Potassium Channel Types in Arterial Chemoreceptor Cells and Their Selective Modulation by Oxygen

Potassium Channel Types in Arterial Chemoreceptor Cells and Their Selective Modulation by Oxygen MARfA DOLORES GANFORNINA and Josg LOPEZ-BARNEO From the Departamento de Fisiologfa y Bioflsica, Facuhad de Medicina, Universidad de Sevilla, 41009 Sevilla, Spain ABSTRACT Single K + channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na + and Ca 2+ channels. We have found three classes of voltage-gated K + channels that differ in their single-channel conductance (~/), dependence on internal Ca 2+ (Cai2+), and sensitivity to changes in 02 tension (Po2). Ca2+-activated K + channels (Kca channels) with ~/ ~ 210 pS in symmetrical K + solutions were observed when [Ca2+]i was > 0.1 IzM. Small conductance channels with ~/= 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (Pop,n) was unaffected when exposed to normoxic (Po2 = 140 mmHg) or hypoxic (P02 - 5-10 mmHg) external solutions. A third channel type (referred to as Ko 2 channel), having an intermediate ,/( ~ 40 pS), was the most frequently recorded. Ko~ channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in < 500 ms, and their Pope, reversibly decreases upon exposure to low P02. The effect of low Po2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, Popen diminishes to ~ 40% of the control value. The time course of ensemble current averages of Ko 2 channels is remarkably similar to that of the O2-sensitive K + current. In addition, ensemble average and macroscopic K + currents are affected similarly by low P02. These observations strongly suggest that Ko 2 channels are the main contributors to the macroscopic K + current of glomus cells. The reversible inhibition of Ko 2 channel activity by low P02 does not desensitize and is not related to the presence of F-, ATP, and GTP--¢-S at the internal face of the membrane. These results indicate that Ko 2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K + channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule. INTRODUCTION Although it has been known for decades that the mammalian carotid bodies participate in the regulation of breathing by adjusting the ventilator), rate to the level Address reprint requests to Dr. J. L6pez-Barneo, Departamento de Fisiologfa y Biofisica, Facultad de Medicina, Avenida S~tnchez Pizjmfn, 4, 41009 Sevilla, Spain. j. GEN. PHYSIOL. C) The Rockefeller University Press • 0022-1295/92/09/0401/26 $2.00 Volume 100 September 1992 401-426 401 on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 402 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992 of oxygen tension (Po2) in arterial blood (De Castro, 1926; Heymans, Bouckaert, and Dautrebande, 1930; Fidone and Gonzfilez, 1986; Fitzgerald and Lahiri, 1986) the mechanisms involved in the process of 02 sensing have remained unknown. There has been a general consensus that type I (or glomus) cells, the most numerous in the carotid body, are the elements responsible for chemotransduction since they make synapses with afferent nerve endings, have cytosolic granules containing catecholamines, and secrete dopamine in response to hypoxia and high external K ÷ (Fidone, Gonzfilez, and Yoshizald, 1982; Fishman, Greene, and Platika, 1985; Almaraz, Gonz~ilez, and Obeso, 1986; Rigual, Gonzfilez, Gonzfilez, and Fidone, 1986; Obeso, Fidone, and Gonzfilez, 1987). Direct proof for the chemoreceptive properties of type I ceils has come, however, from recent electrophysiological experiments. It has been shown that type I cells can fire action potentials repetitively and, as in other electrically excitable cells, they generate voltage-dependent Na +, Ca z+, and K + currents (Duchen, Caddy, Kirby, Patterson, Ponte, and Biscoe, 1988; L6pez-Barneo, L6pez-L6pez, Urefia, and Gonzfilez, 1988; Urefia, L6pez-L6pez, Gonzfilez, and L6pez-Barneo, 1989a). Furthermore, it has also been found that the voltage-gated K ÷ current of type I cells is reversibly attenuated by lowering environmental Po2, whereas Na s and Ca e+ currents remain unaltered (L6pez-Barneo et al., 1988). These findings, confirmed by the parallel work of other investigators on several mammalian species (Delpiano and Hescheler, 1989; Hescheler, Delpiano, Acker, and Pietruschka, 1989; Peers, 1990; Stea and Nurse, 1991), have provided a framework for understanding the basic mechanisms underlying sensory transduction in the carotid body. Inhibition of the O2-sensitive K ÷ current under hypoxic conditions produces an increase in the firing frequency of glomus cells (L6pez-L6pez, Gonzfilez, Urefia, and L6pez-Barneo, 1989), which could lead to Ca 2+ influx, enhanced transmitter release, and activation of the afferent fibers of the sinus nerve. This basic scheme is also supported by work on type I cells loaded with fluorescent Ca 2s indicators showing an increase in cytosolic Ca 2s in response to low Po2 (Biscoe and Duchen, 1990a; Benot, A., J. Urefia, and J. L6pez-Barneo, unpublished observations). The macroscopic K ÷ current recorded in type I cells has a small Ca2S-dependent component that disappears after wash-out of Ca 2+ channels and when internal solutions with high Ca2+-buffering capacity are used. In addition, the Ca2S-indepen - dent component of the current inactivates almost entirely in 200 ms but the degree of inactivation varies among different cells (Urefia et al., 1989a). Therefore, it can be expected that, as in other excitable cells (Marty and Neher, 1985; Hoshi and Aldrich, 1988), glomus cells possess several classes of K s channels with specific biophysical properties (for review, see Rudy, 1988). The present research was undertaken to establish a first classification of K + channels in type I cells and to ascertain whether a specific K s channel class is responsible for the O2 sensitivity of the cells' electrical properties. These questions are of critical importance for elucidating the molecular mechanisms underlying 02 sensing. The identification of the primary site involved in 02 detection is also of interest because it has