Ionic currents in dispersed chemoreceptor cells of the mammalian carotid body

Ionic Currents in Dispersed Chemoreceptor Cells of the Mammalian Carotid Body j. UREI~/A, J. LOPEZ-L6PEZ, C. GONZALEZ, and j. LOPEZ-BARNEO From the Departamento de Fisiologla y Biofisica, Facultad de Medicina, Universidad de Sevilla, 41009 Sevilla, Spain ABSTRACT Ionic currents of enzymatically dispersed type I and type II cells of the carotid body have been studied using the whole cell variant of the patch-clamp technique. Type II cells only have a tiny, slowly activating outward potassium current. By contrast, in every type I chemoreceptor cell studied we found (a) sodium, (b) calcium, and (c) potassium currents. (a) The sodium current has a fast activation time course and an activation threshold at ~-40 mY. At all voltages inactivation follows a single exponential time course. The time constant of inactivation is 0.67 ms at 0 mV. Half steady state inactivation occurs at a membrane potential of ~- 50 mV. (b) The calcium current is almost totally abolished when most of the external calcium is replaced by magnesium. The activation threshold of this current is at ~-40 mV and at 0 mV it reaches a peak amplitude in 6-8 ms. The calcium current inactivates very slowly and only decreases to 27% of the maximal value at the end of 300-ms pulses to 40 mV. The calcium current was about two times larger when barium ions were used as charge carriers instead of calcium ions. Barium ions also shifted 15-20 mV toward negative voltages the conductance vs. voltage curve. Deactivation kinetics of the calcium current follows a biphasic time course well fitted by the sum of two exponentials. At -80 mV the slow component has a time constant of 1.3 _+ 0.4 ms whereas the fast component, with an amplitude about 20 times larger than the slow component, has a time constant of 0.16 _+ 0.03 ms. These results suggest that type I cells have predominantly fast deactivating calcium channels. The slow component of the tails may represent the activity of a small population of slowly deactivating calcium channels, although other possibilities are considered. (c) Potassium current seems to be mainly due to the activity of voltage-dependent potassium channels, but a small percentage of calcium-activated channels may also exist. This current activates slowly, reaches a peak amplitude in 5-10 ms, and thereafter slowly inactivates. Inactivation is almost complete in 250-300 ms. The potassium current is reversibly blocked by tetraethylammonium. Under current-clamp conditions type I cells can spontaneously fire large action potentials. These results indicate that type I cells are excitable and Address reprint requests to Dr. J. L6pez-Bameo, Departamento de Fisioiogla y Biofisica, Facultad de Medicina, Avenida de S/mchez Pizju~.n, 4, 41009 Sevilla, Spain. Dr. J L6pez-L6pez and Dr. C. Gonz~lez' permanent address is Departamento de Bioquimica y Biologia Molecular y Fisiologla, Facuitad de Medicina, Universidad de Valladolid, C/Ram6n y Cajal 5, 47005 Valladolid, Spain. J. GgN. PHYSIOL. ~ The Rockefeller University Press 9 0022-1295/89/05/0979/21 $2.00 Volume 93 May 1989 979-999 979 980 THE JOURNAL OF GENERAL PHYSIOLOGY" 9 VOLUME 93 9 1989 have a variety of ionic conductances. We suggest a possible participation of these conductances in chemoreception. INTRODUCTION The mammalian carotid bodies are paired organs that sense the level of oxygen tension (pO~) in arterial blood. This sensory information is sent to the central nervous system where it induces an adequate ventilatory response (De Castro, 1928; Heymans et al., 1930; Fitzgerald and Lahiri, 1986). Among the different structures of the receptor complex (glomus or type I cells, subtentacular or type II cells, and nerve endings) type I cells, the most numerous in the carotid body, are presumably the primary chemoreceptors. Type I cells are rich in secretory granules containing dopamine, norepinephrine, and other neurotransmitters and they establish chemical synapses with nerve endings. A decrease in pO~ ultimately results in transmitter release by type I cells and excitation of the afferent fibers of the carotid sinus nerve (see for reviews Eyzaguirre and Zapata, 1968; Fidone and Gonz~lez, 1986). The mechanism involved in the transduction of the hypoxic stimulus is unknown. It has been