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. 2000 May 1;524(Pt 3):725–735. doi: 10.1111/j.1469-7793.2000.00725.x

Modulation of Kir4.1 and Kir5.1 by hypercapnia and intracellular acidosis

Haoxing Xu 1, Ningren Cui 1, Zhenjiang Yang 1, Zhiqiang Qu 1, Chun Jiang 1
PMCID: PMC2269897  PMID: 10790154

Abstract

  1. CO2 chemoreception may be mediated by the modulation of certain ion channels in neurons. Kir4.1 and Kir5.1, two members of the inward rectifier K+ channel family, are expressed in several brain regions including the brainstem. To test the hypothesis that Kir4.1 and Kir5.1 are modulated by CO2 and pH, we carried out experiments by expressing Kir4.1 and coexpressing Kir4.1 with Kir5.1 (Kir4.1-Kir5.1) in Xenopus oocytes. K+ currents were then studied using two-electrode voltage clamp and excised patches.

  2. Exposure of the oocytes to CO2 (5, 10 and 15 %) produced a concentration-dependent inhibition of the whole-cell K+ currents. This inhibition was fast and reversible. Exposure to 15 % CO2 suppressed Kir4.1 currents by ∼20 % and Kir4.1-Kir5.1 currents by ∼60 %.

  3. The effect of CO2 was likely to be mediated by intracellular acidification, because selective intracellular, but not extracellular, acidification to the measured hypercapnic pH levels lowered the currents as effectively as hypercapnia.

  4. In excised inside-out patches, exposure of the cytosolic side of membranes to solutions with various pH levels brought about a dose-dependent inhibition of the macroscopic K+ currents. The pK value (-log of dissociation constant) for the inhibition was 6.03 in the Kir4.1 channels, while it was 7.45 in Kir4.1-Kir5.1 channels, an increase in pH sensitivity of 1.4 pH units.

  5. Hypercapnia without changing pH did not inhibit the Kir4.1 and Kir4.1-Kir5.1 currents, suggesting that these channels are inhibited by protons rather than molecular CO2.

  6. A lysine residue in the N terminus of Kir4.1 is critical. Mutation of this lysine at position 67 to methionine (K67M) completely eliminated the CO2 sensitivity of both the homomeric Kir4.1 and heteromeric Kir4.1-Kir5.1.

  7. These results therefore indicate that the Kir4.1 channel is inhibited during hypercapnia by a decrease in intracellular pH, and the coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivity to CO2/pH and may enable cells to detect both increases and decreases in PCO2 and intracellular pH at physiological levels.

CO2 central chemoreceptors play an important role in autonomic respiration. An increase in PCO2 level may augment the tonic activity of these chemoreceptors that project to the respiratory central pattern generators, leading to an increase in respiratory motor output (Mitchell & Berger, 1975; von Euler, 1986). These chemoreceptors were firstly demonstrated in the ventralateral medulla (Mitchell et al. 1963; Schlaefke et al. 1970; Loeschcke, 1973) and later found in several other brainstem nuclei (Schlaefke et al. 1979; Dean et al. 1989, 1990; Nattie et al. 1993; Richerson, 1995; Kawai et al. 1996; Pineda & Aghajanian, 1997; Oyamada et al. 1998; Wang et al. 1998; Wellner-Kienitz et al. 1998). The effect of CO2 on neurons in brainstem chemoreceptive areas exists following the blockade of synaptic transmission, indicating that the effect is postsynaptic (Dean et al. 1990; Kawai et al. 1996; Oyamada et al. 1998). It is possible that CO2 sensing in these neurons is conducted by certain molecules that are CO2 sensitive and couple the change in PCO2 levels to a change in membrane excitability.

The inward rectifier K+ (Kir) channel appears to be one of these potential CO2 sensing molecules. Certain genetically unidentified Kir channels have been shown to be inhibited by pH and CO2 around physiological levels in brainstem neurons and renal epithelial cells (Schlatter et al. 1994; Zhou & Wingo, 1994; Pineda & Aghajanian, 1997). It is known that these K+ channels are responsible for the maintenance of membrane potential. In fact, there is experimental evidence indicating that inhibition of Kir channels produces depolarization (Pineda & Aghajanian, 1997). Molecular structures and biophysical properties of numerous Kir channels have been well studied in molecular clonings over the past 5 years. Some of the cloned Kir channels show a similar CO2/pH sensitivity to those in brainstem neurons (Coulter et al. 1995; Tsai et al. 1995; Doi et al. 1996; Qu et al. 1999). Although these observations suggest the involvement of Kir channels in hypercapnia, their specific expression has not been confirmed in the brainstem. Therefore, it is necessary to know which members of the Kir family that are expressed predominantly in the brainstem are CO2/pH sensitive.

The Kir4.1 channel is a member of the Kir family which is known to be mainly expressed in the brainstem (Bredt et al. 1995). This channel is inhibited by ATP (Bredt et al. 1995), and is proton sensitive (Yang & Jiang, 1999). Two other Kir channels with a high sequence similarity to brain Kir4.1 (i.e. Kir1.2 and Kir4.2 cloned from the kidney and liver, respectively) are also inhibited by low pH (Shuck et al. 1997; Pearson et al. 1999). However, the pH sensitivity of these Kir4 channels is low (pK 6.0–6.2) which may go against their participation in CO2 detection (Shuck et al. 1997; Yang & Jiang, 1999). Brain Kir4.1 is coexpressed with brain Kir5.1, a subunit which does not produce a functional channel as a homomultimer (Pessia et al. 1996). Kir5.1, however, has a molecular motif identical to a critical sequence in pH sensing of Kir2.3 (Qu et al. 1999), which may provide the heteromeric K+ channels with a pH sensing mechanism in addition to those existing in Kir4.1. Lysine 67 in Kir4.1 may be involved in the pH sensing mechanisms since a corresponding residue (Lys80) in Kir1.1 plays a critical part in the channel sensitivity to pH (Fakler et al. 1996). To test these possibilities, we performed experiments in which brain Kir4.1 was coexpressed with brain Kir5.1, and CO2 and pH sensitivity of the heteromeric K+ channels were studied.

