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Postnatal development of E-4031-sensitive potassium current in rat carotid chemoreceptor cells

¹Division of Pediatric Pulmonology, Department of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, and Children's Hospital Research Institute; and ²Arkansas Children's Hospital, Little Rock, Arkansas

Submitted 24 November 2003 ; accepted in final form 28 November 2004

	ABSTRACT

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The O₂ sensitivity of dissociated type I cells from rat carotid body increases with age until 14–16 days. Hypoxia-induced depolarization appears to be mediated by an O₂-sensitive K⁺ current, but other K⁺ currents may modulate depolarization. We hypothesized that membrane potential may be stabilized in newborn type I cells by human ether-a-go-go-related gene (HERG)-like K⁺ currents that inhibit hypoxia-induced depolarization and that a decrease in this current with age could underlie, in part, the developmental increase in type I cell depolarization response to hypoxia. In dissociated type I cells from 0- to 1- and 11- to 16-day-old rats, using perforated patch-clamp and 70 mM K⁺ extracellular solution, we measured repolarization-induced inward K⁺ tail currents in the absence and presence of E-4031, a specific HERG channel blocker. This allowed isolation of the E-4031-sensitive HERG-like current. E-4031-sensitive peak currents in type I cells from 0- to- 1-day-old rats were 2.5-fold larger than in cells from 11- to 16-day-old rats. E-4031-sensitive current density in newborn type I cells was twofold greater than in cells from 11- to 16-day-old rats. Under current clamp conditions, E-4031 enhanced hypoxia-induced depolarization in type I cells from 0- to- 1-day-old but not 11- to 16-day-old rats. With use of fura 2 to measure intracellular Ca²⁺, E-4031 increased the cytosolic Ca²⁺ concentration response to anoxia in cells from 0- to- 1-day-old but not cells from 11- to 16-day-old rats. E-4031-sensitive K⁺ currents are present in newborn carotid body type I cells and decline with age. These findings are consistent with a role for E-4031-sensitive K⁺ current, and possibly HERG-like K⁺ currents, in the type I cell hypoxia response maturation.

human ether-a-go-go-related gene; potassium channel; hypoxia

A current view of carotid body O₂ transduction is that hypoxia inhibits one or more type I cell K⁺ channels, leading to cell membrane depolarization, Ca²⁺ influx via voltage-gated Ca²⁺ channels, and release of a transmitter that depolarizes afferent nerve endings (for review, see Ref. 46). Although theories differ with respect to the identity and location(s) of the O₂ sensor(s), the mechanism of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) rise and the roles of various O₂-sensitive K⁺ conductances, they converge at the point of type I cell membrane depolarization, which appears to be a key step in the type I cell O₂ transduction cascade (33, 46).

Postnatal maturation of O₂ sensitivity can be demonstrated in enzymatically dissociated type I cells from rats 0 to 14 days (4, 55). In addition to an age-related increase in the [Ca²⁺]_i response to hypoxia, the magnitude of hypoxia-induced membrane potential (E_m) depolarization of dissociated rat type I cells increases severalfold between 1–3 and 12–16 days of age (13). In rat type I cells functional expression of hypoxia-sensitive Ca²⁺-activated K⁺ (K_Ca) currents increases between 4 days and 5 wk of age (27), and hypoxia-induced inhibition of a background K⁺ current increases severalfold between 1–3 and 11–14 days of age (31). Taken together, these results suggest that the developmental increase in the carotid chemoreceptor response to hypoxia depends, at least in part, on a developmentally regulated increase in type I cell excitability.

The reduced magnitude of hypoxia-induced depolarization in newborn type I cells is likely due, at least in part, to low O₂ sensitivity of background K⁺ currents that subsequently increases with age. Here we consider the additional possibility that immature type I cell membrane potential may also be buffered by an E_m-stabilizing K⁺ conductance that declines with maturation. A possible candidate is the K⁺ channel encoded by the human ether-a-go-go-related gene (HERG) (2, 17, 24). Although the best known example of a HERG-mediated K⁺ current is the rapidly activating delayed rectifier K⁺ current (I_Kr) in cardiac myocytes (47; reviewed in Ref. 49), HERG-like K⁺ channels are also involved in resting membrane potential regulation, secretion, and suppression of excitability in cell types of neural crest origin, including neuroblastoma cell lines, neuroendocrine tumor cells, and lactotrophs (2, 6, 7).