been argued that the attenuation of the macroscopic K + current on lowering Po2 could be a secondary phenomenon rather than an initial step in the process of chemotransduction (Biscoe and Duchen, 1989, 1990a, b). In this paper we present a systematic analysis of the single K s channel types found in glomus cells. We show that in excised membrane patches there are three different on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 GANFORNINA AND LrPEZ-BARNEO Single K + Channel Modulated by 02 in Glomus Cells 403 K + channel types and that only one of them (referred to as "Ko~ channel") is selectively and reversibly modulated by hypoxia. The characteristics of these channels fully account for the properties of the macroscopic O2-sensitive K + current. Our experimental results indicate that Ko 2 channels are directly regulated by 02 and strongly suggest that the 02 sensor of chemoreceptor cells is in, or closely associated with, the plasma membrane. In the following article we focus on a more detailed description of the kinetic properties of the Ko 2 channel and propose a minimal model that.explains the effects of hypoxia on channel gating. A brief report of part of the work presented in this paper has been published (Ganfornina and L6pez-Barneo, 1991). METHODS Cell Preparation Experiments were performed on type I cells isolated from rabbit carotid bodies. The procedures followed for enzymatic cell dispersion and culture were the same as described TABLE I Composition of Solutions External NaCl KCI CaCle MgCI2 HEPES Glucose Standard Na 140 2.7 2.5-5 2 10 5 140 K -- 140 2.5-5 2 10 5 80 K 60 80 2.5-5 2 10 5 Internal KCI K-glutamate KF HEPES EGTA MgCI~ Standard K 30 80 20 10 10 2.58 130 K 130 -- -- 10 10 2.58 All values are given in millimolar. Tetrodotoxin at a concentration of 0.6 IxM was added to the standard Na and 80 K solutions. Solutions with variable [Ca 2+] were made by mixing CaCI2, MgClz, and EGTA. The final free [Ca 2÷] was calculated with a computer program that takes into account the affinity constants of EGTA for Ca 2+ and Mg 2+ at different pH (Tabares, Urefia, and L6pez-Barneo, 1989). In those solutions with 10 mM EGTA and no Ca 2+ added the estimated [Ca ~+] was < 10 -9 M. previously (L6pez-Barneo et al., 1988; Urefia et al., 1989a). Cells were plated on slivers of glass coverslips treated with poly-l-lysine and used for recording between 12 h and 2 d after dissociation. During the experiments a coverslip was transferred to a small chamber of ~ 0.2 ml volume with continuous flow of solutions that could be replaced in 10-15 s. Solutions The composition of solutions used in the experiments is shown in Table I. Solutions were adjusted to a pH of 7.3 (internal) or between 7.35 and 7.4 (external) and had an osmolarity of 290-300 mosmol/kg. In the text and in the figure legends solutions are given as external// internal with specification of the final [Ca z÷] or [EGTA] used. Experiments were performed at on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 404 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992 room temperature (22-25°C). During the experiments the external solutions were equilibrated with either air, N2, or a mixture of both, in order to obtain the desired Po2 in the recording chamber (see below). Recording Techniques The data presented in this article are mainly based on single K + channel currents recorded from membrane patches of glomus cells using the patch clamp technique (Hamill, Marry, Neher, Sakmann, and Sigworth, 1981). In some experiments whole-cell K + currents were studied before establishing the outside-out patch configuration following the methodology previously reported (Urefia et al., 1989a). For single-channel recording we used fire-polished glass pipettes fabricated from borosilicate glass (Kimax 51) that once filled with solution had a resistance of 4-10 MI'~. The recording bandwidth of the amplifier was 10 kHz, however its output signal was low-pass filtered by an 8-pole Bessel filter (model 902; Frequency Devices Inc., Haverhill, MA) with cutoff frequencies between 1 and 2 kHz, giving an effective cutoff frequency of 0.95-1.29 kHz (Colquhoun and Sigworth, 1983). The time resolution of our recording system and its influence on the measurement of the amplitude and duration of single-channel events is further explained in the accompanying paper. Data Acquisition and Analysis An IBM-PC-AT computer interfaced to the analog electronics was used for data acquisition, display, and analysis. In most experiments we recorded ionic currents in response to voltage steps. In these cases the current signal was digitized on-line by an input-output interface built in our laboratory (Urefia, Mateos, and Lrpez-Barneo, 1989b). A sweep was defined by either 500 or 1,000 digital points. Single-channel currents generated in response to long-lasting (> 220 ms) or stationary depolarizations were initially stored on video tape. The segments of the signal required for figures or analysis were replayed on a chart recorder or converted into digital form using a GPIB-PC card plugged into the computer expansion slots. The sample frequency varied according to the experimental protocol and is given in the figure legends. Leakage and uncompensated capacity currents were digitally subtracted using scaled templates constructed by fitting smooth functions to either records with no openings or to the average of 20 consecutive current sweeps generated by 20-mV hyperpolarizations from a holding potential of -80 mV. Ensemble averages were obtained from original traces after capacity and leakage subtraction. Single-channel current amplitude was measured by averaging values obtained from 20-40 well-resolved single events. We used a 50% amplitude criterion to detect opening and closing transitions (Colquhoun and Sigworth, 1983). Unless otherwise noted, no correction for unresolved events was performed. The number of active channels in a patch (N) was determined by observing for long time periods the maximum number of simultaneous current steps that appeared