METHODS

Oocytes from female frogs (Xenopus laevis) were used in the present studies. All experimental procedures were subject to the Animal Welfare Assurance of Georgia State University (no. A97008). Frogs were anaesthetized by immersion in 0.3 % 3-aminobenzoic acid ethyl ester. A few lobes of ovaries were removed after a small abdominal incision (∼5 mm) was made. The surgical incision was closed and the frogs were allowed to recover from the anaesthesia. Oocytes were removed not more than three times from any animal, with adequate time for healing between each procedure. Following the last collection the anaesthetized frogs were killed by decapitation. Xenopus oocytes were treated with 2 mg ml−1 of collagenase (Type I, Sigma Chemicals, St Louis, MO, USA) in OR2 solution (composition (mm): NaCl 82, KCl 2, MgCl2 1 and Hepes 5, pH 7.4) for 90 min at room temperature. After three washes (10 min each) of the oocytes with OR2 solution, cDNAs (25–50 ng in 50 nl double distilled water) were injected into the oocytes. The oocytes were then incubated at 18°C in ND-96 solution containing (mm): NaCl 96, KCl 2, MgCl2 1, CaCl2 1.8, Hepes 5 and sodium pyruvate 2.5, with 100 mg l−1 geneticin added (pH 7.4).

Brain Kir4.1 (BIRK10) and brain Kir5.1 (BIRK9) cDNAs were generously provided by Dr Adelman (Oregon Health Science University, OR, USA; Bond et al. 1994). A vector for eukaryotic expression (pcDNA3.1, Invitrogen Inc., Carlsbad, CA, USA) was used to express Kir4.1 and coexpress Kir4.1 and Kir5.1 channels in the Xenopus oocytes. The Kir4.1 cDNA was removed from the pBF vector at EcoRI restriction sites on each end of the cDNA, and the Kir5.1 cDNA was cut out from the pBF vector at EcoRI and KpnI sites. These cDNAs were subsequently subcloned into corresponding EcoRI, and EcoRI and KpnI sites in the pcDNA3.1, respectively. For coexpression of Kir4.1 and Kir5.1, a tandem dimer of these two cDNAs was constructed using the overlapping extension technique (Qu et al. 1999), in which the full-length Kir4.1 and Kir5.1 sequences were obtained using Pfu DNA polymerase (Stratagene, La Jolla, CA, USA) chain reaction (PCR). The PCR products were joined to each other at the 3′ end of Kir4.1 and 5′ end of Kir5.1 and then cloned in the pcDNA3.1 as detailed by Pessia et al. (1996). Site-specific mutations were produced using a site-directed mutagenesis kit (Stratagene). The tandem dimer of Kir4.1-Kir5.1 cDNA was used as a template for mutagenesis in Kir4.1-Kir5.1. The orientation of the constructs and correct mutations were confirmed with DNA sequencing. Expression of Kir4.1 was examined following Kir4.1 cDNA injection. Coexpression of Kir4.1-Kir5.1 currents was studied by co-injecting Kir4.1 and Kir5.1 cDNAs in a 1:1 ratio or by injecting the tandem dimer of Kir4.1-Kir5.1.

Whole-cell currents were studied on the oocytes 2–4 days after injection. Two-electrode voltage clamp was performed using an amplifier (Geneclamp 500, Axon Instruments Inc., Foster City, CA, USA) at room temperature (∼25°C). The extracellular solution contained (mm): KCl 90, MgCl2 3 and Hepes 5 (pH 7.4). Cells were impaled using electrodes filled with 3 M KCl. The potential leakage of KCl from the recording electrodes was not corrected because of the large volume of oocytes. The electrode (1.0–2.0 MΩ) which was connected to the HS-2 x1L headstage (input resistance, 1011Ω) served to record voltage, and the other electrode (0.3–0.6 MΩ) which was connected to the HS-2 x10MG headstage (maximum current, 130 μA) was used for current recording. Oocytes were accepted for further experiments only if they did not show evident leakage. Current records were low-pass filtered (Bessel, 4-pole filter, 3 dB at 5 kHz), digitized at 5 kHz (12-bit resolution) and stored on computer disk for later analysis (pCLAMP 6.0.3, Axon Instruments) (Jiang et al. 1994; Zhu et al. 1999). Junction potentials between bath and pipette solutions were appropriately nulled.

