Characterization of ion channels and O2 sensitivity in gill neuroepithelial cells of the anoxia-tolerant goldfish (Carassius auratus)

Abstract

The neuroepithelial cell (NEC) of the fish gill is an important model for O₂ sensing in vertebrates; however, a complete picture of the chemosensory mechanisms in NECs is lacking, and O₂ chemoreception in vertebrates that are tolerant to anoxia has not yet been explored. Using whole cell patch-clamp recording, we characterized four types of ion channels in NECs isolated from the anoxia-tolerant goldfish. A Ca²⁺-dependent K⁺ current (I_KCa) peaked at ~20 mV, was potentiated by increased intracellular Ca²⁺, and was reduced by 100 μM Cd²⁺ A voltage-dependent inward current in Ba²⁺ solution, with peak at 0 mV, confirmed the presence of Ca²⁺ channels. A voltage-dependent K⁺ current (I_KV) was inhibited by 20 mM tetraethylammonium and 5 mM 4-aminopyridine, revealing a background K⁺ current (I_KB) with open rectification. Mean resting membrane potential of -45.2 ± 11.6 mV did not change upon administration of hypoxia (Po₂ = 11 mmHg), nor were any of the K⁺ currents sensitive to changes in Po₂ during whole cell recording. By contrast, when the membrane and cytosol were left undisturbed during fura-2 or FM 1-43 imaging experiments, hypoxia increased intracellular Ca²⁺ concentration and initiated synaptic vesicle activity. 100 μM Cd²⁺ and 50 μM nifedipine eliminated uptake of FM 1-43. We conclude that Ca²⁺ influx via L-type Ca²⁺ channels is correlated with vesicular activity during hypoxic stimulation. In addition, we suggest that expression of I_KCa in gill NECs is species specific and, in goldfish, may contribute to an attenuated response to acute hypoxia.NEW & NOTEWORTHY This study provides the first physiological characterization of oxygen chemoreceptors from an anoxia-tolerant vertebrate. Neuroepithelial cells (NECs) from the gills of goldfish displayed L-type Ca²⁺ channels and three types of K⁺ channels, one of which was dependent upon intracellular Ca²⁺ Although membrane currents were not inhibited by hypoxia during patch-clamp recording, this study is the first to show that NECs with an undisturbed cytosol responded to hypoxia with increased intracellular Ca²⁺ and synaptic vesicle activity.

Keywords: chemoreceptor; goldfish; hypoxia; ion channel; neuroepithelial cell.

Fig. 1.

Identification and passive membrane properties of neuroepithelial cells (NECs) isolated from the goldfish gill. A: a single NEC is shown under bright-field illumination and stained with neutral red (NR) 24 h after dissociation (upper left) and immediately after fixation (upper right). Using fluorescence microscopy, the same NEC was subsequently labeled by antibodies against serotonin (5-HT, lower left) and the synaptic vesicle protein, SV2 (lower right). Scale bar = 5 µm. B: frequency distribution of the number of cells vs. cell diameter measured from dissociated cells (n = 102) stained with NR and labeled with 5-HT and SV2, demonstrating a mean diameter of 10.2 ± 1.3 µm. C: frequency distribution of the number of cells vs. whole cell membrane capacitance (C_m, n = 108) indicated that 90% of cells were between 2 and 4 pF with a mean of 3 ± 0.9 pF. D: frequency distribution of the number of cells vs. resting membrane potential (V_m) under current-clamp (I = 0 pA, n = 65) indicated that 69% of cells had a V_m ≥ –40 mV with a mean of –45.2 ± 11.6 mV.

Fig. 2.

Whole cell currents in isolated neuroepithelial cells (NECs) indicate Ca²⁺-activated K⁺ (K_Ca) channels and voltage-gated Ca²⁺ channels. A: traces are shown from stepwise depolarization under voltage clamp, averaged from multiple cells. Traces at left (n = 6) show currents evoked in normal extracellular solution with peak conductance at 20 mV. Traces at right (n = 8) show currents evoked in solution containing 10 mM Ba²⁺ as a charge carrier, 20 mM TEA, and 5 mM 4-AP. Initial activation of inward current occurred at approximately –30 mV, with peak conductance at 0 mV. B: current-voltage relationships show means ± SE. Whole cell current in normal extracellular solution demonstrates that a component of outward K⁺ current was Ca²⁺-activated (I_KCa; ●); and in Ba²⁺, TEA, and 4-AP solution demonstrating an inward current through Ca²⁺ channels (○).

Fig. 3.

Preloading NECs with Ca²⁺ induced a transient increase in current through K_Ca. A: upper traces were generated from the step protocol in the lower panel. The upper trace shows a recording from a single cell, while the middle trace shows the average of 10 cells. Total whole cell current (I_Total) was generated with a control step from a holding potential of –60 mV to 100 mV (lower panel, dotted line). The transient Ca²⁺-activated K⁺ current (I_KCa) was evoked by preloading NECs with Ca²⁺ using an initial 100-ms step to 0 mV, followed immediately by a step to 100 mV (lower panel, solid line). B: preloading cells with Ca²⁺ resulted in a significant increase in means ± SE outward current immediately following the step to 100 mV (*P < 0.05, paired t test, n = 10).