at strongly depolarized voltages. In patches where one or two simultaneous openings were observed, the probability that this number could be smaller than the actual value of N was statistically tested by the binomial distribution method as indicated by Patlak and Horn (1982). In those recordings where the estimated value of N was one, average channel open probability (Pop,n) was calculated by dividing the time spent in the open state by the total duration of the recording. In patches with more than one channel, Pop~n was estimated by the formula: Popen = (N'i'T)-t'f:l(t) "dt (1) where i is the single-channel current amplittrde, T is the duration of the pulse or of the observation period in stationary conditions, and I(t) is the net current during the recording on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 GANFORNINA AND L6PEZ-BARNEO Single K + Channel Modulated by 02 in Glomus Cells 405 period. In channels activated during depolarizing pulses, we obtained an ensemble current average (l(a)) given by: I (a) = N'i'Po(t) (2) where Po(t) is the open probability as a function of time. Significance of differences between mean values obtained (e.g., for different Po2) was determined with a Student's t test for paired samples. Unless otherwise indicated, the level of significance (cx) of the test was set at 0.05. A > v 0 > 2.0 1.5 1.0 0.5 0 B 1.0 '~ I 0.8 i i > 0.6 o 0.4 I I > I I I 0.2 0 ~- 0 0.3 0.6 0.9 1.2 0 V bottery (V) I I I 30 60 90 % 02 i 120 C D N2 N2 N 2 PO2(mmHg) lOO 50 o Air Air Air 1 min FIGURE 1. Performance and calibration of the O2-sensing electrode. (A) Output voltage of the current-to-voltage converter as a function of the negative polarizing potential and 02 tension. 150 mmHg (filled circles), 87 mmHg (open circles), and 0--5 mmHg (filled squares). At a polarizing voltage between -0.6 and -0.8 V the output voltage is linearly related to the Po 2 in the solution (range between dashed lines). (B) Output voltage vs. %02 relative to air at a polarizing voltage of -0.7 V. (C and D) Responses of the 02 electrode in the chamber (C) and during instantaneous immersions in solutions equilibrated with N2 and air (D). Measurement of Oxygen Tension Because some experimental protocols required repetitive exposure of a membrane patch to external solutions with a reproducible Po2 value, we built an O2-measuring electrode to estimate Po2 values in the vicinity of the current-recording pipette. We used a negatively polarized 100-v.m-thick platinum wire insulated by an O2-impenneant enamel except at the end section (Tsacopoulos and Lehmenkiihler, 1977; Tsacopoulos, Poitry, and Borsellino, 1981). Current generated in the wire in response to variable PO 2 values was recorded by an on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 406 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 " 1992 operational amplifier wired as an 1/V converter. The negative pole of a d.c. battery was connected to the noninverting input of the I/V converter to maintain the polarizing voltage at a constant value. The major characteristics as well as the performance of the O2-measuring electrode are illustrated in Fig. 1. Plot A shows the changes in the output voltage of the 1/V converter (Vou0 as a function of the negative potential applied to the platinum electrode (Vbatte~y) for three different Po~ values. In the Vbatte~y range between -0.6 and -0.8 V, Vout was proportional to the 02 concentration; thus we used -0.7 V as the most appropriate value to polarize the platinum electrode. At this polarizing voltage, which was used in all the experiments, the output voltage of the recording electrode is linearly related to the O2 concentration and therefore a calibration curve could be done using solutions equilibrated with known concentrations of 02 (Fig. 1 B ). During the experimental protocol Vout was continuously monitored and stored on tape. The response of the electrode is illustrated in Fig. 1, C and D. When immersed in solutions equilibrated with N2 or air, the change in voltage was almost instantaneous (D), switching to similar solutions when placed in the experimental chamber produced a fast change (~ 80% of the maximum) in a few seconds but complete equilibration required >40 s (C). The voltage signal from the Oz-sensing electrode was unaffected by changes in pH or by modifications in the ionic composition of the solutions. Most of the experiments reported here and in the accompanying report were based on repetitive exposure of the K ÷ channels to a reproducible Poz value. Given that our recording chamber is in contact with the air, we found that the most easily reproducible Poz level in the vicinity of the cells (Po2 = ~ 5-10 mmHg) was obtained by bubbling the test solution with N 2. RESULTS There Are Three Major K + Channel Types in Glomus Cells Single K + channel currents recorded from membrane patches with well-resolved single-channel events allowed the classification of the K + channels of type I cells into three major classes. To facilitate comparison, single-channel currents representative of the various K + channel types are shown in isolation in Fig. 2. Table II summarizes the major properties of each channel population. The traces of Fig. 2 are from three different inside-out patches with two functional channels. In all cases the membrane was exposed to asymmetrical K + solutions and the current was recorded at various membrane potentials (Vm). The three sets of recordings display openings and closures of the channels that appear as current steps of fixed amplitude. Fast transitions are partially filtered due to the limited recording bandwidth. Opening of each channel type produced current steps of clearly different amplitude, but in all cases channel opening was favored by membrane depolarization. It will be shown below that besides distinct single-channel conductance values the three channel populations also differ in their kinetic properties, Ca z+ dependence, and sensitivity to changes in Po2 (see Table II). For the sake of clarity abbreviations are used for