Macroscopic Kir currents were recorded in excised patches at room temperature (∼25°C) as described previously (Zhu et al. 1999). In brief, the oocyte vitelline membranes were mechanically removed after exposure to hypertonic solution (400 mosmol kg−1) for 5 min. The stripped oocytes were placed in a Petri dish containing regular bath solution (see below for composition). Recordings were performed using solutions containing equal concentrations of K+ applied to the bath and recording pipettes. The bath solution contained (mm): KCl 40, potassium gluconate 75, potassium fluoride 5, sodium vanadate 0.1, potassium pyrophosphate 10, ethylene glycol-bis-β-aminoethylether-N,N,N′, N′-tetraacetic acid (EGTA) 1, adenosine diphosphate (ADP) 0.2, piperazine-N, N′-bis-2-ethanesulfonic acid (Pipes) 10, glucose 10 and spermine 0.1 (FVPP solution, pH 7.4) (Kubokawa et al. 1995; Amico et al. 1998; Huang et al. 1998). The pipette was filled with the same FVPP solution used in the bath or a solution containing (mm): KCl 40, potassium gluconate 110, ADP 0.2, EGTA 1, Hepes 10, glucose 10 and MgCl2 2 (pH 7.4). This bath solution was chosen after several others had been tested with respect to channel rundown in excised patches. In our previous studies, we have found that macroscopic currents recorded from giant inside-out patches are very well maintained, showing less than 10 % reduction over a recording period of 20 min in such a bath solution (Zhu et al. 1999). This period is sufficient for all our patch recording protocols, which were generally completed within 10 min. Fire-polished patch pipettes (2–4 MΩ) were made from 1.2 mm borosilicate capillary glass (Sutter P-94/PC puller). Giant inside-out patches were employed to study macroscopic currents in cell-free conditions using recording pipettes of 0.5–1.0 MΩ.

Intracellular pH (pHi) and extracellular pH (pHo) were measured using ion-selective microelectrodes. Details of procedures were described in our previous reports (Jiang et al. 1992). In brief, two single-barrelled microelectrodes were employed. One of them (ion selective) was exposed to hexamethyldisilazan vapour (Fluka Chemie AG, Switzerland) for 30 min and then baked at 120°C for 8 h. The tip of the ion-selective microelectrode was filled with H+ liquid exchanger (Hydrogen Ionophore 1 – Cocktail A, Fluka Chemie AG, Switzerland) and the remainder of the microelectrode was backfilled with phosphate buffer (pH 7.00) for both pHi and pHo measurements. This ionophore is greatly selective for H+, e.g. H+: K+, Na+ or Ca2+ > 1 000 000:1. The other microelectrode was filled with 3 M KCl. Electrodes were used only if they had high frequency response (90 % response time ≤ 5 s) and showed an excellent sensitivity (voltage change of more than 55 mV when pH decreased from 8.0 to 7.0). A high input-resistance amplifier (Duo773, World Precision Instruments, Inc., Sarasota, FL, USA) was used for pH measurements. The ion-selective electrode was connected to the high input-resistance channel (1015Ω), and the KCl electrode to the other channel (1012Ω). Voltage was removed by subtracting records between these two channels. Serial calibrations of ion-selective microelectrodes were made with potassium phosphate buffer at pH 6.0, 7.0 and 8.0 (Fisher Scientific, Pittsburgh, PA, USA).

Experiments were performed in a semi-closed recording chamber (BSC-HT, Medical System, Greenvale, NY, USA), in which oocytes were placed on a supporting nylon mesh, and the perfusion solution bathed both the top and bottom surface of the oocytes. The perfusate and the superfusion gas entered the chamber from two inlets at one end and flowed out at the other end. There was a 3 mm × 15 mm gap on the top cover of the chamber, which served as the gas outlet and access to the oocytes for recording microelectrodes. The perfusate contained (mm): KCl 90, MgCl2 3, Hepes 5 (pH 7.4). At baseline, the chamber was ventilated with atmospheric air. Exposure of the oocytes to CO2 was carried out by switching to a perfusate that had been bubbled for at least 30 min with a gas mixture containing CO2 (5, 10 or 15 %) balanced with 21 % O2 and N2, and superfused with the same gas. The high dissolvability of CO2 resulted in a detectable change in intra- or extracellular acidification as fast as 10 s in these oocytes. Thus, in most experiments only the superfusion air was switched to CO2, in which similar results were obtained. Intracellular pH was measured at ∼100 μm inside the cell. Since the frog Ringer solution contains 5–10 mm buffer as opposed to the 20–30 mm in mammalian Ringer solution, a large decrease in intracellular pH was seen during hypercapnia. Extracellular pH was measured in the immediate vicinity of the oocytes at a horizontal level similar to the intracellular measurement. Current responses to CO2 were studied before, during (4–5 min) and after CO2 exposure. Extracellular acidification was done using a buffer containing 10 mm Pipes. The pH of these solutions was adjusted to desired levels immediately before experiments. Pipes buffer was used because of its buffering range from pH 5.8 to pH 7.4. This buffer does not cause intracellular acidification, as shown in our previous studies (Qu et al. 1999). In intracellular acidification experiments, 20 mm KCl was replaced with the same concentration of KHCO3 (pH adjusted to 7.4), so that the K+ concentration remained the same in these experiments. Intracellular pH levels were measured when oocytes were exposed to these perfusates. To determine the effect of molecular CO2 on Kir channel activity, Kir4.1-Kir5.1 and Kir4.1 currents were studied in inside-out patches with 10 mm HCO3− added to the FVPP bath solution. The bath solution was bubbled with 15 % CO2 and adjusted to pH 7.38 after 30 min bubbling, so that the pHi level was generally the same as control. The CO2 bubbling was maintained throughout the experiments.

A parallel perfusion system was used to exchange internal solutions to patches at a rate of ∼1 ml min−1 with no dead space (Zhu et al. 1999). Low pH exposures were performed using the same bath solutions that had been adjusted to various pH levels as required by experimental protocols with KOH, HCl or gluconic acid.

Data are presented as means ± standard error of the mean (s.e.m.). ANOVA or Student's t test was used to compare CO2 and pH effects before and during exposures. Differences were considered to be statistically significant if P was equal to or smaller than 0.05.