Compared with voltage-dependent K⁺ channels, HERG-like K⁺ current exhibits peculiar gating kinetics; that is, inactivation kinetics are faster than activation kinetics during depolarization. Similarly, recovery from inactivation (deinactivation) kinetics is much faster than deactivation kinetics during repolarization (reviewed in Ref. 49). In addition, HERG-like K⁺ currents are selectively blocked by class III antiarrhythmic drugs such as E-4031 and can be isolated from other K⁺ currents in native cells as an "E-4031-sensitive current" (5, 49, 56).

HERG-like K⁺ currents in the rabbit carotid body purportedly regulate the resting membrane potential of the type I cells (42). In other cell types, HERG-like currents have been shown to be present at immature stages of development and decline or disappear with age (2, 15). Furthermore, HERG-like currents are frequently expressed in tumors that develop hypoxic microenvironments, particularly neural crest-derived tumors (2, 3, 16, 17), and HERG-like current was increased in neuroblastoma cells in vitro by exposure to chronic hypoxia (24). The latter observations are particularly intriguing because the fetus is relatively hypoxic before birth and carotid body neurosensory elements develop from the neural crest (43).

The above considerations led us to hypothesize that the resting E_m of immature carotid body type I cells may be stabilized by HERG-like currents, which decrease during postnatal maturation. Using the selective blocker of HERG-like K⁺ current, E-4031, we show here the presence of an E-4031-sensitive K⁺ current in immature and mature carotid body type I cells. In addition, the magnitude of the E-4031-sensitive K⁺ current was severalfold greater in type I cells from newborns vs. 14-day-old rats, and both E_m depolarization and the [Ca²⁺]_i rise in response to hypoxia were increased by E-4031 in type I cells from newborns but not in cells from 11- to 14-day-old rats. These results have been reported in part previously (30).

	METHODS

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Carotid body isolation.   Carotid body type I cells were isolated from two age groups (0–1 and 11–16 days old) of Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) with techniques described previously (55). Each rat was killed with anesthesia (isoflurane) followed by decapitation, and the head was placed in ice-cold saline. All procedures were approved by Animal Care and Use Committee of University of Arkansas Medical Sciences, Little Rock, AR. The carotid bifurcations were dissected and placed in ice-cold PBS (Sigma). The carotid bodies were dissected from the bifurcations and placed in enzymatic solution comprised of trypsin (0.4–0.5 mg/ml, Sigma) and type I collagenase (0.4–0.5 mg/ml, Worthington Biochemical, Lakewood, NJ) in low Ca²⁺-PBS. The carotid bodies were incubated with the enzyme solution and dispersed by gentle trituration with a fire-polished glass pipette. Dissociated cells were plated on poly-D-lysine-coated glass coverslip and incubated in growth media composed of Ham's F-12 (Life Technologies) with 10% fetal calf serum, 33 mM glucose, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.08 units/ml insulin at 37°C until use. Clusters of type I cells were studied between 3 and 8 h after plating.

Electrophysiology.   Electrophysiological recordings were made from cells 3–8 h after plating, by using the perforated whole cell configuration of the patch-clamp technique. Patch pipettes were pulled from 1.5 mm diameter borosilicate glass capillaries (Warner Instrument) on a Flaming-Brown micropipette puller (model P-97; Sutter Instrument, Novato, CA). Pipette resistance was 3–5 M when filled with intracellular solution. The bath was grounded to an Ag-AgCl pellet with 3 M KCl agar bridge. Liquid junction potentials (8.6 mV) were not offset. Voltage- and current-clamp experiments were conducted using a HEKA EPC9 patch-clamp amplifier (HEKA Elecktrik, Lambrecht, Germany). Offset potentials were nulled directly before formation of seal. Whole cell capacitance and series resistance were corrected. Only cells with access resistance <100 M were used. Current traces are shown without correction for leakage currents. All whole cell patch-clamp experiments were performed at 32°C.