recently shown that either low pO~ or high external potassium can induce the release of dopamine from type I cells, which is dependent on external calcium and is inhibited by calcium channel blockers (Fidone et al., 1982; Obeso et al., 1987). These data suggest that, in concordance with the mechanism of stimulussecretion coupling in many secretory systems, membrane depolarization may play a part in the response of type I cells to hypoxia. The present research work was undertaken to characterize the electrical properties of the cellular elements of the carotid body and to test the hypothesis that membrane ionic conductances could be altered by changes in pO~. From an electrophysiological viewpoint type I cells are practically unknown. Some previous electrophysiological studies have been performed using intraceUular recording microelectrodes, but they are somewhat confusing and inconclusive because they were probably done on cells damaged by the microelectrode impalement. It has been reported that type I cells are nonexcitable and their electrical parameters are unaltered by hypoxia (Eyzaguirre et al., 1983; Acker and Pietruschka, 1977). These experiments were, however, done on cells with an average resting potential (-20 mV) that is low enough to produce complete inactivation of the voltage-dependent membrane ionic conductances. We have performed experiments in acutely dispersed cells of the carotid body subjected to whole-cell patch clamp, which is a technique that permits accurate electrical measurements in small cells (Hamill et al., 1981). Our data demonstrate that type I cells have a variety of voltage-dependent ionic channels and that they are able to generate large sodiumand calcium-dependent action potentials. Type II cells are unexcitable and on depolarization only generate a small outward current. This article concentrates on the full description of the sodium, calcium, and potassium currents of type I cells. The following article illustrates that in these cells potassium channel activity is reversibly decreased by hypoxia, which may be the membrane mechanism responsible for chemotransduction. Some of this work has already appeared in a short report (L6pez-Barneo et al., 1988). URENA ~rAL. Ionic Currents of Glomus Cells 981 METHODS Cell Dissociation and Culture Experiments were performed on cells dissociated from rabbit carotid bodies. The protocol used for cell dissociation was worked out at the University of Valladolid in collaboration with Dr. Benito Herreros. Animals were anesthetized and the whole region of the carotid artery bifurcation was removed. Four carotid bodies were dissected under a microscope, cleaned, and then each one of them was cut into two or three small pieces. The tissue was placed in a vial with 2 ml of a Ca ~+- and Mg~+-free Tyrode solution of the following composition, in millimolar: 140 NaCI, 4.7 KCi, 5 Na pyruvate, 3 sucrose, 5 glucose, and 10 HEPES. This solution also contained trypsin (2 mg/ml), collagenase (2 mg/ml), and DNase (0.5 mg/ml). Every 10 min the tissue was triturated. After 20 min at 37~ the vial was centrifuged at 800 g for 5 rain. The pellet was resuspended in another 2 ml of the same solution with collagenase (4 mg/ml), DNase (0.5 mg/ml), and albumin (5 mg/ml), and kept at 37~ for 20 min. The tissue was triturated every 10 min. At the end of the incubation period the preparation was washed twice with the Ca ~+ and Mg2+-free solution to remove the enzymes, and the final pellet was resuspended in 5 ml of minimum essential medium supplemented with giutamine (1%), penicillin-streptomicin (2%), and fetal calf serum (5%). Cells were plated on slivers of glass cover slips treated with poly-/-lysine and kept in a CO~ incubator at 37~ until use (4-48 h after plating). After the cells were dissociated, most of them had a round shape and two distinct populations could be easily distinguished. Type I cells had a diameter between 9 and 13 #m and a typical birefringence appearance under the light microscope. A second population of cells, classified as putative type II cells, had a smaller and more uniform diameter (between 5 and 7 um). These two cell types also had clear differences in their electrical properties (see Results). After 48 h in culture many cells began to show growing processes and acquired a bipolar or an irregular