RESULTS

Baseline properties of Kir4.1 and coexpressed Kir4.1 and Kir5.1

Whole-cell currents were studied in the two-electrode voltage-clamp mode using an extracellular solution containing 90 mm K+. Depolarizing and hyperpolarizing command pulses were given to the cell in a range from -160 mV (-120 mV in some cells) to 100 mV in 20 mV increments at a holding potential of 0 mV. Under such conditions, inward rectifying currents were observed 2–6 days after injection of cDNAs of brain Kir4.1 alone or Kir4.1 with brain Kir5.1 (Kir4.1-Kir5.1) in a 1:1 ratio. The Kir4.1 currents showed a fast activation at hyperpolarization with no obvious time delay (Fig. 1, top panel). The Kir4.1-Kir5.1 currents, on the other hand, reached the maximal amplitude with a long period of delay (Fig. 1, bottom panel). The time constant for these currents was 836 ± 92 ms (mean ±s.e.m., n = 6) at -120 mV and 934 ± 98 ms (n = 6) at -80 mV. At -120 mV hyperpolarization, steady-state currents averaged -15.9 ± 2.1 μA (n = 19) for Kir4.1, and -12.8 ± 1.3 μA (n = 38) for Kir4.1-Kir5.1. All these currents were clearly different from the endogenous inward rectifying currents in oocytes with an injection of the expression vector pcDNA3.1 alone (-1.6 ± 1.4 μA, n = 6). These Kir currents were sensitive to micromolar concentrations of Ba2+ and Cs+, and millimolar concentrations of Na+. The IC50 (concentration giving 50 % of maximum inhibition) of Ba2+ was ∼30 μM for Kir4.1, and ∼10 μM for Kir4.1-Kir5.1. These results are therefore consistent with previous reports on these K+ channels indicating that Kir4.1 and Kir5.1 are coexpressed as heteromers in Xenopus oocytes (Pessia et al. 1996).

Figure 1. Expression of inward rectifier K+ channels in Xenopus oocytes.

Figure 1

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Currents were recorded using two-electrode voltage clamp. A high concentration (90 mm) of K+ was applied to the extracellular solution. Membrane potential (Vm) was held at 0 mV. A series of command pulses from -160 mV to 100 mV were applied to the cell in 20 mV increments. Ionic currents were recorded from Xenopus oocytes 3 days after injection of Kir4.1 cDNA (Kir4.1, top panel) and a tandem dimer of Kir4.1-Kir5.1 (Kir4.1-Kir5.1, bottom panel). Both of these currents were sensitive to micromolar concentrations of Ba2+. The sensitivity of Kir4.1-Kir5.1, however, was higher than Kir4.1 in comparison with baseline currents (Control). Washout of Ba2+ led to a complete recovery of these currents.

Inhibition of K+ currents by CO2

Effects of CO2 on these K+ currents were studied under the same conditions as described above. Oocytes were positioned 100–200 μm beneath the surface of the perfusate in a semi-closed recording chamber (see Methods). Exposure of these oocytes to 15 % CO2 for 4–8 min produced inhibition of Kir4.1 and Kir4.1-Kir5.1 currents. Inhibition of the inward rectifying K+ currents became evident 20–30 s into the CO2 exposure. After the maximum effect was reached, the inhibition was maintained through the rest of the exposure period and a few minutes after (Fig. 2A and B). The time profile of the inhibition for Kir4.1-Kir5.1 currents was slightly shorter than that for Kir4.1 currents. In Kir4.1-Kir5.1 channels the maximal inhibition occurred in 2–3 min, while it took 3–4 min to show the maximal effect in Kir4.1 channels. The degree of the inhibition was different. At the maximal inhibition (measured at -120 mV), 57.0 ± 3.2 % (n = 7) of the Kir4.1-Kir5.1 currents were suppressed, whereas the Kir4.1 currents were reduced by only 23.7 ± 5.6 % (n = 9, P < 0.001). The effect of CO2 on these currents was reversible. A complete recovery was seen in most oocytes (> 70 %) studied with one to two exposures. The magnitude of the inhibition depended on CO2 concentrations. Inhibition of these Kir currents was evident with 5 % CO2. Higher concentrations of CO2 (10 and 15 %) induced much stronger inhibition of these currents (Fig. 2C and D).

Figure 2. CO2 reversibly inhibits Kir4.1 and Kir4.1-Kir5.1 currents.

Figure 2

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Currents were recorded from oocytes 2–3 days after cDNA injection using 90 mm K+ in the bath solution. The oocytes were placed 100–200 μm beneath the surface of the bath solution. Currents were recorded in the voltage-clamp mode with -120 and 60 mV command pulses. The duration of the pulses was 2 s for Kir4.1 (A) and 4 s for Kir4.1-Kir5.1 (B). The profile of the current amplitude shows that both Kir4.1 and Kir4.1-Kir5.1 currents started to decrease almost immediately after the perfusion gas was switched to 15 % CO2, reached maximum levels at about 3–4 min in Kir4.1-Kir5.1 and 5–6 min in Kir4.1, and remained at this level until the perfusion gas was switched back to room air. Note that there is a gap of 0.5–4 min between records. C and D, concentration-dependent inhibition of Kir4.1 and Kir4.1-Kir5.1 currents during CO2 exposures. Three levels of CO2 (5, 10 and 15 %) were examined for the dose-dependent inhibition of Kir4.1-Kir5.1 currents. Graded CO2 produced a graded inhibition of these currents. Data are presented as means and s.e.m..