HERG-like currents were recorded in 70 mM KCl extracellular buffered salt solution (BSS), in the absence and presence of E-4031. Control traces were recorded in the absence of E-4031 until stable currents were obtained (for 5 min). From a holding potential of 0 mV, repolarization-induced inward K⁺ currents were recorded by hyperpolarizing cell membrane potentials from 0 mV to –120 mV for 250 ms and returned to 0 mV; three to five sweeps were recorded at 10-s sweep intervals. Under these recording conditions the peak inward current during hyperpolarization likely contains not only HERG-like but also other K⁺ currents. We therefore repeated the above protocol in the presence of E-4031 (Alomone, Jerusalem, Israel), a class III antiarrhythmic methanesulfonanilide and selective blocker for HERG K⁺ channels, to quantify the E-4031-sensitive component of the repolarization-induced peak inward current. After the current restabilized, E-4031 was applied by switching to an identical BSS superfusion solution containing E-4031. At 1-min intervals, the above protocol was repeated and, typically by 3–7 min after switching to E-4031-containing solution, the maximum reduction in current was observed. Once the maximum reduction was achieved, the perfusion solution without E-4031 was restored to observe recovery. Peak repolarization-induced tail current was measured from the average of three to five sweeps and was defined as the absolute amplitude of the maximal current during repolarization. Similarly, peak E-4031-sensitive current was measured from three to five averaged sweeps and defined as absolute amplitude of the maximal subtraction current during repolarization (peak repolarization-induced tail current in presence of E-4031 – peak tail current without E-4031).

In current-clamp (I = 0) mode, the resting membrane potential was recorded until stable in BSS containing 4.5 mM KCl (details below). The first challenge, BSS with 20 mM KCl, was applied for 2 min and washed out for another 2 min with normoxic BSS. After full recovery to stable baseline, the cell was superfused with anoxic BSS for 2 min. After recovery in normoxic BSS, the superfusate was switched to normoxic BSS with 1 µM E-4031 for 5 min, then to anoxic BSS with 1 µM E-4031 for 2–3 min, then back to normoxic BSS. With the exception of example tracings shown in Fig. 5, membrane potential results were not corrected for junction potential.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Effects of K+ and anoxia on membrane potential in absence and presence of E-4031. Representative recordings of membrane potential obtained from current clamp mode (I = 0) from carotid type I cells of 0- to 1- (left) and 11- to 16-day-old rats (right) (corrected for liquid junction potential).

where R_o is the measured fluorescence ratio, R_min is the fluorescence ratio at 0 Ca²⁺, R_max is the fluorescence ratio at saturating Ca²⁺, K_d is the dissociation constant for fura 2, 224 nM (25), and is the ratio of 380-nm fluorescence intensity at 0 Ca²⁺ to 380-nm fluorescence intensity at saturating Ca²⁺ concentrations. Calibration was performed using cell-free solutions as described previously (55). Autofluorescence, which was determined by using the fura 2 filter set and treating the cells as above but without loading with fura 2, was negligible and did not vary with age. Image pair acquisition frequency was every 6 s for 1 min before and during a challenge with KCl or 0% O₂. During recovery periods an image pair was acquired every 30 s to minimize photobleaching.

For Ca²⁺ measurements, cells were superfused with BSS bubbled with 21% O₂-5% CO₂ (baseline), challenged with BSS containing 20 mM K⁺, followed by three consecutive challenges of 0% O₂-5% CO₂ (without sodium dithionite). The second and third hypoxia challenges were conducted in the presence of 1 µM E-4031. Approximately 5 min of recovery time separated each challenge. Control experiments were identical except without E-4031. Cells that did not exhibit a [Ca²⁺]_i response to KCl were rejected.