each channel type. Kc, denotes the large Ca2+-activated K + channels, SK are channels of small conductance, and Ko 2 refers to the O2-sensitive K + channels. Ca2+-dependent K + Channels (Kca Channels) Kca channel activity was clearly observed in inside-out membrane patches (a total of 24 patches) when [Ca 2+] in the solution facing the cytosolic side of the membrane was >0.1 ~M. These channels were active during maintained depolarizations and on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 GANFORNINA AND LOPEZ-BARNEO Kca V::oV Single K + Channel Modulated by 02 in Glomus Cells SK Ko2 0 2 ~ -- 02 01 02 01 __01 C --C C 407 20 ~15p A ....... ~ 2pA 50 ms FIGURE 2. Representative recordings of the activity of the three major K ÷ channel types found in type I cells. The data were obtained from different inside-out excised patches, containing two channels each, depolarized at the indicated membrane potentials. Kca = Ca~+-activated channels; SK = small conductance channels; K% = O~-sensitive channels. In all figures upward deflections from the zero current level (c) indicate outward current. K~ and SK channels were recorded under steady depolarizations and K% channels upon 200-ms step depolarizations from -80 mV. Effective cutoff frequency = 0.95 kHz and sampling interval = 500 Ixs. Solutions: standard Na, TIX//130 K, 10 EGTA. K~ channels were recorded with an internal solution containing 1 IxM free Ca 2+. therefore their single-channel current-voltage (i-Vm) relation and Popen were studied after steady-state changes in the membrane potential. Single Kca channel activity as a function of the membrane potential is illustrated in Fig. 3 A. The traces are from a patch bathed in symmetrical K + solutions that contained at least three active channels. Single-channel current amplitude varied in parallel with the electrochemical driving force for K + ions, and the number of active channels as well as the time TABLE II Classification of K ÷ Channels in Gloraus Cells Channel Conductance in 130 K//130 K Conductance in 2.7 K//130 K Ca 02 type Inside-out Outside-out Inside-out Outside-out dependence sensitivity K~ 206.7 (10) -- 83.7 (3) -- Yes No SK 16 (6) -- 6 (2) -- No No K% 41.5 (1) 41.5 (2) 17.6 (18) 20.1 (14) No Yes 21 (10)* 19.5 (5)* Average slope conductance values are given in picosiemens. The number of patches is in parentheses. Kc~ = Ca-activated channel; SK = small conductance channel; K% - oxygen-sensitive channel. For the K% channel values are given in control and hypoxic ( ) conditions. Potassium concentrations are in millimolar and indicated as external//internal. on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 408 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992 spent in the open state increased with depolarization. The average i-Vm relations obtained from data pooled from several patches exposed to symmetrical and asymmetrical K ÷ concentrations ([K÷]) are plotted in Fig. :3 B. In symmetrical [K ÷] (filled symbols) the i-Vm plot is linear between -50 and +60 mV, and the reversal potential was 0 mV, as expected for K+-selective channels. A linear regression fit to the data points yields a value of 206.7 pS (n = 10 patches) as the average singleA Vm ~- o2 --01 (mV) +30 --C ~ --03 --0 2 --01 +20 --C -10 ........ C --0 2 -20 -~--C --01 --0 2 1 O0 ms B I -90 15 9 -30 • i(pA) I I 30 90 FIGURE 3. Single-channel current-voltage relation of the Kca channels. (A) Unitary events recorded in an inside-out excised patch with at least three simultaneously open channels at various stationary membrane potentials. Effective cutoff frequency = 0.95 kHz and sampling interval = 500 I~s. Solutions: 130 K, 0.01 ~M Ca2+//130 K, 1 I~M Ca 2+. (B) Plot of single-channel current (i) as a function of the membrane potential (Vm) measured in symmetrical (filled symbols, n = 10 inside-out patches) and asymmetrical (open symbols, n = 3 inside-out patches) K + concentrations. The points indicate the mean ± SD values and the straight lines the linear regression fits to the data. Slope conductances are: 206.7 pS (filled symbols, r = 0.99) and 83.7 pS (open symbols, r = 0.98). Solutions: 130 K, 0.01 I~M Ca2+//130 K, 1 I~M Ca z+ (symmetrical [K+]); standard Na, TFX//130 K, 1 I~M Ca 2+ (asymmetrical [K+]). channel conductance. In asymmetrical [K ÷] (open symbols) the average slope conductance, measured between -30 and +50 mV, is 83.7 pS (n = 3 patches). The possible modulatory effect of Po2 on the activity of Kca channels was investigated in inside-out excised patches that were initially exposed to variable internal Ca ~+ concentrations ([Ca2+]i) to test the Ca 2+ dependence of channel activation, and thereafter to various Po~ levels keeping [Ca2+]i unaltered. An example on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 GANFORNINA AND LrPEZ-BARNEO Single K + Channel Modulated by 02 in Glomus Cells 409 of this experimental protocol is illustrated in Fig. 4. The membrane was bathed in symmetrical high K + solutions with a [Ca2+]i of 1 ~M and held at a potential of +20 inV. Under these conditions opening of the two active channels included in the patch produced an outward current that disappeared completely after switching to a solution with 0.01 o,M Ca ~+. This effect was perfectly reversible on reintroduction of 1 ~M Ca 2+ in the chamber (Fig. 4 A ). When at a fixed [CaZ+]i of 1 v,M the same patch was exposed to low Po2, no appreciable changes in single-channel activity or unitary A 02 -- 01 -- C -- 0.01 #.M Ca 2+ 1 /,t,M Ca 2+ i l B 02-- l 0 t -- C -- 10 pA 140 mmHg N 2 Air FIGURE 4. Dependence of Kca channels on internal Ca ~+ and lack of effect of changes in Po2. (A) Activity at +20 mV of two Kca channels in an inside-out patch exposed to 1 I~M internal Ca 2+ and reversible inhibition when [Ca2+]i is decreased to 0.01 IxM. (B) At the same membrane potentials and [Ca2+]i = 1 I~M channel activity was unaffected by lowering Po2. The signal from the O~-measuring electrode is shown in the lower panel. Effective cutoff frequency = 0.95 kHz. Solutions: 130 K, 0.01 I~M Ca~÷//130 K, 