This inhibition did not show voltage dependence in a voltage range from -120 to 100 mV in Kir4.1 currents. Similar results were also found in the coexpressed Kir4.1-Kir5.1 currents.

Effects of intra- and extracellular acidifications on K+ currents

To determine whether Kir channel inhibition is caused by molecular CO2 or the consequent decrease in intra- and extracellular pH (pHi and pHo, respectively) during hypercapnia, experiments were carried out using acidified buffers. We first measured pHi and pHo using ion-selective microelectrodes. Baseline pHi in oocytes was 7.19 ± 0.12 (n = 6) with pHo 7.40. When the oocytes were exposed to 15 % CO2, pHi was reduced to 6.58 ± 0.15 (n = 4) and pHo to 6.18 ± 0.07 (n = 4).

If the decrease in pHo during hypercapnia were a cause of Kir inhibition, a similar inhibition of these Kir currents would occur with the perfusion of oocytes using an acidified buffer in the absence of CO2. To examine this possibility, we studied the Kir currents at low pHo using the membrane-impermeant Pipes buffer. At pHo 6.2, the measured pHi averaged 7.16 ± 0.08 (n = 3). Thus, extracellular pH was selectively reduced without changing pHi. Extracellular acidification to pH 6.2 with the Pipes buffer, however, failed to produce any significant change in the Kir4.1 and Kir4.1-Kir5.1 currents (2.3 ± 1.7 %, n = 4 and -2.8 ± 2.3 %, n = 3, respectively), similar to the effect on pH-insensitive Kir2.1 currents (-2.4 ± 1.7 %, n = 4; P > 0.05) (also see Coulter et al. 1995; Fakler et al. 1996; Zhu et al. 1999).

To understand the effect of intracellular acidification on K+ channel inhibition, we studied Kir4.1 and Kir4.1-Kir5.1 currents by selectively reducing pHi without changing pHo. In these experiments, oocytes were bathed with a solution that contained 90 mm KHCO3 to replace KCl, in which the pHo was adjusted to 7.35 ± 0.12 (n = 4). Exposure to this solution reduced pHi to 6.58 ± 0.15 (n = 4) similar to that during 15 % CO2 exposure. Under such conditions, Kir4.1 currents were inhibited by 24 ± 4.1 % (n = 4, Fig. 3A–C), and Kir4.1-Kir5.1 currents by 50.5 ± 4.5 % (n = 7, Fig. 3D–F), suggesting that a drop in intracellular pH during hypercapnia inhibits these Kir currents.

Figure 3. Intracellular acidification without changing extracellular pH inhibits both Kir4.1 and Kir4.1-Kir5.1 currents.

Figure 3

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A, Kir4.1 currents were recorded from an oocyte 3 days after Kir4.1 cDNA injection in a solution containing 90 mm KCl (pH 7.4). B, when the oocyte was exposed to a solution in which KCl was replaced with KHCO3 (90 mM), the intracellular pH was reduced to 6.58 and extracellular pH remained at 7.37. Under such conditions, Kir4.1 currents were inhibited by 20 %. C, this inhibition was reversible. Recovery was seen after washout of KHCO3 solution. D-F, the coexpressed Kir4.1-Kir5.1 currents were also inhibited by intracellular acidification. This inhibition was much stronger, as the currents were reduced by ≈70 % at pHi 6.6 (E).

Inhibition of K+ currents by protons in excised patches

To further examine the pH effect on these Kir currents and to rule out the possibility of mediation of cytosol-soluble factors in the Kir inhibition, channel activity was studied in cell-free excised patches. The expression of inward rectifier currents was first identified under the voltage-clamp mode in oocytes that had received a Kir cDNA injection. Giant inside-out patches were then obtained from the oocytes. Macroscopic currents were studied with either pulse command potentials from -140 mV to 100 mV in 20 mV increments or ramp command potentials from 100 mV to -100 mV applied to these patches. Symmetric concentrations of K+ (150 mm) were applied to both sides of the patch membranes. To prevent channel rundown, vanadate, fluoride and pyrophosphate were used in the internal solution, which served to block phosphatases and phosphodiesterase. These factors plus the lack of Mg2+ and ATP make this solution unfavourable for the turnover of protein phosphorylation and dephosphorylation.

The macroscopic Kir4.1 currents showed a moderate inward rectification with no delay in channel activation (Fig. 4). Exposure of the internal membranes of the patches to low pH solutions caused inhibition of the inward rectifying currents. This inhibition was fast, fully reversible and dependent on pH levels. At pH 5.8, >70 % of the inward rectifying currents were suppressed (Fig. 4). The relationship of the current amplitude to pHi levels can be described using the Hill equation with pK 6.03 and Hill coefficient (h)2.2 (n = 5) (Fig. 5).

Figure 4. Inhibition of Kir currents in excised patches.

Figure 4

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Top panel, Kir4.1 currents were recorded from an inside-out patch with symmetric K+ concentrations on both sides of the oocyte membrane. Pulse command potentials from -140 mV to 100 mV were applied to the patch with an increment of 20 mV at a holding potential of 0 mV. Exposure of the internal membrane to solutions with various pH levels produced a graded inhibition of inward rectifying currents. This inhibition was fast (40–60 s each exposure) and fully reversible. These currents started to be inhibited at pH 6.2, and there were still ≈25 % currents seen at pH 5.8. Bottom panel, the coexpressed Kir4.1-Kir5.1 currents are more sensitive to pH than the Kir4.1 currents. The amplitude of these Kir4.1-Kir5.1 currents increased with an increase in pH and reached the maximal level at pH 8.0. At baseline (pH 7.5) currents were inhibited by 50 %. Further acidification caused more inhibition of these currents. Currents were inhibited by 80 % at pHi 7.0, and almost completely abolished at pH 6.5. Similar to Kir4.1, the inhibition of Kir4.1-Kir5.1 currents by protons was reversible. The current amplitude returned to the baseline level within ≈1 min after washout. Note that activation patterns in the Kir4.1 and Kir4.1-Kir5.1 currents are similar to those recorded in whole-cell recordings.

