Ningren Cui

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

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.

Effects of intra‐ and extracellular acidifications on single channel Kir2.3 currents

1 The inward rectifier K+ channel Kir2.3 is inhibited by hypercapnia, and this inhibition may be mediated by decreases in intra‐ and extracellular pH. To understand whether Kir2.3 has two distinct pH sensors and whether cytosol‐soluble factors are involved in the modulation of this channel during intracellular acidification, single channel currents were studied by expressing Kir2.3 in Xenopus oocytes. 2 In excised inside‐out patches, Kir2.3 currents had a high baseline channel open‐state probability (Po, at pH 7.4) with a strong inward rectification. Single channel conductance at hyperpolarizing membrane potential was about 17 pS with 150 mM K+ applied to both sides of the membrane. The channel showed a substate conductance of about 8 pS. 3 Reduction of intracellular pH (pHi) produced a fast and reversible inhibition of single channel Kir2.3 currents in inside‐out patches. The extent of this inhibition is concentration dependent. A clear reduction in Kir2.3 currents was seen at pHi 7.0, and channel activity was completely suppressed at pHi 6.2 with mid‐point inhibition (pK) at pH 6.77. 4 The effect of low pHi on Kir2.3 currents was due to a strong inhibition of Po and a moderate suppression of single channel conductance. The pK values for these single channel properties were pH 6.78 and 6.67, respectively. 5 The decrease in Po with low pHi resulted from an increase in the channel mean closed time without significant changes in the mean open time. Substate conductance was not seen during low pHi. 6 Decrease in extracellular pH (pHo) also caused inhibition of single channel activity of Kir2.3 currents in excised outside‐out patches. This effect, however, was clearly different from that of pHi: the pK (pH 6.70) was about 0.1 pH units lower; more than 50 % channel activity was retained at pHo 5.8; and low pHo affected mainly single channel conductance. 7 These results therefore indicate that (1) there are two distinct pH sensors in Kir2.3, (2) different mechanisms are involved in the modulation of Kir2.3 through these two pH sensors, and (3) cytosol‐soluble factors do not appear to be engaged in this modulation.

Identification of a critical motif responsible for gating of Kir2.3 channel by intracellular protons.

Protons are involved in gating Kir2.3. To identify the molecular motif in the Kir2.3 channel protein that is responsible for this process, experiments were performed using wild-type and mutated Kir2.3 and Kir2.1. CO2 and low pHi strongly inhibited wild-type Kir2.3 but not Kir2.1 in whole cell voltage clamp and excised inside-out patches. This CO2/pH sensitivity was completely eliminated in a mutant Kir2.3 in which the N terminus was substituted with that in Kir2.1, whereas a similar replacement of its C terminus had no effect. Site-specific mutations of all titratable residues in the N terminus, however, did not change the CO2/pH sensitivity. Using several chimeras generated systematically in the N terminus, a 10-residue motif near the M1 region was identified in which only three amino acids are different between Kir2.3 and Kir2.1. Mutations of these residues, especially Thr53, dramatically reduced the pH sensitivity of Kir2.3. Introducing these residues or even a single threonine to the corresponding positions of Kir2.1 made the mutant channel pH-sensitive. Thus, a critical motif responsible for gating Kir2.3 by protons was identified in the N terminus, which contained about 10 residues centered by Thr53.

Distinct Histidine Residues Control the Acid-induced Activation and Inhibition of the Cloned KATP Channel

The modulation of KATP channels during acidosis has an impact on vascular tone, myocardial rhythmicity, insulin secretion, and neuronal excitability. Our previous studies have shown that the cloned Kir6.2 is activated with mild acidification but inhibited with high acidity. The activation relies on His-175, whereas the molecular basis for the inhibition remains unclear. To elucidate whether the His-175 is indeed the protonation site and what other structures are responsible for the pH-induced inhibition, we performed these studies. Our data showed that the His-175 is the only proton sensor whose protonation is required for the channel activation by acidic pH. In contrast, the channel inhibition at extremely low pH depended on several other histidine residues including His-186, His-193, and His-216. Thus, proton has both stimulatory and inhibitory effects on the Kir6.2 channels, which attribute to two sets of histidine residues in the C terminus.

