Biophysical and molecular mechanisms underlying the modulation of heteromeric Kir4.1-Kir5.1 channels by CO2 and pH

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4. 1-Kir5.1 were studied in inside-out patches. These Kir4.1-Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P(open)) approximately 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P(open) without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at approximately 1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1-Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P(open) and reduced channel sensitivity to intracellular protons. In the presence of 10 microM PIP2, the Kir4.1-Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1-Kir5.1. In excised patches, interestingly, the Kir4.1-Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.     

Protein kinase C dependent inhibition of the heteromeric Kir4.1-Kir5.1 channel

Heteromultimerization of Kir4.1 and Kir5.1 leads to a channel with distinct functional properties. The heteromeric Kir4.1-Kir5.1 channel is expressed in the eye, kidney and brainstem and has CO(2)/pH sensitivity in the physiological range, suggesting a candidate molecule for the regulation of K(+) homeostasis and central CO(2) chemoreception. It is known that K(+) transport in renal epithelium and brainstem CO(2) chemosensitivity are subject to modulation by hormones and neurotransmitters that activate distinct intracellular signaling pathways. If the Kir4.1-Kir5.1 channel is involved in pH-dependent regulation of cellular functions, it may also be regulated by some of the intracellular signaling systems. Therefore, we undertook studies to determine whether PKC modulates the heteromeric Kir4.1-Kir5.1 channel. The channel expressed using a Kir4.1-Kir5.1 tandem dimer construct was inhibited by the PKC activator PMA in a dose-dependent manner. The channel inhibition was produced via reduction of the P(open). The effect of PMA was abolished by specific PKC inhibitors. In contrast, exposure of oocytes to forskolin (a PKA activator) had no significant effect on Kir4.1-Kir5.1 currents. The channel inhibition appeared to be independent of PIP(2) depletion and PKC-dependent internalization. Several consensus sequences of potential PKC phosphorylation sites were identified in the Kir4.1 and Kir5.1 subunits by sequence scan. Although the C-terminal peptides of both Kir4.1 and Kir5.1 were phosphorylated in vitro, site-directed mutagenesis of individual residues failed to reveal the PKC phosphorylation sites suggesting that the channel may have multiple phosphorylation sites. Taken together, these results suggest that the Kir4.1-Kir5.1 but not the homomeric Kir4.1 channel is strongly inhibited by PKC activation.     

Modulation of Kir4.1 and Kir4.1-Kir5.1 channels by extracellular cations

This work demonstrates that extracellular Na⁺ modulates the cloned inwardly rectifying K⁺ channels Kir4.1 and Kir4.1-Kir5.1. Whole-cell patch clamp studies on astrocytes have previously indicated that inward potassium currents are regulated by external Na⁺. We expressed Kir4.1 and Kir4.1-Kir5.1 in Xenopus oocytes to disclose if Kir4.1 and/or Kir4.1-Kir5.1 at the molecular level are responsible for the observed effect of [Na⁺]_o and to investigate the regulatory mechanism of external cations further. Our results showed that Na⁺ has a biphasic modulatory effect on both Kir4.1 and Kir4.1-Kir5.1 currents. Depending on the Na⁺-concentration and applied voltage, the inward Kir4.1/Kir4.1-Kir5.1 currents are either enhanced or reduced by extracellular Na⁺. The Na⁺ activation was voltage-independent, whereas the Na⁺-induced reduction of the Kir4.1 and Kir4.1-Kir5.1 currents was both concentration-, time- and voltage-dependent. Our data indicate that the biphasic effect of extracellular Na⁺on the Kir4.1 and Kir4.1-Kir5.1 channels is caused by two separate mechanisms.     