Figure 5. Comparison of pH sensitivity of Kir4.1 with Kir4.1-Kir5.1.

Figure 5

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The amplitude of these Kir currents is expressed as a function of intracellular pH (pHi) using the Hill equation: y = 1/(1 + (pK/x)h), where y is the current amplitude, pK the midpoint pH value for channel inhibition, x the pHi, and h the Hill coefficient. The values of pK and h here are 6.03 and 2.2, respectively, for Kir4.1 (n = 5) and 7.45 and 2.1, respectively, for Kir4.1-Kir5.1 (n = 4). Note that Kir2.1 (IRK) shows little or no pH sensitivity in the range pH 7.4 to pH 5.8 (n = 4). Data are presented as means ±s.e.m..

When Kir4.1 and Kir5.1 were coexpressed in the oocytes, the macroscopic currents showed an activation pattern similar to that seen in whole-cell recordings, i.e. the currents were slowly activated with hyperpolarization (Fig. 4). At baseline (pH 7.4), the amplitude of these currents was small. Increases in pH levels in the internal solution augmented the Kir4.1-Kir5.1 currents, which reached their maximal levels above pH 8.0, suggesting that a significant amount of the currents is inhibited at pH 7.4. These channels were shut off at pH 6.5 (Fig. 4). The current amplitude can be expressed as a function of pHi with pK of 7.45 and h of 2.1, about 1.4 pH units higher than that of the Kir4.1 currents (Fig. 5). The inhibition of these Kir channels was specific, as another Kir, Kir2.1 (IRK1), was basically unaffected by intracellular protons in a range of pH 7.4–6.5 (Fig. 5) (also see Coulter et al. 1995; Fakler et al. 1996).

Lack of direct effect of CO2 on Kir currents

To determine whether CO2 has a direct effect on channel activity independent of pH, Kir4.1 and Kir4.1-Kir5.1 currents were studied in inside-out patches with 10 mm HCO3− added to the FVPP solution in the bath. The bath solution was adjusted to pH 7.38 after 30 min bubbling with 15 % CO2, so that the pH level was generally the same as control. Under such conditions (isohydric hypercapnia), Kir4.1 currents averaged 128 ± 49 pA at baseline and 130 ± 47 pA during exposure (P > 0.05, n = 5), and Kir4.1-Kir5.1 currents were 94 ± 30 pA at baseline and 88 ± 24 pA during exposure (P > 0.05, n = 5), indicating that CO2 has no direct effect on these channels.

Critical role of lysine 67 in pHi sensing

Our results in these studies have suggested that pH-sensing mechanisms are likely to be located in the Kir channel proteins. A lysine residue in Kir1.1 (Lys80) is known to play a key role in pH sensing of Kir1 channels (Fakler et al. 1996; McNicholas et al. 1998). This lysine residue exists at the corresponding position of Kir4.1 (Lys67). Also, a short motif centred on a threonine residue which was originally found in Kir2.3 (Thr53) to be critical in channel sensitivity to intracellular acidification (Qu et al. 1999) can be seen in Kir5.1. To understand the molecular mechanisms underlying pH sensitivity in Kir4.1-Kir5.1 and Kir4.1 channels, we studied Lys67 in Kir4.1 and Thr68 in Kir5.1 using site-directed mutagenesis. After replacement of Lys67 (K67) with methionine (M) (a counterpart residue found in the pH-insensitive Kir2.1) or glutamine (Q), we found that 15 % CO2 inhibited the mutant Kir4.1 channel by only 2.3 ± 3.9 % (n = 7, K67M) and 1.5 ± 2.8 % (n = 4, K67Q) similar to the CO2 effect on Kir2.1 (2.9 ± 3.2 %, n = 4; P > 0.05) (Fig. 6). The K67M mutation in Kir4.1 also eliminated the CO2 sensitivity of the heteromeric Kir4.1-Kir5.1 (4.9 ± 1.7 %, n = 9; P > 0.05 in comparison with the inhibition of Kir2.1). Interestingly, the K67M mutation also largely eliminated time-dependent activation of Kir4.1-Kir5.1 currents (Fig. 6C).

Figure 6. Effects of Lys67 mutation on CO2 sensitivity of Kir4.1 and Kir4.1-Kir5.1 currents.

Figure 6

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Whole-cell currents were recorded from oocytes as described in Fig. 1. A, inward rectifier K+ currents were seen in an oocyte that had received an injection of K67M mutant Kir4.1. B, although the K67M mutation did not affect the baseline currents, channel sensitivity to 15 % CO2 (5 min) was abolished. C, the effect of the K67M mutation on Kir4.1-Kir5.1 currents was studied in another oocyte in which a cDNA of the tandem dimer of Kir4.1-Kir5.1 was injected with the K67M mutation on Kir4.1. Kir4.1-Kir5.1 currents were observed 3 days after the injection. Note that the Kir4.1-Kir5.1 currents with the K67M mutation lost their time-dependent activation as seen in the wild-type Kir4.1-Kir5.1. D, these Kir4.1-Kir5.1 currents lost their hypercapnic sensitivity, as no change in the current amplitude was seen after 7 min exposure to 15 % CO2.