Gating of inward rectifier K+ channels by proton-mediated interactions of N- and C-terminal domains.

Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO2/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO2 and pH i . These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K+ channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator.

Suppression of Kir2.3 Activity by Protein Kinase C Phosphorylation of the Channel Protein at Threonine 53

Kir2.3 plays an important part in the maintenance of membrane potential in neurons and myocardium. Identification of intracellular signaling molecules controlling this channel thus may lead to an understanding of the regulation of membrane excitability. To determine whether Kir2.3 is modulated by direct phosphorylation of its channel protein and identify the phosphorylation site of protein kinase C (PKC), we performed experiments using several recombinant and mutant Kir2.3 channels. Whole-cell Kir2.3 currents were inhibited by phorbol 12-myristate 13-acetate (PMA) in Xenopus oocytes. When the N-terminal region of Kir2.3 was replaced with that of Kir2.1, another member in the Kir2 family that is insensitive to PMA, the chimerical channel lost its PMA sensitivity. However, substitution of the C terminus was ineffective. Four potential PKC phosphorylation sites in the N terminus were studied by comparing mutations of serine or threonine with their counterpart residues in Kir2.1. Whereas substitutions of serine residues at positions 5, 36, and 39 had no effect on the channel sensitivity to PMA, mutation of threonine 53 completely eliminated the channel response to PMA. Interestingly, creation of this threonine residue at the corresponding position (I79T) in Kir2.1 lent the mutant channel a PMA sensitivity almost identical to the wild-type Kir2.3. These results therefore indicate that Kir2.3 is directly modulated by PKC phosphorylation of its channel protein and threonine 53 is the PKC phosphorylation site in Kir2.3.

An alternative approach to the identification of respiratory central chemoreceptors in the brainstem

Central chemoreceptors (CCRs) play a crucial role in autonomic respiration. Although a variety of brainstem neurons are CO(2) sensitive, it remains to know which of them are the CCRs. In this article, we discuss a potential alternative approach that may allow an access to the CCRs. This approach is based on identification of specific molecules that are CO(2) or pH sensitive, exist in brainstem neurons, and regulate cellular excitability. Their molecular identity may provide another measure in addition to the electrophysiologic criteria to indicate the CCRs. The inward rectifier K(+) channels (Kir) seem to be some of the CO(2) sensing molecules, as they regulate membrane potential and cell excitability and are pH sensitive. Among homomeric Kirs, we have found that even the most sensitive Kir1.1 and Kir2.3 have pK approximately 6.8, suggesting that they may not be capable of detecting hypocapnia. We have studied their biophysical properties, and identified a number of amino acid residues and molecular motifs critical for the CO(2) sensing. By comparing all Kirs using the motifs, we found the same amino acid sequence in Kir5.1, and demonstrated the pH sensitivity in heteromeric Kir4.1 and Kir5.1 channels to be pK approximately 7.4. In current clamp, we show evidence that the Kir4.1-Kir5.1 can detect P(CO(2)) changes in either hypercapnic or hypocapnic direction. Our in-situ hybridization studies have indicated that they are coexpressed in brainstem cardio-respiratory nuclei. Thus, it is likely that the heteromeric Kir4.1-Kir5.1 contributes to the CO(2)/pH sensitivity in these neurons. We believe that this line of research intended to identify CO(2) sensing molecules is an important addition to current studies on the CCRs.

Allosteric modulation of the mouse Kir6.2 channel by intracellular H+ and ATP.