The disruption of central CO2 chemosensitivity in a mouse model of Rett syndrome

People with Rett syndrome (RTT) have breathing instability in addition to other neuropathological manifestations. The breathing disturbances contribute to the high incidence of unexplained death and abnormal brain development. However, the cellular mechanisms underlying the breathing abnormalities remain unclear. To test the hypothesis that the central CO(2) chemoreception in these people is disrupted, we studied the CO(2) chemosensitivity in a mouse model of RTT. The Mecp2-null mice showed a selective loss of their respiratory response to 1-3% CO(2) (mild hypercapnia), whereas they displayed more regular breathing in response to 6-9% CO(2) (severe hypercapnia). The defect was alleviated with the NE uptake blocker desipramine (10 mg·kg(-1)·day(-1) ip, for 5-7 days). Consistent with the in vivo observations, in vitro studies in brain slices indicated that CO(2) chemosensitivity of locus coeruleus (LC) neurons was impaired in Mecp2-null mice. Two major neuronal pH-sensitive Kir currents that resembled homomeric Kir4.1 and heteromeric Ki4.1/Kir5.1 channels were identified in the LC neurons. The screening of Kir channels with real-time PCR indicated the overexpression of Kir4.1 in the LC region of Mecp2-null mice. In a heterologous expression system, an overexpression of Kir4.1 resulted in a reduction in the pH sensitivity of the heteromeric Kir4.1-Kir5.1 channels. Given that Kir4.1 and Kir5.1 subunits are also expressed in brain stem respiration-related areas, the Kir4.1 overexpression may not allow CO(2) to be detected until hypercapnia becomes severe, leading to periodical hyper- and hypoventilation in Mecp2-null mice and, perhaps, in people with RTT as well.     

Modulation of the heteromeric Kir4.1-Kir5.1 channel by multiple neurotransmitters via Galphaq-coupled receptors

The heteromeric Kir4.1-Kir5.1 channel is a candidate sensing molecule for central CO(2) chemoreception. Since central CO(2) chemoreception is subject to neural modulations, we performed studies to test the hypothesis that the Kir4.1-Kir5.1 channel is modulated by the neurotransmitters critical for respiratory control, including serotonin (5-HT), substance-P (SP), and thyrotropin releasing hormone (TRH). The heteromeric Kir4.1-Kir5.1 channel was strongly inhibited by SP, TRH, and 5-HT when expressed in Xenopus oocytes, whereas these neurotransmitters had no effect on the homomeric Kir4.1 channel. Such an inhibition was dose-dependent and relied on specific G(alphaq)-protein-coupled receptors and protein kinase C (PKC). No direct interaction of the channel with G-proteins was found. Channel sensitivity to CO(2)/pH was not compromised with the inhibition by these neurotransmitters, as the channel remained to be inhibited by acidic pH following an exposure to the neurotransmitters. The firing rate of CO(2)-sensitive brainstem neurons cultured in microelectrode arrays was augmented by SP or a 5-HT2A receptor agonist, which was blocked by PKC inhibitors suggesting that PKC underscores the inhibitory effect of SP and 5-HT in cultured brainstem neurons as well. Immunostaining showed that both Kir4.1 and Kir5.1 proteins were co-localized in the cultured brainstem neurons. These results therefore indicate that the heteromeric Kir4.1-Kir5.1 channel is modulated by the neurotransmitters critical for respiratory control, suggesting a novel neuromodulatory mechanism for the chemosensitivity of brainstem neurons to elevated PCO(2) and acidic pH.     

Protein kinase C dependent inhibition of the heteromeric Kir4.1-Kir5.1 channel

Heteromultimerization of Kir4.1 and Kir5.1 leads to a channel with distinct functional properties. The heteromeric Kir4.1-Kir5.1 channel is expressed in the eye, kidney and brainstem and has CO₂/pH sensitivity in the physiological range, suggesting a candidate molecule for the regulation of K⁺ homeostasis and central CO₂ chemoreception. It is known that K⁺ transport in renal epithelium and brainstem CO₂ chemosensitivity are subject to modulation by hormones and neurotransmitters that activate distinct intracellular signaling pathways. If the Kir4.1-Kir5.1 channel is involved in pH-dependent regulation of cellular functions, it may also be regulated by some of the intracellular signaling systems. Therefore, we undertook studies to determine whether PKC modulates the heteromeric Kir4.1-Kir5.1 channel. The channel expressed using a Kir4.1-Kir5.1 tandem dimer construct was inhibited by the PKC activator PMA in a dose-dependent manner. The channel inhibition was produced via reduction of the P_open. The effect of PMA was abolished by specific PKC inhibitors. In contrast, exposure of oocytes to forskolin (a PKA activator) had no significant effect on Kir4.1-Kir5.1 currents. The channel inhibition appeared to be independent of PIP₂ depletion and PKC-dependent internalization. Several consensus sequences of potential PKC phosphorylation sites were identified in the Kir4.1 and Kir5.1 subunits by sequence scan. Although the C-terminal peptides of both Kir4.1 and Kir5.1 were phosphorylated in vitro, site-directed mutagenesis of individual residues failed to reveal the PKC phosphorylation sites suggesting that the channel may have multiple phosphorylation sites. Taken together, these results suggest that the Kir4.1-Kir5.1 but not the homomeric Kir4.1 channel is strongly inhibited by PKC activation.     

