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(Circulation Research. 2000;87:537.)
© 2000 American Heart Association, Inc.

Reports

Splice Variants Reveal the Region Involved in Oxygen Sensing by Recombinant Human L-Type Ca²⁺ Channels

Ian M. Fearon, Gyula Varadi, Sheryl Koch, Idit Isaacsohn, Stephen G. Ball, Chris Peers

From the Institute for Cardiovascular Research (I.M.F., S.G.B., C.P.), The University of Leeds, Leeds, UK; Institute of Molecular Pharmacology and Biophysics (I.I., S.K., G.V.), University of Cincinnati College of Medicine, Cincinnati, Ohio.

Correspondence to Dr Ian Fearon, Institute for Cardiovascular Research, The University of Leeds, Leeds LS2 9JT, UK. E-mail cvsimf{at}leeds.ac.uk

	Abstract

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Regulation of vascular smooth muscle Ca²⁺ channels by oxygen tension contributes importantly to hypoxic vasodilatation. We previously described the inhibitory effects of hypoxia on the recombinant human cardiac L-type Ca²⁺ channel _1C subunit (hHT isoform) expressed in HEK 293 cells. We now demonstrate that hypoxia inhibits only one of the three naturally occurring splice variants of this channel that differ only in the C-terminal domain, permitting identification of a 71-amino acid insert in the C-terminal region of the channel that confers oxygen sensitivity. Selective restriction of the spliced insert allowed determination of a 39-amino acid region essential for oxygen sensing. This represents the first identification of the structural region of an ion channel required for sensing changes in oxygen tension.

Key Words: L-type Ca²⁺ channel • _1C subunit • hypoxia • inhibition

	Introduction

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Regulation of ion channels by oxygen tension was first observed in chemoreceptive type I cells of the carotid body, where K⁺ channels were inhibited by hypoxia.¹ Similar inhibitory effects of hypoxia on K⁺ channels have since been demonstrated in a variety of cells.² Native L-type Ca²⁺ channels in carotid body type I and vascular smooth muscle cells are also regulated by hypoxia.^{3 4 5 6} We have previously shown that hypoxia reversibly inhibits the recombinant human cardiac L-type Ca²⁺ channel _1C subunit when expressed in human embryonic kidney (HEK 293) cells,⁷ indicating that auxiliary subunits are not required for oxygen sensing.

Several studies have examined the mechanism(s) involved in oxygen sensing by ion channels,² and candidate mechanisms include redox modulation or sensing by membrane-bound, heme-containing structures. However, no studies have examined the structural requirements for oxygen sensing by ion channels. In the present study, we studied the oxygen sensitivity of the three splice variants of the human L-type (_1C) Ca²⁺ channel.⁸ Only one, possessing a 71-amino acid C-terminal insert, was oxygen-sensitive. Mutation of the splice insert allowed further determination of the region of the L-type Ca²⁺ channel responsible for oxygen sensing.

	Materials and Methods

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Experiments were conducted in HEK 293 cells, cultured as previously described⁷ and transiently expressing the required L-type Ca²⁺ channel construct. Production of constructs and transient transfection methods are detailed in the online-only data supplement (available at http://www.circresaha.org).

Coverslip fragments with attached cells were continually perfused (4 mL/min, bath volume 80 µL), and whole-cell patch-clamp recordings⁹ were made using pipettes of resistance 4 to 7 M. Perfusate composition was as follows (in mmol/L): NaCl 95, CsCl 5, MgC1₂ 0.6, BaCl₂ 20, HEPES 5, D-glucose 10, and TEA-Cl 20 (21°C to 24°C, pH 7.4). Pipette solution composition was as follows (in mmol/L): CsCl 120, TEA-Cl 20, MgC1₂ 2, EGTA 10, HEPES 10, and ATP 2 (pH 7.2).

Cells were clamped at -80 mV, and whole-cell currents were evoked by 100-ms step depolarizations to various test potentials (0.1 Hz). Series resistance compensation of 70% to 90% was applied. Currents were filtered at 5 kHz and digitized at 10 kHz. Capacitative transients were minimized by analogue means, and corrections for leak current were made by the scaling and subtraction of the average leak current evoked by small hyperpolarizing and depolarizing steps (20 mV). Analysis and voltage protocols were performed with the use of an Axopatch 200A amplifier/Digidata 1200 interface (Clampex software, pCLAMP 6.0.3, Axon Instruments Inc). Results are expressed as mean±SEM, and statistical comparisons were made using paired or unpaired Student’s t tests, as appropriate.

Bath hypoxia was achieved by bubbling the reservoir leading to the bath with 100% N₂. The level of hypoxia was measured as previously described.¹⁰ The time course of the fall in PO₂ in the recording chamber was highly reproducible and was always stable within 30 to 60 seconds of switching solution.

	Results

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We first compared the effects of hypoxia on the three splice variants (hHT, rHT, and fHT) of the human L-type Ca²⁺ channel.⁸ In cells expressing the hHT splice variant, hypoxia (PO₂ 20 mm Hg) caused rapid and reversible reductions in Ba²⁺ current amplitude (Figure 1A; mean degree of inhibition 22.6±1.5%, n=6). By contrast, in cells expressing the rHT splice variant (n=22; eg, Figure 1B) or the fHT splice variant (n=8; Figure 1C), the same degree of hypoxia caused no reduction in Ca²⁺ channel activity in any cell examined.