shape. Solutions During the experiments a cover slip was transferred to a small chamber that had a continuous flow of solution that could be changed in ~15-20 s. The composition of the recording solutions is shown in Table I. In the text and figure legends solutions are indicated as external/ internal. TI'X (500-1,000 nM) was added to the external solution to block Na channels and in most experiments 3 mM Mg-ATP were added to the internal solution to retard wash-out of Ca channels (Kostyuk, 1984; Forscber and Oxford, 1985; Cota 1986). In some experiments designed to record K currents, the internal solution contained a known concentration of free Ca 2+, which was obtained using Ca-EGTA buffers. The concentrations of Ca and EGTA used in these experiments are indicated in the figure legends. Unless otherwise noted, the pH of the external and internal solutions were adjusted to 7.4 and 7.2-7.3, respectively. Experiments were performed at room temperature (20-25~ Recording Techniques Ionic currents of type I cells were recorded using the whole-cell variant of the patch-clamp technique (Hamill et al., 1981). Patch electrodes were usually fabricated from soft hematocrit capillaries (Hirschmann, Federal Republic of Germany) but in several experiments borosilicate glass (Kimax 51) was also used. In our experimental conditions, current recorded with both types of electrodes were indistinguishable. The electrode tip was fire polished on a microforge and electrode resistance varied between 1 and 2 MfL The patch-clamp amplifier used in most experiments was built by us following the standard design (Sigworth, 1983; Mat- 982 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 93 9 1989 teson and Armstrong, 1984; Armstrong and L6pez-Barneo, 1987). The current-to-voltage converter was a Burr-Brown OPA 111 (Tucson, AZ). Frequency response was improved by using low resistance electrodes, electronic compensation of series resistance, and a relatively low feedback resistance (100 Mfl). Current-clamp recordings were done in some cells using a List patch-clamp amplifier (model LPC-7; Adams and List Associates, Ltd., Great Neck, NY). Data Acquisition and Analysis An IBM-PC/AT computer interfaced to the analog electronics was used for pulse generation and for acquisition, display, storage, and analysis of the data. The interface, fabricated in our laboratory, is built on two IBM prototype cards directly connected to the expansion bus of the computer (Urefia et ai., 1989). The pulse generator has an 8 bit digital-to-analog converter (DCA 0808; Analog Devices, Inc., Norwood, MA) and a programmable peripheral interface (PPI 8255A-5; NEC Microcomputers, Inc.) with the ports programmed in output TABLE I Composition of Solutions External NaC1 KCI CaCI~ MgCI2 BaCI~ CdCI~ TEACI HEPES 140 Na 140 2.7 2.5-10 .... 10 140 Na, 10 Ba 140 2.7 -- -- 10 -- -- 10 140 Na, 9 Mg 140 2.7 1 9 -- -- -- 10 140 Na, 0.5 Cd 140 2.7 1.5 -- -- 0.5 -- 10 100 Na, TEA 100 2.7 5 -- -- -- 40 10 Internal NaCI KCI K-glutamate KF CsCi CsFI MgCI~ HEPES EGTA 130 K -- 30 80 20 -- -- 2 10 5-10 130 Cs .... 110 20 2 10 5-10 90 Cs, 40 Na 40 -- -- -- 70 20 2 10 5-10 All values are given in millimolar. mode (Peatman, 1977; Liu and Gibson, 1986). Current signals are digitized in a 12 bit analog-to-digital converter (ADC, AD578; Analog Devices, Inc.). The 12 lines of parallel output of the ADC are transferred to the computer RAM memory by means of a PPI chip with the ports programmed in input mode (Peatman, 1977). The acquisition sampling interval can be changed by the program to values between 20 and 500/zs. Digitized current signals are displayed in an oscilloscope after being converted into analog form. In this system each current trace is defined by 500 samples. In most experiments linear ionic and capacity currents were subtracted using a P/4 procedure (Bezanilla and Armstrong, 1977). During analysis tail currents were fitted with one or the sum of two exponentials using a least-squares procedure. RESULTS The data presented in this paper were obtained from over 150 type I chemoreceptor cells subjected to voltage clamp. These cells had an average capacitance of 7.27 +_ 2.26 pF (mean + SD, n = 50). The electrical properties of type II cells, which generate only a tiny outward current, are presented at the end of the paper. URENA ET AL. lOrtiC Currents