Solutions.   The standard extracellular or bath solution was used with BSS solution containing (mM): 117 NaCl, 4.5 KCl, 23 NaHCO₃, 1 MgCl₂, 2.5 CaCl₂, and 11 glucose; pH adjusted to 7.4 with NaOH. Because of their peculiar kinetics during depolarization, with inactivation occurring as fast as or faster than activation, HERG-like currents cannot be readily identified as outward current at depolarized potentials. Repolarization causes rapid removal of inactivation (deinactivation), resulting in a tail current, which is followed by relatively slow deactivation. For making measurements, HERG-like tail currents are typically amplified at hyperpolarized potentials by raising the concentration of the extracellular K⁺ (1, 22). The high K⁺ (70 mM) extracellular solution designed to increase the amplitude of the inward-rectifying K current (47) contained (in mM) 51.5 NaCl, 70 KCl, 23 NaHCO₃, 1 MgCl₂, 2.5 CaCl₂, and 11 glucose; pH adjusted to 7.4. The intracellular pipette solution for electrical recording conditions contained (in mM) 55 K₂SO₄, 30 KCl, 1 MgCl₂, 1 EGTA, and 10 HEPES; pH was adjusted to 7.2 with NaOH. The pipette tips were filled with this solution then backfilled with the same solution containing 240–360 µg/ml amphotericin B. A 20 mM K solution was made from the above BSS with equimolar substitution for NaCl by KCl. All perfusates were maintained at 32–35°C. Perfusion rate was 1.5 ml/min.

The effect of lowered O₂ tension was studied by switching between normoxic BSS and hypoxic or anoxic BSS superfusate solution. Studies of [Ca²⁺]_i used a small volume closed recording chamber in which stable hypoxia could be achieved in <1 min. Normoxic solutions were equilibrated with 21% O₂, 5% CO₂, and 74% N₂. Hypoxic solution (10 mmHg) was equilibrated with 5% CO₂ and 95% N₂ for at least 30 min before perfusion. Patch-clamp studies were recorded in an open chamber with the surface covered with argon gas to maintain a stable O₂ level in the open chamber. Because of the difficulty of obtaining consistent, reproducible hypoxia challenges in an open recording chamber, anoxic superfusate solution (0 mmHg) was used for patch-clamp studies [0.5 mM sodium hydrosulfite (Na₂S₂O₄) added to BSS equilibrated with 5% CO₂ and 95% N₂]. In separate preliminary studies, O₂ tension was measured with a needle O₂ electrode (Diamond General Development, Ann Arbor, MI) in the recording chamber by use of the same protocols but without cells.

Data analysis.   All values are shown as means ± SE of n number of cells. [Ca²⁺]_i was measured for 30 s before and 2–2.5 min after switching to superfusate containing E-4031. [Ca²⁺]_i was calculated by subtracting average baseline [Ca²⁺]_i during the minute before hypoxia challenge from the peak [Ca²⁺]_i response to hypoxia. Statistical comparisons were performed by paired Student's t-test, unpaired t-test, or one-way ANOVA (Newman-Keuls post hoc test) for multiple comparisons. P value <0.05 was considered statistically significant.

	RESULTS

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Rat carotid chemoreceptors express a HERG-like K current.   In 70 mM KCl extracellular solution repolarization-induced peak inward current in cells from 0- to- 1- and 11- to 16-day-old rats was –471.9 ± 34.0 pA (n = 9) and –272.6 ± 25.5 pA (n = 12), respectively (Fig. 1). The peak tail current in type I cells from 11–16 day rats was 42% less than in cells from 0- to 1-day-old rats (P < 0.0001), indicating an age-related decline in repolarization-induced peak inward current.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Effect of E-4031 on repolarization-induced tail currents. A: typical currents recorded before (control) and after E-4031 treatment (1 µM E-4031) from type I cells of 0- to 1-day-old rats. B: currents recorded from cells of type I cells of 11- to 16-day-old rats. C: average of peak tail current before and after E-4031 treatment in cells from 0- to- 1-day-old (n = 9) and 11- to 16-day-old rats (n = 12). For recovery only, n = 3 in both age groups.