Fig. 4.

I_KCa was reduced by Cd²⁺. A: whole cell currents elicited by step depolarization and averaged from multiple cells (n = 4) are shown. Left: control traces in normal extracellular solution. Right: traces in solution containing 100 µM Cd²⁺. B: current-voltage relationship generated from step depolarizations in A are shown. Values are expressed as means ± SE. Whole cell current in normal solution (●) shows a characteristic I_KCa profile. Blockade of Ca²⁺ channels with Cd²⁺ eliminated I_KCa and revealed a voltage-gated K⁺ current (I_KV) activating at approximately –30 mV (○). Current was significantly reduced at 20 mV (*P < 0.05, Wilcoxon matched-pairs test).

Fig. 5.

Expression of voltage-dependent (K_v) and voltage-independent (K_B) K⁺ channels in goldfish NECs. A: raw current traces elicited by step depolarization and averaged from multiple cells (n = 5). Left: control traces in normal extracellular solution; right: traces in solution containing 20 mM TEA, 5 mM 4-AP, and 100 µM Cd²⁺. B: current-voltage relationship from traces in A are shown. Values are expressed as means ± SE. Whole cell current in normal solution (●) shows characteristic whole cell current with I_KCa profile. Blocking K_Ca and K_v channels reveals openly rectifying background K⁺ current (I_KB, ○), with significant reduction at 20 mV (*P < 0.05, Wilcoxon matched-pairs test).

Fig. 6.

Hypoxia did not affect whole cell current or membrane potential in goldfish NECs under voltage- or current-clamp. A: means ± SE current-voltage relationship at 10-mV intervals from –100 to 100 mV in normal extracellular solution (n = 6). B: means ± SE current-voltage relationship generated with step depolarizations in solution containing 20 mM TEA, 5 mM 4-AP, and 100 µM Cd²⁺ (n = 4) to isolate background K⁺ current (I_KB). There were no differences in whole cell current between normoxia (●), hypoxia (closed red circles), and return to normoxic solution (○) in A or B (Wilcoxon matched-pairs test). C: in current-clamp recording of a single cell in normal solution, brief application of hypoxia did not elicit a change in membrane potential (V_m, upper trace). Bottom: summary of data indicating that there was no significant change in means ± SE V_m during hypoxic perfusion compared with normoxia (n = 4, Friedman multiple-comparison test). D: carbon fiber recording of changes in Po₂ within the recording chamber. Po₂ change was measured with time, while superfusing the chamber with N₂-bubbled solution to produce hypoxia. Solution reached a Po₂ of ~11 mmHg within 2 min.

Fig. 7.

Intracellular Ca²⁺ increased in response to hypoxia in isolated neuroepithelial cells (NECs) of goldfish. A: representative recording of a NEC responding to hypoxia by increasing intracellular Ca²⁺ concentration ([Ca²⁺]_i). Upper trace shows 340/380-nm excitation ratio (R_340/380) using fura-2 AM; bottom traces show raw intensities at each wavelength. B: Plot of means ± SE R_340/380 indicates that [Ca²⁺]_i rose significantly during hypoxia compared with normoxia (*P < 0.01, Wilcoxon matched-pairs test; n = 7).

Fig. 8.

High K⁺ solution increased intracellular Ca²⁺ in isolated neuroepithelial cells (NECs) of goldfish. Representative recording of a NEC responding to a 30-s application of solution containing 10 mM KCl by increasing intracellular Ca²⁺ concentration ([Ca²⁺]_i). Upper trace shows 340/380 nm excitation ratio (R_340/380) using fura-2 AM, while bottom traces show raw intensities at each wavelength.

Fig. 9.

Ca²⁺-dependent vesicular activity in isolated goldfish NECs increased in response to hypoxia. A: summary of the experimental procedure. Isolated NECs were perfused with normal extracellular solution for 3 min. Subsequently, NECs were exposed to either normoxia or hypoxia in the presence of 2 µM FM 1-43 for 2 min. Cells were then washed with normal solution for 1 min and imaged. B: images show NECs labeled with neutral red under transmitted light (Trans.) or FM 1-43 uptake by fluorescence imaging. Scale bar = 5 µm. Exposure to hypoxia (n = 19) significantly increased uptake of FM 1-43, as indicated by means ± SE corrected total cell fluorescence (CTCF) (*P < 0.05, ANOVA, Bonferroni) as compared with normoxia (n = 17). The addition of 100 µM Cd²⁺ (n = 21) or 50 µM nifedipine (n = 22) to the recording chamber for the duration of the experiment resulted in no significant change in dye uptake during the hypoxic exposure.