of Glomus Cells 983 Ionic Current Components The major components of ionic current recorded in type I cells are shown in Fig. 1. With the standard high K internal solution, depolarization to 30 mV from a holding potential of -70 mV elicited a fast inward current followed by a slower outward current (Fig. 1 A). At the repolarization of the 8-ms pulse the current changes suddenly in direction and a large inward tail current was recorded. This response pattern was observed in every cell studied although the amplitude of the different current components varied from cell to cell. The outward current was mainly carried by K ions since it disappeared when all the K in the pipette solution was replaced by Cs. In this condition (Fig. 1 B) a fast and sustained inward current, followed by an inward tail, were recorded by a depoA 140 Na.2.5Ca//130 K J f / / .[ "i 140 Na,10Ca//130 Cs ! 140 Na,10 Ca,TTX//130 Cs ; ,i 1 2 ms -i nA 0.5 nA nA FIGURE 1. Major current components in type I cells. (A) Current recorded during a voltage step to 30 mV with return to the holding potential (HP) of -70 mV after 8 ms. (B) Blockade of the outward potassium current by internal cesium. Pulse to 20 mV and HP of -80 inV. Calcium current is recorded in isolation by application of the same voltage step in the presence of TTX (C). Solution composition was as indicated next to each trace. Experiments EN1988J (A) and EN2188J (B and C). larization to 20 mV from a holding potential of -80 inV. The addition of TTX to the external solution (Fig. 1 C) abolished the fast component of the inward current, which indicates that it was due to the activity of Na channels. However, the slowly activating inward current and the tail remained unaltered. These two last current components were the result of the activity of Ca channels since, as will be shown below, they disappeared after replacement of external Ca by Mg, or when 0.5 mM Cd was added to the external solution. These results indicate the existence in type I chemoreceptor cells of voltage-dependent Na, Ca, and K channels. Properties of the Sodium Current Current-voltage relations. Na currents were recorded in isolation in cells where internal K ions were replaced by Cs (or a mixture of Cs and Na), and Ca channels 984 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 93 9 1989 were blocked by external Mg or Cd. A family of Na current records obtained by depolarizations to the indicated membrane potentials is shown in Fig. 2 A. The holding potential was -80 mV and the internal solution contained 40 mM Na. Na current had a voltage-dependent fast activation time course, at 0 mV it reached a peak in ~0.6-0.7 ms, and then it inactivated completely. Current was inward at membrane potentials more negative tharl ENa and outward with more positive membrane potentials. The current-voltage relation for this experiment is shown in Fig. 2 B, where the peak current amplitude is plotted as a function of the pulse membrane potential (VM). Activation threshold was ~- 40 mV, the peak of the I-V curve occurred at a VM A :\ .\ -40 to 60 rnV 9 \ '-. / Io.s ~A 1ms -6O 1 -40 -20 FIGURE 2. Family of sodium curIM'nA ,/ rents recorded during depolariza- -0.s /r tions to -40, -20, 0, 20, 40, and 60 /Sp mV from a HP of -80 mV (A). Peak current amplitude as a function of -0.4 the membrane potential is plotted in B. The continuous line was fitted by -0.2 eye. Solutions (in millimolar): 140 20 Na, 0.5 Cd//90 Cs, and 40 Na. Experiment MZ0288M. --0.2 o/ VM'rnV 5/ of 0 to + 10 mV, and the reversal potential was at +32 mV, which was only 1 mV apart from the ENa value predicted by the Nernst equation. Although inward Na currents were observed in every cell studied the amplitude varied from cell to cell. The maximal Na current amplitude recorded in cells without internal Na was 0.46 _+ 0.2 nA (mean -+ SD, n = 6). Inactivation. In type I cells inactivation of the Na current was, as in other preparations, a voltage-dependent process. Fig. 3 A illustrates the voltage-dependence of steady-state inactivation by measuring the peak Na current elicited by a depolarization to 10 mV as a function of V~ during a 50-ms conditioning prepulse. In the ordinate, current amplitude is normalized with respect to its value in the absence of URENA ET AL. IOniC CurraUs of Glomus Cells 985 prepulse. Half steady-state