Age-difference of E-4031-sensitive current.   The E-4031-sensitive current, obtained by subtraction of currents in the presence of E-4031 from control tracings, exhibited characteristics typical of a HERG-like current. Representative tracings of E-4031-sensitive current for 0–1 and 11–16 days are shown in Fig. 2, A and B (expanded time scale). Peak E-4031-sensitive current was –137.3 ± 14.3 pA (n = 9) in type I cells from 0- to 1-day-old rats vs. –56.7 ± 10.7 pA (n = 12) in cells from 11- to 16-day-old rats (Fig. 2C) (P < 0.0001). Thus peak E-4031-sensitive current was 2.4 times greater in type I cells from 0- to 1-day-old rats compared with 11- to 16-day-old rats.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Age dependence of E-4031-sensitive K+ currents. A: E-4031-sensitive K+ current obtained by subtraction of the currents recorded in the presence of 1 µM E-4031 from the currents recorded under control conditions without E-4031 in type I cells from 0- to- 1- and 11- to 16-day-old rats. B: representative current traces from 0–1 and 11–16 days rescaled to visualize detail. C: average peak E-4031-sensitive current of cells from 0- to- 1-day-old (n = 9) and 11- to 16-day-old rats (n = 12). D: average E-4031-sensitive peak current density.

Effects of E-4031 on intracellular Ca²⁺ response to hypoxia.   The presence of HERG-like currents may counteract depolarizing influences, thereby reducing hypoxia-induced depolarization and blunting the [Ca²⁺]_i rise in response to hypoxia. A postnatal decrease in HERG-like current channel expression with age could underlie, in part, the developmental increase in the type I cell [Ca²⁺]_i response to hypoxia. Examples of [Ca²⁺]_i responses to 0% O₂ challenge are shown in Fig. 3. In type I cells from 11- to 16-day-old rats, after the first hypoxia challenge (no E-4031), the presence of E-4031 during the second and third hypoxia challenges had no significant effect on the [Ca²⁺]_i response (n = 8) (Fig. 4D). In sharp contrast, the presence of E-4031 in the second and third hypoxia challenge in cells from 0- to 1-day-old rats increased the [Ca²⁺]_i response by 2.6-fold over the first (no E-4031) challenge (P < 0.01) (Fig. 4B). Consistent with our previous work (4, 55), the [Ca²⁺]_i rise in response to hypoxia was many fold greater in cells from the 11- to 16-day vs. 0- to- 1-day-old group (Fig. 3 and Fig. 4, A and C).

View larger version (32K): [in this window] [in a new window]   Fig. 3. Intracellular Ca2+ ([Ca2+]i) responses to KCl and hypoxia; representative tracings. A: 1-day-old control. B: 1-day-old rat with E-4031 during 2nd and 3rd hypoxia challenge. C: 14-day-old control. D: 12-day-old rat with E-4031 during 2nd and 3rd hypoxia challenge.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Effects of E-4031 on intracellular Ca2+ response to hypoxia. Average cytosolic Ca2+ rise ([Ca2+]i) response to 3 consecutive acute hypoxia challenges (0% O2). A: type I cells from 0- to 1-day-old rats without exposure to E-4031 (n = 12). B: type I cells from 0- to 1-day-old rats exposed to E-4031 during 2nd and 3rd hypoxia challenge (n = 12). C: type I cells from 11- to 16-day-old rats without exposure to E-4031 (n = 16). D: type I cells from 11- to 16-day-old rats exposed to E-4031 during 2nd and 3rd hypoxia challenge (n = 8). NS, not significant.

Effects of E-4031 on depolarization response to anoxia.   To further elucidate the physiological role of the E-4031-sensitive current on the type I cell response to hypoxia, the effects of E-4031 on the depolarization response to anoxia were determined.

As illustrated by typical current clamp recordings in Fig. 5, application of 20 mM KCl depolarized membrane potential to a similar degree at both ages; 12.5 ± 0.6 mV (n = 6) in cells from 0- to 1-day-old rats and 11.4 ± 0.8 mV (n = 10) in the 11–16 day group [not significant (NS)] (Fig. 6, A and C). Anoxic BSS, in sharp contrast, depolarized type I cells from 0- to 1-day-old rats by only 10.8 ± 1.2 mV (n = 7) compared with 30.7 ± 1.5 mV (n = 9) in the 11- to 16-day group (Figs. 5 and 6C). Thus membrane depolarization in response to anoxia was approximately threefold greater in dissociated type I cells from mature 11- to 16-day-old compared with cells from 0- to 1-day-old rats.