View larger version (17K): [in this window] [in a new window]   Figure 1. Hypoxic sensitivity of recombinant human L-type Ca2+ channel 1C subunits. A, Time-series plot of Ba2+ currents in HEK 293 cell expressing the hHT isoform of the 1C subunit before, during, and after exposure to hypoxic perfusate. Each point shows the peak current amplitude evoked by successive step depolarizations to +10 mV (100 ms, 0.1 Hz) from a holding potential of -80 mV. Period of exposure to hypoxia (PO2, 20 mm Hg) is indicated by the horizontal bar. Inset, Superimposed examples of currents from the same recording, obtained under control conditions (c), during exposure to hypoxia (h), and after recovery (r). B and C, Same as in panel A, except that recordings were made in cells expressing the rHT (B) or the fHT (C) isoform of the 1C subunit. Cartoons to the left of each Figure show the portion of the C-terminal region of the 1C subunit present in the isoform examined.

The hHT splice variant contains a 71-amino acid insert in the C-terminal domain of the channel that is absent in the rHT clone,⁸ and the above data suggest that this region is responsible for oxygen sensing by this channel. Furthermore, when this insert was removed from the hHT clone (creating an hHT(-) clone), the channel was rendered oxygen-insensitive (n=10; Figure 2A). When the insert cleaved from the hHT clone was inserted into the rHT clone (creating an rHT(+) clone), this clone became oxygen-sensitive (n=7; Figure 2B). The mean degree of inhibition in cells expressing the rHT(+) clone was 23.3±2.4%, a value not significantly different from that seen in cells expressing the hHT splice variant (22.6±1.5%, n=6; P=0.82, unpaired Student’s t test). Thus, the 71-amino acid insert is responsible for oxygen sensing by this channel.

View larger version (22K): [in this window] [in a new window]   Figure 2. Hypoxic sensitivity of mutant human L-type Ca2+ channel 1C subunits. A, Time-series plot (as in Figure 1) of Ba2+ currents in HEK 293 cell expressing the hHT(-) mutant 1C subunit before, during, and after exposure to hypoxic perfusate. Period of exposure to hypoxia (PO2, 20 mm Hg) is indicated by the horizontal bar. Inset, Superimposed examples of currents from the same recording, obtained before (c), during (h), and after recovery (r) from hypoxia. B, C, and D, Same as in panel A, except that recordings were made in cells expressing either the rHT(+) (B), the hHT24 (C), or the hHT39 (D) mutant 1C subunits. Cartoons above each Figure show the portion of the C-terminal region of the 1C subunit present in the mutant examined.\.

Given the differential oxygen sensitivity of the hHT and rHT splice variants, studies were carried out to further elucidate the region of the channel involved in oxygen sensing. As demonstrated in Figure 2C, when the distal 24 amino acids of the spliced insert were removed (creating an hHT24 clone), the channel retained its oxygen sensitivity. The mean degree of inhibition at a PO₂ of 20 mm Hg in cells expressing this clone was 28.0±4.9% (n=9), a value not significantly different to that seen in cells expressing the full-length hHT splice variant (22.6±1.5%, n=6; P=0.39, unpaired Student’s t test). In contrast, when 39 amino acids at the proximal end of the spliced insert were removed (creating an hHT39 clone), the channel became oxygen-insensitive (n=5, eg, see Figure 2D). These data indicate that the 39 amino acids at the proximal end of the spliced insert are essential for oxygen sensing by human L-type Ca²⁺ channels.

Previous studies have demonstrated that the effect of hypoxia on L-type Ca²⁺ channels is strongly voltage-dependent.^{3 7} Therefore, it is possible, when examining the effects of hypoxia in time-series experiments in which cells are step-depolarized to a single test potential, that any effect of hypoxia in these studies could be masked by altered voltage dependence. However, current-voltage relationships were created for all constructs used in these experiments and show that in any construct in which hypoxia had no effect on channel activity, the lack of effect was observed at all activating test potentials. Furthermore, in the constructs in which hypoxic inhibition could be seen [hHT, rHT(+), and hHT24 clones], the effects of hypoxia were similarly strongly voltage-dependent: inhibition was greatest at test potentials up to and including those at which currents were maximal and was reduced or even absent at higher activating test potentials (see online-only data supplement, available at http://www.circresaha.org).

	Discussion

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In humans, the L-type (_1C) Ca²⁺ channel exists as several different isoforms.^{8 11} Of these, the hHT variant contains a 71-amino acid insert at position 1786. This is not present in the rHT isoform, although the proteins are otherwise identical. Our data demonstrate that Ba²⁺ currents through the hHT isoform were oxygen-sensitive, whereas those through the rHT variant were oxygen-insensitive. The fHT variant contains a different splice region starting at position 1786, and we found that this also generated oxygen-insensitive currents. Therefore, the spliced insert of hHT is essential for oxygen sensing. To confirm this finding, the spliced insert was removed from the hHT clone, eliminating the channel’s sensitivity to hypoxia. Moreover, when this insert was inserted into the rHT clone, the resulting channel became oxygen-sensitive.

To further elucidate the region involved in oxygen sensing by the Ca²⁺ channel, two mutants in which either the proximal or distal portion of the hHT spliced insert was removed were created. These studies showed that removal of 24 amino acids at the distal end of the insert (amino acids 1832 to 1856) had no effect on hypoxic sensitivity. In contrast, removal of the proximal 39 amino acids of the insert (amino acids 1784 to 1823) resulted in the channel becoming oxygen-insensitive. These data identify a 39-amino acid region in the C-terminal critical to oxygen sensing. The mechanism of this property remains to be elucidated and warrants further investigation into the mechanism whereby this small region of the Ca²⁺ channel plays a role in the physiological response to hypoxia.

	Acknowledgments

 
This work was supported by the British Heart Foundation and National Institutes of Health Grant HL 22619-22.

Received August 2, 2000; revision received August 22, 2000; accepted August 22, 2000.

	References

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