inactivation was at ~-50 mV, and at -25 mV the noninactivated fraction of IN, was < 10%. The inset in Fig. 2 B illustrates that at 0 mV inactivation was complete in <6 ms and that its time course could be well fitted by a single exponential with a time constant of 0.67 ms. The plot in the same figure shows that the time constant of inactivation decreased with membrane depolarization. Closing kinetics. The closing of Na channels was studied in a few cells by the application of short depolarizing pulses (0.5-1 ms), which prevented the development of inactivation, and the recording of tail currents at the instant of repolarization. Na tails were well fitted by single exponentials and at -80 mV the time conA B LK "O O Z 0.5 VM,mV ' InA ; 2'0 VM,mV FIGURE 3. (A) Steady-state inactivation of sodium currents. The plot represents normalized peak current amplitude obtained during a test step depolarization to 10 mV as a function of the membrane potential during a 50-ms conditioning prepulse. (B) Time course of inactivation. The inset is a sodium current recorded by a voltage step to 0 inV. Inactivation is well fitted by an exponential with a time constant of 0.67 ms. The plot represents the time constant of inactivation (ordinate) measured in currents recorded at various membrane potentials (abscissa). Lines were fitted by eye. HP, -80 mV. Solutions (in millimolar): 140 Na and 0.5 Cd//130 Cs. Experiments EN2888J (A) and FE0388J (B). stant was between 60 and 70 #s. Thus, these results indicate that type I chemoreceptor cells have a prominent Na current with properties similar to those found in other electrically excitable cells. Properties of the Calcium Current Identification of the current. Ca currents were recorded in cells dialyzed with the 130 Cs solution. Fig. 4 A shows a trace of inward current in this experimental condition and with 10 mM Ca in the external solution. The pulse current was followed by a large inward tall current. Exposure to a different external solution with less Ca (9 mM Mg, 1 mM Ca) produced a marked reduction of the steady-state pulse current and the tail, although a small component of inactivating inward Na current 986 THE JOURNAL OF GENERAL PHYSIOLOGY. VOLUME 93 9 1989 FIGURE 4. Identificatmn of B the calcium current. (A) 140 Na//130 Cs 140 Na,TTX//130 Cs Inward current recorded with ~,~ /~ ~~,.~a,z,~,,,~'~ ~ ~.9 ~,lCa the 140 mM Na, 10 mM Ca 9 Mg.1 Ca = ;;( ,,r --~:-:: external solution during a volt- " :: ~~i" age step to 0 mV. On replacing / . / calcium with magnesium (9 I 10 Ca : 10 Ca ~ 0.SnA 9 mM Mg, 1 mM Ca) the steady ; inward current and the tail 2ms almost disappear but a small component of transient sodium current remains. In the presence of TFX (B) the isolated calcium current recorded in 10 mM Ca is almost abolished after the introduction of the 9 mM Mg, 1 mM Ca solution in the bath. HP, -80 mV. Solutions were as indicated in the figure. Experiments EN2788J (A) and EN2588J (B). remained. After Na channels were blocked with TIX in another cell (Fig. 4 B), Ca currents could be recorded in total isolation. This current activates slowly and at 0 mV it reaches a maximum in ~8 ms. Fig. 4 B also illustrates the almost complete disappearance of the pulse current and the tail after replacement of external Ca by Mg. In other experiments with low external Ca (1.5 mM) the addition of 0.5 mM Cd to the external solution completely abolished the Ca current (see Fig. 2). Current-voltage relations. Fig. 5 (]eft column, 10 Ca) shows calcium currents recorded at various membrane potentials. Current generated during the pulse, indicated between the arrows, activates more rapidly at more depolarized levels and is followed by a fast tail whose amplitude increases with the amplitude of the depolarization. These tails are due to the flow of Ca ions through the channels that were open during each pulse, and their time course reflects that of the closing of the Ca channels. Current amplitude measured at the end of the pulse as a function of pulse VM'mV 10 Ca -20 .... - ..... ~ F "! t ":.: 13 nA 3ms lO Ba t i/ ! i ./ 2 / i ? . . -. ~ - / .j FIGURE 5. Current through calcium channels carried by calcium and barium ions. Currents are the result of depolarizations to the membrane potentials indicated next to each trace with 10 mM Ca or 10 mM Ba in the external solution. The