View larger version (28K): [in this window] [in a new window]   Fig. 6. A: average cell hypoxia-induced membrane potential (Em) in normoxia and during exposure to 20 mM K+ in type I cells from 0- to 1- and 11- to 16-day-old rats (*P < 0.05 compared with normoxia). B: average cell Em in normoxia, anoxia alone, and anoxia + E-4031 in type I cells from 0- to 1- and 11- to 16-day-old rats (*P < 0.05 compared with normoxia, P < 0.05 compared with anoxia). C: average cell membrane depolarization in response to 20 mM K+ and anoxia in type I cells from 0- to 1- and 11- to 16-day-old rats in current-clamp (I = 0) mode. D: average cell membrane depolarization in response to anoxia in absence or presence of E-4031 in type I cells from 0- to- 1- and 11- to 16-day-old rats. See text for n in each group.

Effects of E-4031 on membrane potential in normoxia.   Application of 1 µM E-4031 alone had a small but discernable effect on resting membrane potential in current clamped type I cells in both age groups (Fig. 7). In normoxia, 1 min after switching to E-4031, the average increase in E_m with E-4031 was 2.2 ± 1.1 mV (n = 8) (P = 0.001) in cells from 0- to 1-day-old rats vs. 2.7 ± 1.7 mV (n = 9) (P = 0.0015) in cells from 11- to 16-day-old rats. This small effect of E-4031 in normoxia did not differ significantly with age. The presence of E-4031 in normoxia, even for >5 min, did not cause large E_m depolarization like that seen with anoxia. Thus application of a specific HERG-like channel antagonist did not "mimic" hypoxia, at least at the relatively high concentration of 1 µM.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Representative recordings of membrane potential obtained from carotid type I cells of 0- to- 1- and 11- to 16-day-old rats in response to E-4031 (1 µM) alone in normoxia.

	DISCUSSION

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Using the selective HERG K⁺ channel blocker E-4031, we found an E-4031-sensitive current in enzymatically dissociated rat carotid body type I cells with the characteristics of HERG-like K⁺ current. HERG-like K⁺ current and current density were of larger magnitude in cells from newborn rats compared with type I cells from mature rats. Furthermore, E-4031 augmented type I cell membrane depolarization and the [Ca²⁺]_i rise in response to anoxia in cells from 0- to 1-day-old rats, but not in cells from the 11- to 16-day-old group. Augmentation of hypoxia-induced E_m depolarization by E-4031, only in cells from newborn rats, suggests that HERG-like K⁺ currents may dampen the ability of hypoxia to induce depolarization in an age-dependent manner. Thus HERG-like current may function to dampen hypoxia-induced depolarization of fetal and newborn carotid body type I cells and then decline with age, thereby contributing to the postnatal increase in type I cell excitability.

Presence of HERG-like K⁺ currents in carotid body type I cells.   Multiple K⁺ channels contribute to the large repolarization-induced tail currents observed under conditions of elevated extracellular K⁺ (Fig. 1). Although characterization of all conductances contributing to the repolarization-induced tail current was beyond the scope of this study, we observed clear evidence of tail-current reduction in the presence of the class III antiarrhythmic drug E-4031, which is a potent and specific pharmacological blocker of HERG K⁺ channels (50, 57). This effect was statistically significant in type I cells from both ages, indicating the presence of an E-4031-sensitive or HERG-like current in rat carotid body.

Developmental changes in type I cell response to hypoxia.   In response to anoxia, current-clamped type I cells from newborn rats depolarized only 10 mV, compared with 30 mV depolarization in cells from 14-day rats. Consistent with our previous report that rat type I cell [Ca²⁺]_i response to elevated extracellular K⁺ was not significantly different from 1 to 21 days (55), in the present study type I cell depolarization in response to high extracellular K⁺ concentration also did not vary with age. Thus rat type I cells depolarize to the same degree in response to a nonspecific depolarizing stimulus such as elevated extracellular K⁺ concentration. The finding that hypoxia-induced depolarization of cells from newborn rats is much smaller than the mature response suggests a developmental change specifically in the mechanisms mediating or modulating depolarization in response to hypoxia. These findings raise the questions of what determines the magnitude of type I cell hypoxia-induced depolarization, which is not fully understood even for mature carotid body, and how these mechanisms change with age.

