Foundation and the Medical Research Council.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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