Regulation of inward rectifier K+ channels by shift of intracellular pH dependence

The mechanistic link between mitochondrial metabolism and inward rectifier K+ channel activity was investigated by studying the effects of a mitochondrial inhibitor, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on inward rectifiers of the Kir2 subfamily expressed in Xenopus oocytes, using two-electrode voltage-clamp, patch-clamp, and intracellular pH recording. FCCP inhibited Kir2.2 and Kir2.3 currents and decreased intracellular pH, but the pH change was too small to account for the inhibitory effect by itself. However, pre-incubation of oocytes with imidazole prevented both the pH decrease and the inhibition of Kir2.2 and Kir2.3 currents by FCCP. The pH dependence of Kir2.2 was shifted to higher pH in membrane patches from FCCP-treated oocytes compared to control oocytes. Therefore, the inhibition of Kir2.2 by FCCP may involve a combination of intracellular acidification and a shift in the intracellular pH dependence of these channels. To investigate the sensitivity of heteromeric channels to FCCP, we studied its effect on currents expressed by heteromeric tandem dimer constructs. While Kir2.1 homomeric channels were insensitive to FCCP, both Kir2.1-Kir2.2 and Kir2.1-Kir2.3 heterotetrameric channels were inhibited. These data support the notion that mitochondrial dysfunction causes inhibition of heteromeric inward rectifier K+ channels. The reduction of inward rectifier K+ channel activity observed in heart failure and ischemia may result from the mitochondrial dysfunction that occurs in these conditions.     

Expression of inwardly rectifying K+ channels in the carotid body of rat

The inwardly rectifying K+ channels, Kir1.1, Kir2.3, Kir4.1-Kir5.1, and Kir4.2-Kir5.1, are candidate chemosensory molecules for CO2/H+. Here, we determined the mRNA expression and immunohistochemical localization of these channels in the carotid body (CB) and petrosal ganglion (PG) of the rat. RT-PCR analysis revealed mRNA expression of Kir4.1 and Kir5.1 in CB, and Kir1.1, Kir4.1, and Kir5.1 in PG. Immunohistochemistry identified the glomus cells in CB to express both Kir4.1 and Kir5.1 protein, while the nerve fibers in CB were immunoreactive for Kir1.1, Kir4.1, and Kir5.1. In the PG, immunoreactivity for Kir1.1, Kir4.1, and Kir5.1 was observed in some ganglion cells. Our findings suggest that Kir channels in the peripheral chemoreceptors play a role in sensing hypercapnic acidosis and maintaining the resting membrane potentials.     

Zacopride selectively activates the Kir2.1 channel via a PKA signaling pathway in rat cardiomyocytes

We recently reported that zacopride is a selective inward rectifier potassium current (IK1 ) channel agonist, suppressing ventricular arrhythmias without affecting atrial arrhythmias. The present study aimed to investigate the unique pharmacological properties of zacopride. The whole-cell patch-clamp technique was used to study IK1 currents in rat atrial myocytes and Kir2.x currents in human embryonic kidney (HEK)-293 cells transfected with inward rectifier potassium channel (Kir)2.1, Kir2.2, Kir2.3, or mutated Kir2.1 (at phosphorylation site S425L). Western immunoblots were performed to estimate the relative protein expression levels of Kir2.x in rat atria and ventricles. Results showed that zacopride did not affect the IK1 and transmembrane potential of atrial myocytes. In HEK293 cells, zacopride increased Kir2.1 homomeric channels by 40.7%±9.7% at 50 mV, but did not affect Kir2.2 and Kir2.3 homomeric channels, and Kir2.1-Kir2.2, Kir2.1-Kir2.3 and Kir2.2-Kir2.3 heteromeric channels. Western immunoblots showed that similar levels of Kir2.3 protein were expressed in rat atria and ventricles, but atrial Kir2.1 protein level was only 25% of that measured in the ventricle. In addition, 5-hydroxytryptamine (5-HT) 3 receptor was undetectable, whereas 5-HT 4 receptor was weakly expressed in HEK293 cells. The Kir2.1-activating effect of zacopride in these cells was abolished by inhibition of protein kinase A (PKA), but not PKC or PKG. Furthermore, zacopride did not activate the mutant Kir2.1 channel in HEK293 cells but selectively activated the Kir2.1 homomeric channel via a PKA-dependent pathway, independent to that of the 5-HT receptor.     