Developmental changes in effects of E-4031.   E-4031-sensitive current was greatest in type I cells from newborn rat carotid bodies and decreased 66% by 2 wk of age, consistent with a possible role in age-dependent modulation of hypoxia-induced cell membrane depolarization. Even after accounting for age-related changes in type I cell size (capacitance), E-4031-sensitive current density was 50% reduced by 2 wk of age compared with type I cells from newborn rats (Fig. 2). Because E-4031 is a highly specific antagonist of HERG-like K⁺ channels, our results suggest that, in the rat carotid body, type I cell HERG-like currents are greatest in the immediate newborn period and decline with age. This conclusion was further supported by our finding that E-4031 increased the magnitude of hypoxia-induced type I cell depolarization 60% in cells from newborns but had no effect in cells from 14-day-old rats (Figs. 5 and 6).

It is noteworthy that E-4031 did not convert hypoxia-induced type I cell depolarization to a "mature" magnitude. This makes it unlikely that an age-dependent depolarization damping effect mediated by HERG-like K⁺ currents could fully explain the small E_m response to hypoxia of type I cells from newborns. It could be argued that a higher concentration of E-4031 may have had a greater effect than the 1 µM concentration we employed in the present study. However, the 1 µM concentration of E-4031 was based on multiple studies in variety of cell types, including neural crest-derived neurons, that report an IC₅₀ for E-4031 of <50 nM (22, 57, 58). Although 1 µM E-4031 is considered to be a high concentration likely to be fully inhibitory, we cannot rule out the possibility that a higher concentration of E-4031 may have produced greater enhancement of hypoxia responses in type I cells.

Although the mechanism of hypoxia-induced depolarization remains unknown, even for mature mammals, numerous studies show that elevated extracellular K⁺ depolarizes isolated type I cells (10, 39, 55), suggesting an important role for resting K⁺ permeability, and type I cells express a variety of known hypoxia-sensitive K⁺ channel types (18, 19, 32, 34, 35, 44, 45, 52). It is therefore likely that one or more other K⁺ channels such as TASK-1 or TASK-3 are the main mediators of hypoxia-induced depolarization. In newborn type I cells, the small magnitude of hypoxia-induced depolarization is most likely due, at least in part, to low O₂ sensitivity of background K⁺ currents that subsequently increases with age (31). The data presented here suggest that additional factors may also play a role, such as damping of depolarization in the immature carotid body by HERG-like K⁺ currents. As noted above, one of the purported functions of HERG-like channels transiently expressed during development or in neural crest-derived tumors is to stabilize or "buffer" E_m in hypoxia (24).

In quail embryos, a HERG-like current, q1(IR), is expressed in neuronal neural crest derivatives during early stages of neuronal development but later disappears and is replaced by other K⁺ currents (2). However, little is known about postnatal development of HERG-like K⁺ currents. Dofetilide, another specific HERG channel antagonist, lengthens electrocardiogram R-R, QT, and QTc intervals in neonatal but not adult mice (54). Thus there is evidence in other tissues that HERG-like currents are functional during early postnatal development but disappear in adults (38). Our findings provide new electrophysiological and functional evidence of postnatal downregulation of HERG-like K⁺ current in another neural crest-derived tissue (carotid body).

Fontana et al. (24) reported that chronic hypoxia profoundly alters the biophysical properties of HERG channels in a human neuroblastoma cell line. Specifically, culture of SH-SY5Y cells (human neuroblastoma clone) in chronic hypoxia for 1 wk caused a marked increase in the magnitude of HERG window current in the E_m range –60 to –10 mV, mainly because of a shift in the activation curve toward hyperpolarized potentials. The physiological effect of increased HERG-like K⁺ current (peak at approximately –40 mV) would be to stabilize resting membrane potential under hypoxic conditions (24). Thus, if HERG-like channel properties are regulated in vivo by chronic hypoxia, it is plausible that HERG-like currents could be present at birth and subsequently decline after the approximately fourfold rise in arterial PO₂ that occurs after birth. However, nothing is known about modulation of HERG-like channel biophysical properties by chronic hypoxia in vivo during postnatal development.