arrows indicate the onset and the end of the voltage steps. All traces are from the same cell. HP, -80 inV. Solutions (in millimolar): 140 Na, 10 Ca (10 Ba), and TrX//130 Cs. Experiment MZ0288K. URENA ET AL. Ionic Currents of Glomus Cells -60 -40 -20 0.4 0.60.8VM,mV 20 40 60 1 I , I IM,nA 987 FmURE 6. Calcium current-voltage relation. The current measured at the end of 8-ms pulses is plotted as a function of the pulse membrane potential. The line was fitted by eye. HP, -80 mV. Solutions (in millimolar): 140 Na, l0 Ca, and TrX//130 Cs. Experiment MZ0288K. membrane potential is plotted in Fig. 6. This I-V plot shows that the activation threshold was at --40 mV and that the maximal inward current was obtained at + ] 0 to + 20 mV. At these I'M values the average amplitude of the Ca current measured with 10 mM external Ca was 0.4 .+ 0.2 nA (mean _+ SD, n = 8). The right column of Fig. 5 shows recordings obtained in the same cell after external Ca was replaced by Ba. The traces illustrate that Ba ions flowed through Ca channels better than Ca ions, as indicated by the larger size of the tails. In the range between 0 and + 20 mV, total Ba conductance was about two times larger than Ca conductance. Ba ions also affected the conductance-voltage relation of Ca channels. Fig. 7 plots the normalized amplitude of tail currents recorded with 10 mM Ca (dots) and 10 mMBa (squares). A comparison of both curves indicates that external Ba shifted the activation curve 15-20 mV in the negative direction. Closing kinetics. It has been shown in previous figures that at the instant of repolarization Ca currents were followed by large tail currents. Examples of tails recorded on repolarization to -80 mV after a pulse to + 40 mV are shown in Fig. 8 using 10 mM external Ca (A) or Ba (C) as charge carriers. The decay of the tail current had a clear biphasic time course with a large fast component and a small slow component. The slow component was fitted by an exponential extrapolated to @ N m E Z c g "0 c 0 0.5- -~o -go -2~ FIGURE 7. Conductance-voltage relation of calcium channels with calcium and barium as charge carriers. Conductance, represented in the ordinate, was measured from the amplitude of tail currents recorded at the instant of repolarization of 10ms pulses. The pulse membrane potential is represented on the abscissa. Recordings are from the same cell bathed in 10 mM Ca (dots) and 10 mM Ba (squares). Lines were drawn by eye. HP, -80 mV. Solu2~0 4J0 6~ tions (in millimolar): 140 Na, l0 Ca VM,m v (10 Ba), and TI'X//130 Cs. Experiment MZ0288K. 994 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 98 9 1989 arations (for example Sah et al., 1988). The current has a fast activation and inactivation, is completely blocked by nanomolar concentrations of TTX, and is selectively carried by Na ions. Activation of 1Na in type I cells is similar to the Na current recorded in GHs cells (Matteson and Armstrong, 1984) but is somewhat slower than the current from the squid axon (Hodgkin and Huxley, 1952; Bezanilla and Armstrong, 1977). The peak of the current-voltage curve occurred at 10 mV, which is similar to Na currents recorded in GHs (Dubinsky and Oxford, 1984; Matteson and Armstrong, 1984) and chromaffin (Fenwick et al., 1982) cells, but it is displaced toward positive voltages if compared with Na currents recorded in neurons (Sah et al., 1988) and neuroblastoma cells (Moolenaar and Spector, 1978). These quantitative differences were also seen in the voltage-dependence of steady-state inactivation. In our experiments half of the Na channels were inactivated at ~ -50 mV, a value similar to that measured in GH3 cells (Dubinsky and Oxford, 1984; Matteson and Armstrong, 1984), but less negative than the one observed in central neurons (-75 mV, Sahet al., 1988). Inactivation of Na currents from type I cells follows a single exponential time course and is relatively fast compared with activation. A single exponential decay has also been observed in GH3 cells (Vandenberg and Horn, 1984) although a biexponential time course was required to fit Na inactivation in rat and frog nerve (Chiu, 1977; Neumcke and Stampfli, 1982). Na current density in type I cells, assuming that there is no membrane infolding, was 0.15 mA/cm ~. This value is within the range of the estimations done in GHs cells (Dubinsky and