Unlike Thr53 in Kir2.3, however, mutation of Thr68 in Kir5.1 to alanine (T68A) had no significant effect on channel sensitivity to 15 % CO2 in the heteromeric Kir4.1-Kir5.1 (69.6 ± 8.3%, n = 3; P > 0.05 in comparison to wild-type Kir4.1-Kir5.1). Another mutation involving this threonine and an adjacent histidine residue within the Kir5.1 N-terminal motif (T68I/H72W) did not affect the CO2 sensitivity (63.0 ± 11.9%, n = 3). Therefore, these results suggest that the critical structure for proton sensing in these Kir channels is likely to be located on Kir4.1.

DISCUSSION

Our current studies have shown that brain Kir4.1 is inhibited by hypercapnia and this is mediated by a decrease in intracellular pH. Interestingly, we have found that the coexpression of Kir4.1 with Kir5.1 significantly enhances the CO2/pH sensitivity of the heteromeric channels, which may allow them to respond to cellular ambient CO2 at near physiological levels.

Baseline Kir4.1 and Kir4.1-Kir5.1 currents

Using the eukaryotic expression vector pcDNA3.1, we have expressed both Kir4.1 and Kir4.1-Kir5.1 in Xenopus oocytes. These currents are clearly different from endogenous currents in the oocytes. They show a clear inward rectification and are highly sensitive to Ba2+, Cs+ and Na+ with a slightly higher sensitivity in Kir4.1-Kir5.1. Kir4.1 shows a fast activation at hyperpolarization, while Kir4.1-Kir5.1 is activated with a time delay of 1–2 s. All of these properties are consistent with those reported originally for these two currents (Bond et al. 1994; Pessia et al. 1996). We have noticed that our Kir4.1-Kir5.1 currents are somewhat smaller than Kir4.1 currents. This may be a result of the proportion of cDNAs injected (1:100 by Pessia et al. 1996) and/or a significant inhibition of the Kir4.1-Kir5.1 currents at baseline pH (pHi 7.2).

Sensitivity of these Kir currents to CO2 and pH

It is known that some members of the Kir1 and Kir2 families are sensitive to pH or CO2. For instance, Kir1.1 (ROMK1) is strongly inhibited by intracellular acidification with a pKvalue of pH 6.8 (Fakler et al. 1996). Kir1.2 (ROMK2) is a truncated form of Kir1.1 with the first 19 amino acids missing in its N terminus. This K+ channel is pHi sensitive as well (Choe et al. 1997; McNicholas et al. 1998). In the Kir2 family, Kir2.3 is inhibited by a drop in pHo (Coulter et al. 1995). Our recent studies have demonstrated that Kir2.3 is inhibited even more strongly by intracellular acidification with pK 6.77 (Zhu et al. 1999). Interestingly, changes in pHi and pHo to 6–8 do not affect Kir2.1, another member of the Kir2 family (Coulter et al. 1995; Fakler et al. 1996; Zhu et al. 1999), suggesting that the proton sensing is related to small variations in amino acid sequences of the channel proteins. The Kir4 family also has members that are pH sensitive. Shuck et al. (1997) have cloned a Kir channel from the kidney (named Kir1.2) which has a 97 % identity in its published 237 amino acid sequence (U73193, GenBank) to brain Kir4.1 that has a total of 379 amino acids. This renal K+ channel is inhibited by low pH. The pH sensitivity of this Kir channel (pK 6.2) is close to that of brain Kir4.1 (pK 6.03). Another member of the Kir4 family Kir4.2 cloned from the liver with 64 % amino acid sequence identity to brain Kir4.1, is also pH sensitive although the pK value of the channel is still unknown (Pearson et al. 1999). In our current studies, we have observed that brain Kir4.1 is sensitive to CO2 and pH, which is consistent with our previous observations (Yang & Jiang, 1999). Surprisingly, we have found that channel sensitivity to CO2 and pH is dramatically enhanced when Kir4.1 is coexpressed with brain Kir5.1. Kir5.1 is known to be able to form heteromeric channels with Kir4.1 leading to a change in channel biophysical properties (Pessia et al. 1996), although whether the coexpression can affect cellular functions of other Kir channels has been a mystery. In these studies, we have demonstrated an important function of Kir5.1 in enhancing CO2/pH sensitivity of Kir4.1. The pK value is increased by ∼1.4 pH units in the coexpressed Kir4.1-Kir5.1 channel, which makes the heteromeric channels sensitive to CO2/pH changes at near physiological levels. While the pK value increases in the coexpressed Kir4.1-Kir5.1 channel, the Hill coefficient does not increase at all (i.e. 2.2 in Kir4.1 and 2.1 in Kir4.1-Kir5.1), suggesting that the enhanced pH sensitivity in the coexpressed Kir4.1-Kir5.1 channel is not a result of additional proton-binding sites introduced by Kir5.1.

The mechanisms of CO2 sensing in these Kir channels

Our data strongly suggest that a decrease in pH levels during CO2 exposure is the primary cause for the inhibition of these Kir channels based on the following considerations. First, the inhibition of both Kir4.1 and Kir4.1-Kir5.1 currents by CO2 exposure is closely associated with concurrent changes in intra- or extracellular pH. Second, a reduction in intracellular pH with HCO3− produces an inhibition of these currents. Third, intracellular acidification without changing PCO2 and HCO3− at pH levels similar to those measured during hypercapnia produces similar inhibitions of the Kir currents. Finally, high PCO2 without changing pH has no effect on these currents. Thus, our studies indicate that the decrease in pHi is the central mechanism underlying the inhibition of Kir4.1 and Kir4.1-Kir5.1 during hypercapnia.