The mechanism by which type I cell E-4031-sensitive K⁺ current decreases during postnatal development is unknown, and we did not measure activation or inactivation curves of the E-4031-sensitive current in type I cells. It is possible that the age-related decline in E-4031-sensitive current was due to downregulation of expression of HERG-like channels or decreasing single-channel conductance. However, the mechanisms regulating HERG-like channel expression and conductance are controversial and poorly understood, and nothing is known about regulation of HERG expression by hypoxia.

HERG-like K⁺ currents in carotid body type I cells.   Our results in rats are consistent with a previous report of HERG-like, dofetilide-sensitive K⁺ current in rabbit carotid bodies, with tail current amplitudes similar to those observed previously (41). Overholt and colleagues (41, 42) reported depolarization of 13 mV with application of 150 nM dofetilide to rabbit type I cells, as well as a dose-dependent increase in carotid body chemosensory discharge using an in vitro preparation. In contrast, we found only minimal effects of E-4031 alone on resting membrane potential. In rat type I cells, the relatively high concentration of 1 µM E-4031 on average caused only a 2- to 3-mV depolarization at both ages, suggesting a minor contribution of HERG-like K⁺ current at resting membrane potential. Overholt et al. also reported that dofetilide increased [Ca²⁺]_i in enzymatically dissociated rabbit type I cells, whereas we found little effect of E-4031 alone on [Ca²⁺]_i in normoxia. Interestingly, preliminary findings from Osanai et al. (40) indicate that dofetilide increased rabbit carotid sinus nerve discharge in a dose-dependent manner but also did so during superfusion with Ca²⁺-free solutions, perhaps suggesting a direct effect on carotid sinus nerve endings.

The reasons for the discrepancies between our results and those of Overholt et al. (41, 42) are not immediately clear. Dofetilide and E-4031 are both highly specific blockers of HERG-like K⁺ channels. All of our results suggest only a minor contribution of an E-4031-sensitive K⁺ current under normoxic, resting conditions in rat type I cells. A species difference is possible, as the studies of Overholt et al. used adult male rabbits whereas our experiments employed neonatal rats. HERG-like currents in rabbit type I cells may exhibit a different range of activation potential such that HERG-like currents predominate at resting E_m in that species (41). It is also possible that the 1 µM E-4031 concentration used in the present study was too low. However, this is unlikely because 1 µM is a relatively high concentration and this concentration clearly inhibited tail currents, enhanced hypoxia-induced depolarization, and enhanced the [Ca²⁺]_i response to hypoxia in type I cells from newborn rats. Thus, unlike rabbits (41), blockade of HERG-like currents of rat carotid bodies did not cause a large depolarization from resting E_m or large increase of [Ca²⁺]_i under baseline conditions and therefore does not mimic hypoxia.

In addition to carotid body type I cells (41), there is evidence that HERG-like K⁺ channels influence resting membrane potential and excitability in other neuronal cells types such as rat microglia, lactotrophs, chromaffin cells, and mammalian neuroblastoma cells (2, 3, 6, 17, 26, 48, 57). In some cell types, where HERG-like currents govern resting membrane potential, HERG channel blockers have been reported to cause substantial cell membrane depolarization from resting E_m (e.g., Ref. 17). Our findings suggest that other ionic conductances predominate at resting membrane potential in type I cells but do not preclude a role for a HERG-like current in modulating depolarization in rat type I cells. A HERG-like window current could dampen depolarizing influences such as hypoxia, even if it were not the predominant contributor to resting membrane potential (24).

Regardless of the potential mechanisms, our results indicate that E-4031 enhanced hypoxia-induced depolarization and the [Ca²⁺]_i rise in response to hypoxia in type I cells from newborn but not 14-day-old rats. These findings are consistent with a development-dependent functional role for E-4031-sensitive K⁺ current, and possibly HERG-like K⁺ currents, in the type I cell hypoxia response maturation.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This research was supported by National Heart, Lung, and Blood Institute Grant R01 HL-54621 (J. L. Carroll).

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. L. Carroll, Pediatric Pulmonary Division, Arkansas Children's Hospital, Slot 512-17, 800 Marshall St., Little Rock, AR 72202 (E-mail: carrolljohnl{at}uams.edu)

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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