1 IJ.M or 0.01 I~M Ca ~+. current amplitude were observed (Fig. 4 B). The signal from the Po2-measuring electrode is shown in the lower panel. Following this same experimental procedure, but using various [Ca2+]i (between 0.04 and 1 ~M) and V~ values (between -30 and +30 mV), exposure to hypoxia did not alter Kca channel open probability (Popen) in the 16 patches tested (paired t test). Fig. 5 summarizes the effects of [Ca2+]i and low Po 2 on Kca channel activity. In Fig. 5 A, single-channel Po~, as a function of Vm is plotted at two [Ca~+]i. In both cases the data points are the mean +_ SE values from on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 416 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992 recordings (panel B) show a peak Pope, at the beginning of the depolarization and a progressive decrease during the pulse. Single-channel Pope, integrated throughout the pulse duration (see Eq. 1) is 0.31 in the control solution but only 0.18 during exposure to low Po2. Thus, hypoxia produces a reversible decrease in channel open probability but, as shown in Fig. 8 B (triangles), it does not modify either the i-Vm relation or the single-channel conductance (see Table II). A summary of the effect of low Po2 on the Popen of Ko 2 channels at different membrane potentials is shown in Fig. 11. The average Popen value during 200-ms pulses was measured in patches with one or two functional Ko~ channels. Low Po2 produced a decrease in single-channel Pope. that was statistically significant at all membrane voltages (paired t test, tx < 0.01). At +30 mV, for example, Popen is ~0.43 in the control solution and ~0.31 in low Po2, but at 0 mV these values are, respectively, ~0.2 and ~0.07. This indicates that the inhibition of K + channel activity by hypoxia is more marked at less depolarized membrane potentials (see also GL 0 O0.6 0.4 0.2 [~] control low PO2 0 +10 +20 +30 w (my) FIGURE 11. Decrease of Ko 2 channel Pop, n by hypoxia. Average open probability during 200-ms pulses (ordinate) was measured from either inside-out or outside-out patches at various membrane potentials. Pop,n values measured in the control and in the low (<5 mmHg) Po2 solutions are represented by the mean +-- SE. Number of experiments were: n = 5 (0 mV); n = 3 (+10 mV); n = 23 (+20 mV); and n = 2 (+30 mV). At all voltages the differences were statistically significant (paired t test, c~ < 0.01). Solutions: standard Na +, TIX//130 KCI, 10 EGTA or standard K, 10 EGTA. accompanying paper). For simplicity, in this set of experiments solutions were bubbled with either N~ or air and thus the low Po 2 values in the vicinity of membrane patches were ~ 5-10 mmHg (see Methods). With this protocol the decrease of Pope. by hypoxia is underestimated since it is known that on exposure to extremely low Po2 the inhibition of K + channel activity is relatively less pronounced than when the channels are exposed to moderately low Po2 values (between 60 and 80 mmHg; L6pez-L6pez et al., 1989; Ganfomina and L6pez-Barneo, 1991). Direct modulation of Ko 2 channels accounts for the properties of the macroscopic O2-sensitive K + current. The kinetic and pharmacological properties of the Ko 2 channels indicate that they are the main channels responsible for the macroscopic O~-sensitive K + current of type I cells. The ensemble average currents shown earlier (see Fig. 10 B) illustrate that the time course exhibited by Ko 2 channels during a depolarization closely resembles the kinetics of the macroscopic K + current which turns on in a few milliseconds and inactivates almost completely in 200--300 ms on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 GANFORNINA AND L6PEZ-BARNEO Single K + Channel Modulated by 02 m Glomus Cells 417 (Lrpez-L6pez et al., 1989; see also Fig. 12). In addition, TEA + reversibly blocks the whole-cell K + current (Urefia et al., 1989a) as well as the Koz channels (Ganfornina and Lrpez-Barneo, 1991). The parallel time courses of Ko~ channel Pope, and the macroscopic K + current were clearly evident when recordings in the whole-cell mode and in outside-out multichannel patches were obtained following the same experimental protocol. Fig. 12 illustrates the reversible inhibition of the macroscopic K + current (traces in C) and the decrease in K + channel Popen in an outside-out patch with at least five channels (traces in A and B ) during a transient exposure to hypoxia. The ensemble averages of Fig. 12 B, which represent the behavior of a few channels, have a time course comparable to that of the whole-cell currents. Control Hypoxia Recovery B C 50 ms pA • r~A FIGURE 12. Comparison of the effect of lowering Po2 on single Ko 2 channel and macroscopic K + currents elicited by depolarization to 0 mV from a holding potential of -80 mV. (A and B ) Representative single sweeps and ensemble averages of current recorded from an outside-out patch, containing at least five channels, in the control solution (Po~ = 150 mmHg; n = 13 consecutive sweeps; Pope, = 0.29), during a 9-min exposure to hypoxia (Po~ < 5 mmHg; n = 18 consecutive pulses; Pop~, = 0.15), and after recovery in the normal Po2 solution (n = 18 consecutive pulses; Po~, = 0.27). Effective cutoff frequency = 0.95 kHz and sampling interval = 500 p.s. (C) Whole-cell currents recorded with the same experimental protocol. Effective cutoff frequency = 10 kHz and sampling interval = 500 Ws. Current calibration bar is 2 pA for A and B, and 0.6 nA for C. Solutions: standard Na, TI'X//130 K, l0 EGTA. With a high Ca 2+ buffer capacity at the internal solution, most of the macroscopic K + current is due to the activity of Ko~ channels since Kca channels cannot be activated and, in addition, the density of SK channels is low and their unitary conductance is small. Therefore, the number of Ko~ channels can be estimated by dividing the peak K ÷ current by the value of the single Ko~ channel current amplitude at the same voltage and correcting for the peak channel Popen (~ 0.8 at +20 mV). Our estimate gives values of 720 -+ 80 (mean -+ SD, n = 18) channels