Oxford, 1984; Matteson and Armstrong, 1984) but it is about 10 times smaller than in the squid axon. Thus, on quantitative grounds the Na current of type I cells resembles the INa of GH s and chromaffin cells, but differs in some aspects from the current existing in neurons and nerve cells. Despite the existence of a relatively low density of Na channels, type I cells, like other secretory cells, can generate large action potentials. The fact that in glomus cells steady-state inactivation of IN, is displaced in the positive direction may contribute to a more efficient use of the Na channels available (see also Matteson and Armstrong, 1984). Resting potentials measured in previous intracellular recordings performed in type I cells were in the neighborhood of - 20 mV (Acker and Pietruschka, 1977; Eyzaguirre et al., 1983). Our results show that >90% ofgs, is inactivated at -25 mV, which explains the unexcitability of the intracellularly recorded cells, which were probably damaged by the microelectrodes. Calcium current. Blockade of Na channels by TTX revealed in all type I cells the existence of a Ca current. This current resembles/ca from other vertebrate preparations in its time course, its sensitivity to external divalent cation block, and the ability of Ba to substitute for Ca as charge carrier (Fenwick et al., 1982; Hagiwara and Ohmori, 1982; Dubinsky and Oxford, 1984; Matteson and Armstrong, 1984). The peak of the current-voltage curve was at 10-20 mV, which is close to the values observed in other cells. In equivalent experimental conditions, the amplitude of the Ca current is somewhat larger in type I cells than in GHs (Hagiwara and Ohmori, 1982; Dubinsky and Oxford, 1984; Matteson and Armstrong, 1984), chromaffin (Fenwick et al., 1982), adenohypophysial (Cota, 1986), and pancreatic beta (Hiriart and Matteson, 1988) cells. With 10 mM external Ca, the peak Ca current amplitude was almost the same as the maximal Na current. URENA ET AL. [Ol'tic Currents of Glomus Cells 995 Indications of the existence of more than one Ca channel type have been observed in a number of secretory cells (Cota, 1986; Matteson and Armstrong, 1986; Hiriart and Matteson, 1988; Tabares et al., 1989), neurons (Carbone and Lux, 1984; Nowycky et al., 1985), and muscle (Bean, 1985). In hypophysial pars intermedia, pancreatic beta cells, and adrenocortical cells, FD and SD Ca channels are distinguished according to their different closing kinetics. FD channels also have a higher threshold and inactivate more slowly than SD channels. Ca tails in most type I cells follow a clear biphasic time course and are well fitted by the sum of a small slow exponential and a fast larger exponential. The time constants of the two components are comparable to those measured in other preparations (Cota, 1986; Matteson and Armstrong, 1986). Although the analysis of the deactivation kinetics of Ca currents suggests the existence in type I cells of two Ca channel types, a number of experimental observations do not support this interpretation. (a) Ca current of type I cells inactivate very slowly with a time course that resembles that of FD channels from other preparations. (b) The time course of tail currents recorded after short and large depolarizations is similar and is not affected by a 500-ms conditioning prepulse. In other cells the slow component of the tails, which represents the closing of SD channels, is markedly reduced after long (>50 ms) depolarizations or by a conditioning depolarizing prepulse. (c) The conductance-voltage relation with Ba ions as charge carriers is displaced 15-20 mV toward negative voltages. Ba also slows down the closing of the channels. These effects of Ba are known to be mediated by their interaction with FD channels. Finally, (d) in the absence of exogenous internal Mg-ATP the Ca current of giomus cells is very labile and disappears almost completely in 5-8 rain. This property is typical of FD channels whereas SD channels seem to be resistant to dilution of cytosolic components (Cota, 1986; Matteson and Armstrong, 1986). All together this evidence indicates that Ca current in type I cells is mainly mediated by FD channels. A small population of SD channels, responsible for the slow component of the tails, may exist but unequivocal proofs for their existence were not found. The possibility