Zhenjiang Yang

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.

Opposite effects of pH on open‐state probability and single channel conductance of Kir4.1 channels

1 A decrease in intracellular pH (pHi) inhibits whole‐cell Kir4.1 currents. To understand channel biophysical properties underlying this inhibition, single channel Kir4.1 currents were studied in inside‐out patches using symmetric concentrations of K+ applied to each side of the plasma membrane. Under such conditions, inward rectifying currents were observed in about 2 of 3 patches. At pH 7.4, these currents showed a single channel conductance of 22 pS with a channel open‐state probability (Popen) of ≈0.9. 2 The effects of intracellular protons on macroscopic Kir4.1 currents were examined in giant inside‐out patches at various pH levels of internal solutions. Current amplitude increased with a modest acidification (pH 7.0 and 6.6), and decreased with further reductions in pHi. The Kir4.1 currents were completely suppressed at pH 5.4. These effects were fast and reversible. 3 Low pHi inhibited Popen and enhanced single channel conductance in a concentration‐dependent manner with pK (midpoint pH value for channel inhibition) of 6.0 and 6.8, respectively. At pH 5.8, Popen was inhibited by 70 % and single channel conductance increased by 35 %. Washout brought both Popen and single channel conductance rapidly back to baseline levels. 4 Theoretical currents were calculated using percentage changes in Popen and single channel conductance at each pH level tested. The trajectory of these currents is very close to that of experimental currents recorded from giant patches. Thus, opposite effects of intracellular protons on Popen and single channel conductance are demonstrated, which are likely to result in changes of macroscopic Kir4.1 currents with low pH.

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.

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.

A single residue contributes to the difference between Kir4.1 and Kir1.1 channels in pH sensitivity, rectification and single channel conductance

1 Kir1.1 and Kir4.1 channels may be involved in the maintenance of pH and K+ homeostasis in renal epithelial cells and CO2 chemoreception in brainstem neurons. To understand the molecular determinants for their characteristic differences, the structure‐function relationship was studied using site‐directed mutagenesis. 2 According to previous studies, Glu158 in Kir4.1 is likely to be the major rectification controller. This was confirmed in both Kir1.1 and Kir4.1. Mutation of Gly210, the second potential rectification controller, to glutamate did not show any additional effect on the inward rectification. 3 More interestingly, we found that Glu158 in Kir4.1 was also an important residue contributing to single channel conductance and pH sensitivity. The E158N Kir4.1 mutant had a unitary conductance of 35 pS and a midpoint pH for channel inhibition (pKa) value of 6.72, both of which were almost identical to those of the wild‐type (WT) Kir1.1. Flickering channel activity was clearly seen in the E158N mutant at positive membrane potentials, which is typical in the WT Kir1.1 but absent in the WT Kir4.1. 4 Reverse mutation in Kir1.1 (N171E) reduced the unitary conductance to 27 pS (23 pS in WT Kir4.1). However, the pH sensitivity of this mutant did not show a marked difference from the WT Kir1.1. Therefore, it is possible that a residue(s) in addition to Asn171 is also involved. Thus we studied several other residues in both M2 and H5 regions. We found that joint mutations of Val140 and Asn171 to residues seen in Kir4.1 greatly reduced the pH sensitivity (pKa 6.08). 5 The V140T mutation in Kir1.1 led to a unitary conductance of ∼70 pS, and the G210E mutation in Kir4.1 caused a decrease in pH sensitivity of 0.4 pH units. 6 These results indicate that the pore‐forming sequences are targets for modulations of multiple channel‐biophysical properties and demonstrate a site contributing to rectification, unitary conductance and proton sensitivity in these Kir channels.

Protons Activate Homomeric Kir6.2 Channels by Selective Suppression of the Long and Intermediate Closures

The ATP-sensitive K+ channels (KATP) play an important role in regulating membrane excitability. These channels are regulated by H+ in addition to ATP, ADP, and phospholipids. To understand how protons affect the single-channel properties, Kir6.2DC36 currents were studied in excised inside-out patches. We chose to study the homomeric Kir6.2 channel with 36 amino acids deleted at the C-terminal end, as there are ADP/ATP-binding sites in the SUR subunit, which may obscure the understanding of the channel-gating process. In the absence of ATP, moderate intracellular acidosis (pH 6.8) augmented Popen with small suppression (by ~10%) of the single-channel conductance. The long and intermediate closures were selectively inhibited, leading to a shortening of the mean closed time without significant changes in the mean open time. Stronger acidification (<pH6.2) caused channel rundown. Although similar changes in the single-channel properties were observed in the presence of 1 mM ATP, the Popen-pH relationship curve was shifted by 0.17 pH units toward lower pH levels. ATP had no effect on the inhibition of single-channel conductance during acidosis. Mutation of His175 eliminated the pH effect on the single-channel kinetics, while the single-channel response to acidic pH was retained in the K185E mutant that greatly reduces the ATP sensitivity. These results indicate that Kir6.2DC36 channel gating by protons and ATP relies on two distinct mechanisms opposite to each other, although the pH sensitivity is modulated by ATP.