Subunit positional effects revealed by novel heteromeric inwardly rectifying K+ channels.

Kir 4.1 is an inward rectifier potassium channel subunit isolated from rat brain which forms homomeric channels when expressed in Xenopus oocytes; Kir 5.1 is a structurally related subunit which does not. Co-injection of mRNAs encoding Kir 4.1 and Kir 5.1 resulted in potassium currents that (i) were much larger than those seen from expression of Kir 4.1 alone, (ii) increased rather than decreased during several seconds at strongly negative potentials and (iii) had an underlying unitary conductance of 43 pS rather than the 12 pS seen with Kir 4.1 alone. In contrast, the properties of Kir 1.1, 2.1, 2.3, 3.1, 3.2 or 3.4 were not altered by coexpression with Kir 5.1. Expression of a concatenated cDNA encoding two or four linked subunits produced currents with the properties of co-expressed Kir 4.1 and Kir 5.1 when the subunits were connected 4-5 or 4-5-4-5, but not when they were connected 4-4-5-5. The results indicate that Kir 5.1 associates specifically with Kir 4.1 to form heteromeric channels, and suggest that they do so normally in the subunit order 4-5-4-5. Further, the relative order of subunits within the channel contributes to their functional properties.     

Modulation of Kir4.2 rectification properties and pHi-sensitive run-down by association with Kir5.1

Inwardly rectifying K+ channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2-Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2-Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to EK were essentially time independent for Kir4.2 indicating rapid block by Mg2+. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity.     

Modulation of Kir4.2 rectification properties and pHi-sensitive run-down by association with Kir5.1

Inwardly rectifying K⁺ channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2-Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2-Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to E_K were essentially time independent for Kir4.2 indicating rapid block by Mg²⁺. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity.     

Differential assembly of inwardly rectifying K+ channel subunits, Kir4.1 and Kir5.1, in brain astrocytes

The inwardly rectifying K+ channel subunit Kir5.1 is expressed abundantly in the brain, but its precise distribution and function are still largely unknown. Because Kir5.1 is co-expressed with Kir4.1 in retinal glial Muller cells, we have compared the biochemical and immunological properties of Kir5.1 and Kir4.1 in the mouse brain. Immunoprecipitation experiments suggested that brain expressed at least two subsets of Kir channels, heteromeric Kir4.1/5.1 and homomeric Kir4.1. Immunolabeling using specific antibodies showed that channels comprising Kir4.1 and Kir5.1 subunits were assembled in a region-specific fashion. Heteromeric Kir4.1/5.1 was identified in the neocortex and in the glomeruli of the olfactory bulb. Homomeric Kir4.1 was confined to the hippocampus and the thalamus. Homomeric Kir5.1 was not identified. Kir4.1/5.1 and Kir4.1 expression appeared to occur only in astrocytes, specifically in the membrane domains facing the pia mater and blood vessels or in the processes surrounding synapses. Both Kir4.1/5.1 and Kir4.1 could be associated with PDZ domain-containing syntrophins, which might be involved in the subcellular targeting of these astrocyte Kir channels. Because heteromeric Kir4.1/5.1 and homomeric Kir4.1 have distinct ion channel properties (Tanemoto, M., Kittaka, N., Inanobe, A., and Kurachi, Y. (2000) J. Physiol. (Lond.) 525, 587-592 and Tucker, S. J., Imbrici, P., Salvatore, L., D'Adamo, M. C., and Pessia, M. (2000) J. Biol. Chem. 275, 16404-16407), it is plausible that these channels play differential physiological roles in the K+ -buffering action of brain astrocytes in a region-specific manner.     