still remains that the slow component of the tails is either a result of the activity of Ca channels that are different from SD channels, or that it represents a particular kinetic property of FD channels in type I cells. Potassium current. An outward K current was recorded in all type I cells studied. This current was recorded with a large amplitude in cells with internal Ca concentrations ranging between 0.5 #M and <10 -1~ M, but in a given cell it decreased in amplitude after wash-out of Ca channels. Thus, a percentage of this current may be due to the activity of Ca-dependent K channels activated by the local rise in cytosolic Ca that follows Ca influx through functional Ca channels (Marty and Neher, 1985). Because of the rapid disappearance of Ca channels in our preparation, even in the presence of 3 mM internal Mg-ATP, we centered on the study of the voltagedependent K current after wash-out of Ca channels. The voltage-dependent K current of type I cells has a typical sigmoid activation time course, at +40 mV it reaches half maximal amplitude in 3.5 ms, and thereafter it slowly inactivates. The current is qualitatively similar to delayed K currents from other preparations (Adrian et al., 1970; Dubinsky and Oxford, 1984; Rorsman and Trube, 1986; Matteson and Carmeliet, 1988). It is, however, slower than K currents 996 TH~ JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 93 9 1989 of the squid axon or neurons dispersed from the squid giant fiber lobe (Hodgkin and Huxley, 1952; Armstrong and L6pez-Barneo, 1987), but its activation time course is similar to current from vertebrate secretory cells (Dubinsky and Oxford, 1984; Rorsman and Trube, 1986). The K current in type I cells inactivated almost completely in 250 ms. This same behavior is found in K currents from a number of preparations including squid neurons (Llano and Bookman, 1986), GHs cells (Matteson and Carmeliet, 1988), and skeletal muscle fibers (Adrian et al., 1970). The voltage-dependent K current of type I cells was, as were other K currents, almost abolished by a large concentration of external TEA. In our experiments the reversibility of the TEA blockade was almost perfect. Possible Participation of Ionic Channels in the Physiology of Type I Cells Although type I cells have been considered the best candidates for being the primary chemoreceptors since the early work on the carotid body, the basic mechanisms involved in this process have remained largely unknown (for a review see Belmonte and Gonz~lez, 1983). Recent investigations have shown that both hypoxia and high external potassium, which presumably causes membrane depolarization, produce secretion of dopamine in the carotid body and that this effect is abolished by Ca channel antagonists (Fidone et al., 1982; Almaraz et al., 1986; Obeso et al., 1987). Thus, type I cells may function in a way similar to other secretory systems in which Ca influx through voltage-gated membrane Ca channels is a critical event leading to secretion. This idea could not be reconciled with the fact that type I cells impaled with microelectrodes were found to be unexcitable (Eyzaguirre et al., 1983), however, the results shown in this article demonstrate that they have an appreciable density of voltage-dependent sodium and calcium channels and that they can repetitively generate action potentials. Na channels have, therefore, an important role in spike generation and by producing a fast depolarization contributes to the opening of Ca channels, which are well suited for fast injection of Ca into the cytosol. The Ca-activated component of the K current may participate in spike repolarization, whereas the slowly inactivating K channels are most probably involved in pacemaking. The findings reported here led us to hypothesize that ionic channels in type I cells might be regulated by environmental 02 tension and that they might therefore be directly implicated in chemotransduction. This hypothesis was experimentally confirmed as illustrated in the following article. The authors wish to thank Mrs. Lola Ganfornina, University of Seville, for her valuable participation in some experiments. This research was partially supported by grants PB86-250 and PB86-325 from Direccion General de Invesdgaci6n Cientitica y T6cnica. J. R. 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