The ATP‐sensitive K+ (KATP) channels are regulated by intracellular H+ in addition to ATP, ADP, and phospholipids. Here we show evidence for the interaction of H+ with ATP in regulating a cloned KATP channel, i.e. Kir6.2 expressed with and without the SUR1 subunit. Channel sensitivity to ATP decreases at acidic pH, while the pH sensitivity also drops in the presence of ATP. These effects are more evident in the presence of the SUR1 subunit. In the Kir6.2 + SUR1, the pH sensitivity is reduced by about 0.4 pH units with 100 μM ATP and 0.6 pH units with 1 mm ATP, while a decrease in pH from 7.4 to 6.8 lowers the ATP sensitivity by about fourfold. The Kir6.2 + SUR1 currents are strongly activated at pH 5.9‐6.5 even in the presence of 1 mm ATP. The modulations appear to take place at His175 and Lys185 that are involved in proton and ATP sensing, respectively. Mutation of His175 completely eliminates the pH effect on the ATP sensitivity. Similarly, the K185E mutant‐channel loses the ATP‐dependent modulation of the pH sensitivity. Thus, allosteric modulations of the cloned KATP channel by ATP and H+ are demonstrated. Such a regulation allows protons to activate directly the KATP channels and release channel inhibition by intracellular ATP; the pH effect is further enhanced with a decrease in ATP concentration as seen in several pathophysiological conditions.

Modulation of the heteromeric Kir4.1–Kir5.1 channels by P CO 2 at physiological levels

Several inward rectifier K+ (Kir) channels are pH‐sensitive, making them potential candidates for CO2 chemoreception in cells. However, there is no evidence showing that Kir channels change their activity at near physiological level of P  CO 2 , as most previous studies were done using high concentrations of CO2. It is known that the heteromeric Kir4.1–Kir5.1 channels are highly sensitive to intracellular protons with pKa value right at the physiological pH level. Such a pKa value may allow these channels to regulate membrane potentials with modest changes in P  CO 2 . To test this hypothesis, we studied the Kir4.1–Kir5.1 currents expressed in Xenopus oocytes and membrane potentials in the presence and absence of bicarbonate. Evident inhibition of these currents (by ∼5%) was seen with P  CO 2 as low as 8 torr. Higher P  CO 2 levels (23–60 torr) produced stronger inhibitions (by 30–40%). The inhibitions led to graded depolarizations (5–45 mV with P  CO 2 8–60 torr). Similar effects were observed in the presence of 24 mM bicarbonate and 5% CO2. Indeed, the Kir4.1–Kir5.1 currents were enhanced with 3% CO2 and suppressed with 8% CO2 in voltage clamp, resulting in hyper‐ (−9 mV) and depolarization (16 mV) in current clamp, respectively. With physiological concentration of extracellular K+, the Kir4.1–Kir5.1 channels conduct substantial outward currents that were similarly inhibited by CO2 as their inward rectifying currents. These results therefore indicate that the heteromeric Kir4.1–Kir5.1 channels are modulated by a modest change in P  CO 2 levels. Such a modulation alters cellular excitability, and enables the cell to detect hypercapnia and hypocapnia in the presence of bicarbonate.

A Threonine Residue (Thr71) at the Intracellular End of the M1 Helix Plays a Critical Role in the Gating of Kir6.2 Channels by Intracellular ATP and Protons

ATP-sensitive K+ (KATP) channels are known to be gated by several intracellular molecules, but the gating mechanisms remain unclear. To understand the relationship of channel gating to ligand binding, we studied Kir6.2 channel gating by ATP and protons, which inhibit and activate the channel, respectively. We have previously shown that a threonine residue (Thr71) is critical for the pH sensitivity of Kir6.2 channel. If this site is involved in channel gating rather than ligand binding, it should affect channel gating by both ATP and proton. To test this hypothesis we performed a mutation analysis. Site-specific mutations of Thr71 to a bulky residue reduced the ATP sensitivity by >100-fold and eliminated the pH sensitivity. Single-channel activity of these mutants was stabilized at the open state with no detectable rundown. Mutations to a small amino acid had little effect on the ATP and pH sensitivities. Mutations to intermediate amino acids reduced but did not abolish the ATP and pH sensitivities. Hydrophobicity is not critical, as both polar and nonpolar amino acids are found in each group. Mutation to a positively charged lysine markedly exacerbated the pH- but not ATP-sensitivity, whereas mutation to glutamate moderately reduced ATP and pH sensitivities. These results indicate that the residue mass is critical for Kir6.2 channel gating, a mass that should be below 120 daltons with no charge. The existence of such a site as Thr71 involved in channel gating by both ATP and proton suggests that channel gating in the KATP channel likely is separate from ligand binding.