pH dependence of the inwardly rectifying potassium channel, Kir5.1, and localization in renal tubular epithelia

The physiological role of the inwardly rectifying potassium channel, Kir5.1, is poorly understood, as is the molecular identity of many renal potassium channels. In this study we have used Kir5.1-specific antibodies to reveal abundant expression of Kir5.1 in renal tubular epithelial cells, where Kir4.1 is also expressed. Moreover, we also show that Kir5.1/Kir4.1 heteromeric channel activity is extremely sensitive to inhibition by intracellular acidification and that this novel property is conferred predominantly by the Kir5.1 subunit. These findings suggest that Kir5.1/Kir4.1 heteromeric channels are likely to exist in vivo and implicate an important and novel functional role for the Kir5.1 subunit.     

CO(2) inhibits specific inward rectifier K(+) channels by decreases in intra- and extracellular pH

Hypercapnia has been shown to affect cellular excitability by modulating K(+) channels. To understand the mechanisms for this modulation, four cloned K(+) channels were studied by expressing them in Xenopus oocytes. Exposures of the oocytes to CO(2) for 4-6 min produced reversible and concentration-dependent inhibitions of Kir1.1 and Kir2.3 currents, but had no effect on Kir2.1 and Kir6.1 currents. Intra- and extracellular pH (pH(i), pH(o)) dropped during CO(2) exposures. The inhibition of Kir2.3 currents was mediated by reductions in both intra- and extracellular pH, whereas the suppression of Kir1.1 resulted from intracellular acidification. In cell-free excised inside-out patches with cytosolic-soluble factors washed out, a decrease in pH(i) produced a fast and reversible inhibition of macroscopic Kir2.3 currents. The degree of this inhibition was similar to that produced by hypercapnia when compared at the same pH(i) level. Exposure of cytosolic surface of patch membranes to a perfusate bubbled with 15% CO(2) without changing pH failed to inhibit the Kir2.3 currents. These results therefore indicate that (1) hypercapnia inhibits specific K(+) channels, (2) these inhibitions are caused by intra- and extracellular protons rather than molecular CO(2), and (3) these effects are independent of cytosol-soluble factors.     

Role of conserved glycines in pH gating of Kir1.1 (ROMK)

Gating of inward rectifier Kir1.1 potassium channels by internal pH is believed to occur when large hydrophobic leucines, on each of the four subunits, obstruct the permeation path at the cytoplasmic end of the inner transmembrane helices (TM2). In this study, we examined whether closure of the channel at this point involves bending of the inner helix at one or both of two highly conserved glycine residues (corresponding to G134 and G143 in KirBac1.1) that have been proposed as putative "gating hinges" for potassium channels. Replacement of these conserved inner helical glycines by less flexible alanines did not abolish gating but shifted the apparent pKa from 6.6 +/- 0.01 (wild-type) to 7.1 +/- 0.01 for G157A-Kir1.1b, and to 7.3 +/- 0.01 for G148A-Kir1.1b. When both glycines were mutated the effect was additive, shifting the pKa by 1.2 pH units to 7.8 +/- 0.04 for the double mutant: G157A+G148A. At this pKa, the double mutant would remain completely closed under physiological conditions. In contrast, when the glycine at G148 was replaced by a proline, the pKa was shifted in the opposite direction from 6.6 +/- 0.01 (wild-type) to 5.7 +/- 0.01 for G148P. Although conserved glycines at G148 and G157 made it significantly easier to open the channel, they were not an absolute requirement for pH gating in Kir1.1. In addition, none of the glycine mutants produced more than small changes in either the cell-attached or excised single-channel kinetics which, in this channel, argues against changes in the selectivity filter. The putative pH sensor at K61-Kir1.1b, (equivalent to K80-Kir1.1a) was also examined. Mutation of this lysine to an untitratable methionine did not abolish pH gating, but shifted the pKa into an acid range from 6.6 +/- 0.01 to 5.4 +/- 0.04, similar to pH gating in Kir2.1. Hence K61-Kir1.1b cannot function as the exclusive pH sensor for the channel, although it may act as one of multiple pH sensors, or as a link between a cytoplasmic sensor and the channel gate. K61-Kir1.1b also interacted differently with the two glycine mutations. Gating of the double mutant: K61M+G148A was indistinguishable from K61M alone, whereas gating of K61M+G157A was midway between the alkaline pKa of G157A and the acid pKa of K61M. Finally, closure of ROMK, G148A, G157A, and K61M all required the same L160-Kir1.1b residue at the cytoplasmic end of the inner transmembrane helix. Hence in wild-type and mutant channels, closure occurs by steric occlusion of the permeation path by four leucine side chains (L160-Kir1.1b) at the helix bundle crossing. This is facilitated by the conserved glycines on TM2, but pH gating in Kir1.1 does not absolutely require glycine hinges in this region.     