per cell, which corresponds to two to four channels per square micrometer. This relatively on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 418 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 - 1992 high density may explain why we obtained multichannel patches in ~ 50% of the experiments (87 of 168) even though we used relatively high resistance pipettes (> 8 MI~). It was shown in a previous report that the modulation by 02 of the macroscopic K + current of type I cells is independent of internal Ca 2÷ or the presence of exogenous nucleotides (L6pez-Barneo et al., 1988; L6pez-L6pez et al., 1989). Our results at the single-channel level confirm and extend these observations since reversible inhibition A Control B Hypoxia _J I C ~, control ~. I1 pA 2 pA 100 ms FIGURE 13. Lack of desensitization after maintained exposure to low Po2. (A and B) Single-channel currents recorded from an inside-out patch with two Ko~ channels during 350-ms depolarizations to +20 mV from -80 mV. The cell was exposed for 30 min to low Po2 ( < 5 mmHg) before excising the patch from which the sweeps were obtained during alternating exposures to control and hypoxic solution as explained in Fig. 10. (C) Superimposed ensemble averages of traces recorded in control (n = 25 sweeps, Pop~n = 0.52) and hypoxic (n = 16 sweeps, Po~n = 0.28) conditions. Effective cutoff frequency = 0.95 kHz and sampling interval = 500 ~s. Solutions: standard Na, TIX//130 K, 10 EGTA. of Ko~ channel activity was observed in excised patches without Ca 2+ or nucleotides added to the internal solution. We also tested whether 02 could act through the activation of a membrane-bound G protein, a family of proteins that are irreversibly activated by GTP-,/-S (Gilman, 1987) or by A1F 4 formed from fluoride (a normal component of some of our internal solutions) and aluminum (which could be released from the micropipette glass) (Sternweis and Gilman, 1982; Bigay, Deterre, Pfister, and Chabre, 1985). The reversible modulation of Ko 2 channels by changes in Poe was on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 GANFORNINA AND LOPEZ-BARNEO Single K + Channel Modulated by 02 in Glomus Cells 419 unaltered in solutions free of F- (n = 51) or when up to 200 v,m GTP-',/-S was added to the internal solution (n = 22). Thus, the results suggest that soluble cytosolic mediators or membrane-bound G proteins do not participate in the effect of 02 on the Ko 2 channel, and that Oz may interact with an intrinsic sensor closely associated with the channel protein. Repetitive exposure to low Po2 does not produce desensitization. In many examples of ligand-receptor interaction, repeated or permanent exposure to the agonist produces an attenuation of the physiological response. This phenomenon, called "desensitization," has been typically studied in some ligand-activated channels and it is well known that, after withdrawal of the agonist, recovery to the resting conditions is slow (see Hille, 1984). Although desensitization is a term applicable to ligandreceptor interactions, it is a phenomenon that could play a part in the physiological adaptation observed in some sensory receptors (Stebbens, Brown, and Peterson, 1984). It was described in our previous work that chemosensory transduction in the type I cell is a nonadapting, or slowly adapting, process since reversible attenuation of the macroscopic K + current can be repeatedly observed in a given cell (L6pez-Barneo et al., 1988; see also Ganfornina, 1991). Fig. 13 shows single-channel current sweeps recorded during alternating exposure to control (A) and low Po2 (B) solutions in a patch with two Ko 2 channels excised from a cell that had been preincubated in extreme hypoxia (Po2 = 5 mmHg) for 30 min. To facilitate comparison, ensemble averages in the two experimental conditions are shown superimposed in Fig. 13 C. Reversible inhibition of Ko 2 channel activity by low Po2 can be observed repeatedly after long-lasting exposure to extreme hypoxia, further suggesting that the O2-Ko~ channel interaction does not desensitize. DISCUSSION In this article we describe the properties of three types of K + channels in chemoreceptor cells of the carotid body that can be distinguished by their biophysical characteristics. We also demonstrate that in excised membrane patches only the activity of a specific K + channel class, the Ko~ channel, is reversibly inhibited by lowering environmental Po~. Our findings explain the modulation by O~ of the macroscopic K + current of glomus cells and strongly suggest that the O2-sensing mechanism resides in the plasma membrane. K + Channel Types in Glomus Cells Both cell-attached and excised membrane patches of rabbit glomus cells contain three major K+-selective channels (Kca, SK, and Ko~ channels). A Ca~+-independent and high-conductance CIchannel encountered in rat type I cells (Stea and Nurse, 1989) was not studied. The three classes of K ÷ channels differ in their single-channel conductance and kinetics as well as in their dependence on internal Ca 2+ and 02 sensitivity. K + channels are extraordinarily diverse (see for reviews Rudy, 1988; Adams and Nonner, 1989) and it is well known that different subpopulations coexist in a given membrane (Dubois, 1983; Conti, Hille, and Nonner, 1984; Marty and Neher, 1985; Hoshi and Aldrich, 1988; Llano, Webb, and Bezanilla, 1988). In this respect, the single K + channels identified in type I cells share most of their properties on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 420 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992 with those classified in bovine chromaffin (Marry and Neher, 1985) and mouse neuroblastoma (Quandt, 1988) cells. Equivalent single-channel currents arc also present in pheochromocytoma cells (Hoshi and Aldrich, 1988). Interestingly, all these cell types have a close embryological origin. The Kca channels have, in symmetrical high K + solutions, an average conductance of 206.7 pS. These channels are similar to the maxi-K + Ca2+-dependent channels of other preparations (Marty, 