In cell-free excised patches when cytosolic soluble factors are generally washed out, we have found that the pH sensitivity of Kir4.1 and Kir4.1-Kir5.1 is retained in a manner similar to that seen in whole-cell recordings. These observations suggest that the inhibition of the Kir channels by low pHi is unlikely to be caused by changes in concentrations of cytosolic soluble factors such as second messengers and Mg2+. The inhibition does not seem to be mediated by polyamines either, since the inward but not outward rectifying currents are predominantly affected (Baukrowitz et al. 1999). Our results do not support the idea that protein phosphorylation is responsible for the inhibition of these Kir currents by intracellular protons. Several blockers of phosphatase and phosphodiesterase such as vanadate, fluoride and pyrophosphate were used in the intracellular solution. These chemicals tend to inhibit protein dephosphorylation. Meanwhile, there was no Mg2+ and ATP in this intracellular solution. Under such conditions, the turnover of protein phosphorylation and dephosphorylation should not occur at least in the time frame of our low pH exposures (0.5–2.0 min). Thereby, it is unlikely that the Kir inhibition during low pH is a result of protein phosphorylation, and the inhibition appears to be related to amino acid sequences and the tertiary structures of Kir channel proteins.

Indeed, our studies have begun to reveal the molecular basis for the channel inhibition by protons. We have found that Lys67 in Kir4.1 is critical in the modulation. Channel sensitivity to CO2 is completely eliminated when this lysine residue is mutated to methionine or glutamine. A lysine residue found at the same position in Kir1.1 and Kir1.2 channels has been demonstrated to play a crucial role in pHi sensing of these channels (Fakler et al. 1996; McNicholas et al. 1998). Interestingly, our data have shown that this lysine residue is not only critical in pHi sensing of the homomeric Kir4.1 but also responsible for proton detection in the heteromeric Kir4.1-Kir5.1, as the K67M mutation also abolishes CO2 sensitivity of Kir4.1-Kir5.1. In Kir2.3, we have previously demonstrated a short motif in the N terminus, which consists of approximately eight amino acids centred around Thr53. Mutation of Thr53 in Kir2.3 greatly reduces channel sensitivity to CO2 and pHi (Qu et al. 1999). The same motif including the threonine residue at position 68 (Thr68) is also found in Kir5.1. However, our data indicate that Thr68 is not involved in pH sensing, nor in the enhancement of pH sensitivity of the heteromeric Kir4.1-Kir5.1. These results suggest that the enhanced pH sensitivity in the heteromeric Kir4.1-Kir5.1 is not due to introducing an additional motif for pHi sensing to the Kir4.1-Kir5.1.

It is known that lysine is titratable only at extremely high pH levels with pK 10.5. Previous studies have suggested that residues adjacent to Lys80 in Kir1.1 may be involved in reducing its pK value to a physiological pH range (Fakler et al. 1996; Choe et al. 1997). If this is also the case in the heteromeric Kir4.1-Kir5.1, the presence of Kir5.1 may have largely changed the pK value of this lysine residue. Hence, the influence of amino acids in Kir5.1 on the pK value of Lys67 appears to be much stronger than residues in Kir4.1, suggesting a close interaction of these two subunits and a plausible explanation of the enhanced pH sensitivity in the heteromeric Kir4.1-Kir5.1. Since evidence for protonation of this lysine residue is lacking, there is still a possibility that the lysine is only a part of the gating mechanisms rather than the proton-binding site. With an interruption of the gating mechanism, Kir4.1 and Kir4.1-Kir5.1 may not be able to close during hypercapnia or intracellular acidification. Since the binding versus gating has been a common problem in studies of all ligand-gated ion channels (Colquhoun, 1998), the demonstration of a convergent site for channel gating by intracellular protons in Kir4.1 and Kir4.1-Kir5.1 in our current studies may contribute to the understanding of the gating mechanisms in these K+ channels.

Functional implications

Inward rectifier K+ channels are important players in the maintenance of plasma membrane excitability and the control of intra- and extracellular K+ ionic homeostasis. Inhibition of these K+ channels produces depolarization and increases membrane excitability. Neurons in brainstem chemoreceptive areas are indeed inhibited by intracellular protons and tend to have a long lasting pH change in the cytosol in response to hypercapnia (Pineda & Aghajanian, 1997; Ritucci et al. 1997; Wieman et al. 1998). In the brainstem where the Kir4.1 channels are expressed, the inhibition of Kir4.1 channels by high CO2 or low pH can have a major impact not only on cells expressing these channels but also on local neuronal networks. Depolarization and increase in membrane excitability caused by hypercapnia in these cells may lead to a spread of the excitation to other brainstem neurons such as those that are responsible for cardio-respiratory controls in the neuronal networks. As a result, the hypercapnic information can be coupled to a corresponding change in excitability of the cardio-respiratory system (von Euler, 1986). Thus, expression of these Kir channels in brainstem neurons that have direct or indirect connections with cardio-respiratory neuronal networks may enable these cells as well as the cardio-respiratory system to detect PCO2 levels in the cerebral spinal fluid and blood circulation. Therefore, the studies of the CO2/pH sensitivity in these Kir channels constitute a significant step towards the understanding of their potential functions in CO2 and pH sensing in nerve cells in the brainstem.

Acknowledgments

This work was supported by the NIH (RO1 HL58410-01) and the Grant-in-Aid Award (9950528 N) from the American Heart Association. We would like to thank Dr John Adelman for his generosity in sharing with us the Kir4.1 and Kir5.1 cDNAs.

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