Identification of a heteromeric interaction that influences the rectification,gating, and pH sensitivity of Kir4.1/Kir5.1 potassium channels

Heteromultimerization between different potassium channel subunits can generate channels with novel functional properties and thus contributes to the rich functional diversity of this gene family. The inwardly rectifying potassium channel subunit Kir5.1 exhibits highly selective heteromultimerization with Kir4.1 to generate heteromeric Kir4.1/Kir5.1 channels with unique rectification and kinetic properties. These novel channels are also inhibited by intracellular pH within the physiological range and are thought to play a key role in linking K+ and H+ homeostasis by the kidney. However, the mechanisms that control heteromeric K+ channel assembly and the structural elements that generate their unique functional properties are poorly understood. In this study we identify residues at an intersubunit interface between the cytoplasmic domains of Kir5.1 and Kir4.1 that influence the novel rectification and gating properties of heteromeric Kir4.1/Kir5.1 channels and that also contribute to their pH sensitivity. Furthermore, this interaction presents a structural mechanism for the functional coupling of these properties and explains how specific heteromeric interactions can contribute to the novel functional properties observed in heteromeric Kir channels. The highly conserved nature of this structural association between Kir subunits also has implications for understanding the general mechanisms of Kir channel gating and their regulation by intracellular pH.     

Inwardly rectifying potassium channels (Kir) in central nervous system glia: a special role for Kir4.1 in glial functions

Glia in the central nervous system (CNS) express diverse inward rectifying potassium channels (Kir). The major function of Kir is in establishing the high potassium (K+) selectivity of the glial cell membrane and strongly negative resting membrane potential (RMP), which are characteristic physiological properties of glia. The classical property of Kir is that K+ flows inwards when the RMP is negative to the equilibrium potential for K+ (E(K)), but at more positive potentials outward currents are inhibited. This provides the driving force for glial uptake of K+ released during neuronal activity, by the processes of "K+ spatial buffering" and "K+ siphoning", considered a key function of astrocytes, the main glial cell type in the CNS. Glia express multiple Kir channel subtypes, which are likely to have distinct functional roles related to their differences in conductance, and sensitivity to intracellular and extracellular factors, including pH, ATP, G-proteins, neurotransmitters and hormones. A feature of CNS glia is their specific expression of the Kir4.1 subtype, which is a major K+ conductance in glial cell membranes and has a key role in setting the glial RMP. It is proposed that Kir4.1 have a primary function in K+ regulation, both as homomeric channels and as heteromeric channels by co-assembly with Kir5.1 and probably Kir2.0 subtypes. Significantly, Kir4.1 are also expressed by oligodendrocytes, the myelin-forming cells of the CNS, and the genetic ablation of Kir4.1 results in severe hypomyelination. Hence, Kir, and in particular Kir4.1, are key regulators of glial functions, which in turn determine neuronal excitability and axonal conduction.     

Control of pH and PIP2 Gating in Heteromeric Kir4.1/Kir5.1 Channels by H-Bonding at the Helix-Bundle Crossing

Inhibition by intracellular H⁺ (pH gating) and activation by phosphoinositides such as PIP₂ (PIP₂ gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP₂ gating and pH gating are controlled by an intrasubunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP₂- and pH-gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP₂ gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP₂ affinities. This indicates that Kir channel PIP₂ affinity has little impact on either the PIP₂- or pH-gating kinetics.