1981; Barrett et al., 1982; Quandt, 1988), and, in excised patches, they arc unaffected by changes in Po~. These observations confirm our previous experiments showing that part of the macroscopic K + current, presumably a Ca2+-dcpendent component, disappears after wash-out of Ca 2+ channels (Urefia et al., 1989a) and that under these conditions, and with l0 mM EGTA added to the internal solution, the K + current is still reversibly attenuated by lowering Po2 (L6pez-Barneo et al., 1988; L6pez-L6pez et al., 1989). It has been reported that hypoxia specifically inhibits the Ca2+-dependent component of the macroscopic K + current recorded in dialyzed carotid body cells from newborn rats (Peers, 1990). The discrepancy between these data and our wholc-ccU and single-channel results may reflect a difference between animal species; however, wc also believe that the conclusion reached by Peers (1990) may have been biased by the experimental protocol used in the isolation of the Ca2+-activated K + current. We know, for example, that millimolar concentrations of Cd ~+ and Co 2+, which could produce a decrease of the Ca2+-activatcd K + current due to blockade of Ca 2+ channels, can also produce a large and reversible inhibition of the Ca2+-independent and O~-sensitive component of the K + current (Ganfornina, M.D., and J. L6pez-Barneo, unpublished results). We followed the terminology of Marty and Neher (1985) to denote a second population of K+-selective channels that have a small conductance (SK channels). In asymmetrical K + solutions the unitary conductance of carotid body SK channels (~ 6-7 pS) is similar to the values reported in chromaffin and neuroblastoma cells (Marty and Ncher, 1985; Quandt, 1988). Hoshi and Aldrich (1988) have also found in pheochromocytoma cells two populations of K + channels (Ky and Kx) with the same unitary conductance value. Although we did not study in detail the kinetic properties of SK channels, in accord with previous work (Marty and Nchcr, 1985; Quandt, 1988), they behaved as Ca2+-independent and slowly activating channels. In glomus cells inactivation of SK channels, if any, must be also very slow since channel activity could be recorded for minutes at depolarized membrane potentials. SK channels are only moderately voltage dependent (Popen = 0.2 at +20 and 0.05 at -70 mV) and their Popen at negative voltages suggests that they may contribute to the gcncration of the rcsting potential of the cells. In excised patches SK channels wcrc unaltcrcd by changcs in Po2, but we cannot discount that in situ they could be subjected to modulation. This idea is based on the fact that in some inside-out patches the activity of SK channels appeared abruptly several minutcs after excision of the membrane, which could be explained by the dilution of some soluble mediator that blocks the channels or that favors their closed conformation. Wc have coined the term Ko 2 to designate the K+-sclectivc and O2-scnsitive channels of type I cells. These channels were the most frequently observed, probably because they are densely packed in the glomus cell membrane. Our estimate is two to on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 GANFORNINA AND LrPEZ-BARNEO Single K + Channel Modulated by 02 in Glomus Cells 421 four channels per square micrometer. In asymmetrical K + solutions the unitary conductance of the Ko 2 channel is ~ 20 pS. This value is in excellent agreement with the conductance of fast activating (FK) channels in chromaffin, neuroblastoma, and pheochromocytoma cells (Marty and Neher, 1985; Hoshi and Aldrich, 1988; Quandt, 1988) as well as of delayed rectifier and A-type K + channels described in a number of preparations (Cooper and Shrier, 1985, 1989; Kasai, Kameyama, Yamaguchi, and Fukuda, 1986; Llano et al., 1988). K% channels are not influenced by changes in internal Ca 2+ but they are steeply dependent on membrane voltage; the activation threshold is at ~-50 to -40 mV, and at +20 mV the peak/)open is 0.8. This last parameter is similar to values reported for other mammalian inactivating K ÷ channels (Marty and Neher, 1985; Cooper and Shrier, 1989). Although the activation and inactivation kinetics are studied in more detail in the accompanying article, here we show that K% channels have a fast activation. As the membrane is more depolarized the number of active channels increases and the latency to the first opening decreases. During maintained depolarizations Ko 2 channels inactivate completely in a few hundred milliseconds. These properties are perfectly compatible with the characteristics of the macroscopic K + current of type I cells (Urefia et al., 1989a) and strongly suggest that the Ko~ channels are the main contributors to this current. This is also supported by the close parallelism existing between the time courses of the whole-cell K + current and the ensemble averages from patches containing only Ko~ channels. Modulation of Ko 2 Channels by 02 Tension A distinct property of Ko~ channels is that their Popen decreases on exposure to low Po2. The inhibition of channel activity by hypoxia is reversible and concentration dependent (Ganfornina and L6pez-Barneo, 1991), and is a process that does not undergo desensitization. These properties fit perfectly with those encountered in the modulation of the macroscopic K + current by Po2 (L6pez-Barneo et al., 1988; L6pez-L6pez et al., 1989). The correspondence between the 02 modulation of whole-cell and single-channel currents is quite remarkable and can be clearly seen when ensemble averages from patches containing Ko 2 channels in isolation are compared with the O2-sensitive K ÷ current (see, for example, Fig. 12). The major effect of lowering Po2 is a decrease in the Pope, of the channels leaving unaltered unitary conductance. Interestingly, the magnitude of the decrease of Pope, in hypoxic conditions in the voltage range between +10 and +30 mV (~25% of the control value) is the same as the inhibition of the macroscopic K + current by low Po2 (L6pez-Barneo et al., 1988). The action of low Po2 is more pronounced at less depolarized membrane voltages, which is an observation that, as discussed in the accompanying paper, could be expected if the lack of Oz favors closed or inactivated conformations of the channels. Delpiano and Hescheler (1989) have reported in cell-attached patches of glomus cells from rabbit embryos the existence of a K ÷ channel reversibly inhibited by lowering Po2. This channel was recorded during stationary membrane depolarizations and the unitary conductance value was 137 pS with high K ÷ in the pipette solution. These results are difficult to compare with our own since in Delpiano and Hescheler's work the O2-sensitive channel was not characterized and it does not on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 422 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992 include data about other possible channel types. The kinetics and conductance of Delpiano and Hescheler's channel are comparable to those of the Kca channel, which, as shown before, is insensitive to changes in Po2 in excised patches. Although we have observed in situ the same types of K + channels as in excised patches, we did not attempt an initial classification of K + channels in the cell-attached configuration because the membrane potential, cytosolic Ca 2+ concentration, and other variables are unknown. In addition, we have also observed that type I cells are electrically very compact and therefore current flowing through a cell-attached patch can induce modifications in the membrane potential of the cell (Ganfornina, 1991). Nevertheless, we cannot eliminate the possibility that there could be a change in the O2-sensing mechanisms of carotid body cells during development (Hertzberg, Hellstr6m, Lagercrantz, and Pequignot, 1990). In this respect it is interesting to note that whereas embryonic glomus cells seem to lack Na + channels (Hescheler et al., 1989), in the adult tissue large Na + currents can be recorded (Urefia et al., 1989a). The fact that the modulation of Ko 2 channels by O2 is maintained for long periods of time in excised patches strongly suggests that the O2-Ko 2 channel interaction occurs through an intrinsic sensor of the plasma membrane which may be part of the channels or a molecule closely associated with them. This interaction seems to be direct without the participation of soluble cytosolic mediators. We have sought for the possible involvement of membrane-diffusible G proteins, which after being activated can directly regulate ionic channel activity (Logothetis, Kurachi, Galper, Neer, and Claphan, 1987; Brown and Birnbaumer, 1988), with negative results. Both GTP-~/-S and F-, agents that irreversibly activate G proteins (Gilman, 1987), were ineffective in preventing the reversibility of the inhibition of Ko 2 channels by lowering Po2. Nonetheless, we cannot discount that the Ko 2 or other channels of glomus cells might be modulated by cytosolic mediators in situ. Exposure to hypoxia alters the content of cGMP and cAMP in the carotid body (Wary, Cheng, Dinger, and Fidone, 1989; P6rez-Garcia, Almaraz, and Gonz~lez, 1990) and these agents are known to regulate a broad number of ionic channels. The Ko 2 channel may belong to a family of 02 sensors broadly distributed in nature. Apart from the well-known 02 transport functions of heme proteins, there are, from bacteria to mammalian cells, examples of heme-linked enzymes, the activity of which is regulated by environmental 02 (Goldberg, Dunning, and Bunn, 1988; Gilles-Gonz,41ez, Ditta, and Helinski, 1991). Cross, Henderson, Jones, Delpiano, Hentschel, and Acker (1990) have recently proposed that the activity of a NADPHoxidase in glomus cells (containing a b-type cytochrome) could be regulated by 02, and that this enzyme could determine the redox state of thiol groups of proteins and influence the properties of ionic channels. Physiological Significance of the Ko 2 Channel The Ko 2 channel represents the first known example of an ionic channel regulated by 02. A similar type of regulation has been sought, but not found, in septal neurons (L6pez-L6pez et al., 1989) and in the small dopaminergic interneurons of the sympathetic ganglia (Stea and Nurse, 1991), which are developmentally related to glomus cells. Our results demonstrate the specificity of the Ko 2 channels located in an O2-responsive cell and strongly suggest that they represent the initial step in on September 22, 2014jgp.rupress.orgDownloaded from Published September 1, 1992 GANVORNINA AND L6PEZ-BARNEO Single K + Channel Modulated by Oe in Glomus Cells 423 chemotransduction and thus confer upon glomus cells their unique chemoreceptor properties. Due to our experimental requirements (see Methods), the Poz values of the hypoxic solutions used in this and the accompanying paper are much lower than the ones that can be attained under physiological conditions; however, we have shown before that the Popen of Ko~ channels is reversibly modified by changes of Poz in a physiological range (Ganfornina and L6pez-Barneo, 1991). The lack of appreciable desensitization in the O2-Ko 2 channel interaction may ensure that changes of the physico-chemical variable (02 tension) would be translated into a maintained electrophysiological response. This characteristic could be related to the fact that single-fiber chemoreceptor afferent discharges can be maintained for long periods during sustained hypoxia (Nielsen, Bisgard, and Vidruk, 1988). The significance of Ko 2 channels for respiratory physiology is obvious but they may have a broader functional, and perhaps pathophysiological, relevance. Similar types of channels may exist in lung alveolus and in the fine branches of the pulmonary artery and participate in the regulation of regional pulmonary perfusion, or in small vessels of brain and heart tissues where they may contribute to the autoregulation of blood flow. The authors wish to thank Drs. R. Aldrich, T. Hoshi, and W. Zagotta (Stanford University) for comments on the manuscript. 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