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.     

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg²⁺. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K⁺-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K⁺-flux is convergent with the gating of PIP₂ because, at a saturating concentration, PIP₂ greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K⁺-flux gating.     

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 CO(2) 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 CO(2). 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 approximately 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% CO(2). Indeed, the Kir4.1-Kir5.1 currents were enhanced with 3% CO(2) and suppressed with 8% CO(2) 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 CO(2) 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.     

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.     

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.     

Kir4.1 K+ channels are regulated by external cations

The inwardly rectifying potassium channel (Kir), Kir4.1 mediates spatial K⁺-buffering in the CNS. In this process the channel is potentially exposed to a large range of extracellular K⁺ concentrations ([K⁺]_o). We found that Kir4.1 is regulated by K⁺_o. Increased [K⁺]_o leads to a slow (mins) increase in the whole-cell currents of Xenopus oocytes expressing Kir4.1. Conversely, removing K⁺ from the bath solution results in a slow decrease of the currents. This regulation is not coupled to the pH_i-sensitive gate of the channel, nor does it require the presence of K67, a residue necessary for K⁺_o-dependent regulation of Kir1.1. The voltage-dependent blockers Cs⁺ and Ba²⁺ substitute for K⁺ and prevent deactivation of the channel in the absence of K⁺_o. Cs⁺ blocks and regulates the channel with similar affinity, consistent with the regulatory sites being in the selectivity-filter of the channel. Although both Rb⁺ and NH₄⁺ permeate Kir4.1, only Rb⁺ is able to regulate the channel. We conclude that Kir4.1 is regulated by ions interacting with specific sites in the selectivity filter. Using a kinetic model of the permeation process we show the plausibility of the channel’s sensing the extracellular ionic environment through changes in the selectivity occupancy pattern, and that it is feasible for an ion with the selectivity properties of NH₄⁺ to permeate the channel without inducing these changes.     

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.     

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.     

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K⁺ channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the “helix-bundle crossing”. However, in the inwardly rectifying (Kir) potassium channel family, the role of this “hinge” residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper “hinge” residues are in close contact with the base of the pore α-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the “lower” gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue.     

Differential expression and distribution of Kir5.1 and Kir4.1 inwardly rectifying K+ channels in retina

Kir5.1 is an inwardly rectifying K⁺ channel subunit whose functional role has not been fully elucidated. Expression and distribution of Kir5.1 in retina were examined with a specific polyclonal antibody. Kir5.1 immunoreactivity was detected in glial Müller cells and in some retinal neurons. In the Kir5.1-positive neurons the expression of glutamic acid decarboxylase (GAD₆₅) was detected, suggesting that they may be GABAergic-amacrine cells. In Müller cells, spots of Kir5.1 immunoreactivity distributed diffusely at the cell body and in the distal portions, where Kir4.1 immunoreactivity largely overlapped. In addition, Kir4.1 immunoreactivity without Kir5.1 was strongly concentrated at the endfoot of Müller cells facing the vitreous surface or in the processes surrounding vessels. The immunoprecipitant obtained from retina with anti-Kir4.1 antibody contained Kir5.1. These results suggest that heterotetrameric Kir4.1/Kir5.1 channels may exist in the cell body and distal portion of Müller cells, whereas homomeric Kir4.1 channels are clustered in the endfeet and surrounding vessels. It is possible that homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels play different functional roles in the K⁺-buffering action of Müller cells. inwardly rectifying potassium channel; heteromerization; glial Müller cells; amacrine cells; potassium siphoning     

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.     

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.     

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.     

Kir5.1 underlies long-lived subconductance levels in heteromeric Kir4.1/Kir5.1 channels from Xenopus tropicalis

The inwardly-rectifying potassium channel subunit Kir5.1 selectively co-assembles with members of the Kir4.0 subfamily to form novel pH-sensitive heteromeric channels with unique single channel properties. In this study, we have cloned orthologs of Kir4.1 and Kir5.1 from the genome of the amphibian, Xenopus tropicalis (Xt). Heteromeric XtKir4.1/XtKir5.1 channels exhibit similar macroscopic current properties to rat Kir4.1/Kir5.1 with a faster time-dependent rate of activation. However, single channel analysis of heteromeric XtKir4.1/XtKir5.1 channels reveals that they have markedly different long-lived, multi-level subconductance states. Furthermore, we demonstrate that the XtKir5.1 subunit is responsible for these prominent subconductance levels. These results are consistent with a model in which the slow transitions between sublevel states represent the movement of individual subunits. These novel channels now provide an excellent model system to determine the structural basis of subconductance levels and contribution of heteromeric pore architecture to this process.     

Voltage-dependent inhibition of outward Kir2.1 currents by extracellular spermine

Outward currents through inward rectifier Kir2.1 channels play crucial roles in controlling the electrical properties of excitable cells. Extracellular monovalent and divalent cations have been shown to reduce outward K⁺ conductance. In the present study, we examined whether spermine, with four positive charges, also inhibits outward Kir2.1 currents. We found that extracellular spermine inhibits steady-state outward Kir2.1 currents, an effect that increases as the voltage becomes more depolarizing, similar to that observed for intracellular spermine. However, several lines of evidence suggest that extracellular spermine does not inhibit outward currents by entering the cytoplasmic pore. Site-directed mutagenesis studies support that extracellular spermine directly interacts with the extracellular domain. In addition, we found that the voltage-dependent decay of outward Kir2.1 currents was necessary for inhibition by extracellular spermine. Further, a region at or near the selectivity filter and the cytoplasmic pore are involved in the voltage-dependent decay and thus in the inhibition of outward currents by extracellular spermine. Taken together, the data suggest that extracellular spermine bound to the mouth of the extracellular pore may induce an allosteric effect on voltage-dependent decay of outward currents, a process in which a region in the vicinity of the selectivity filter and cytoplasmic pore are involved. This study reveals that the extracellular pore domain, the selectivity filter and the cytoplasmic pore are in communication and this coupling is involved in modulating K⁺ conduction in the Kir2.1 channel.     

Mislocalization of K channels causes the renal salt wasting in EAST/SeSAME syndrome

The Kir4.1/Kir5.1 channel mediates basolateral K⁺ recycling in renal distal tubules; this process is critical for Na⁺ reabsorption at the tubules. Mutations in Kir4.1 are associated with EAST/SeSAME syndrome, a genetic disorder characterized by renal salt wasting. In this study, we found that MAGI-1 anchors Kir4.1 channels (Kir4.1 homomer and Kir4.1/Kir5.1 heteromer) and contributes to basolateral K⁺ recycling. The Kir4.1 A167V mutation associated with EAST/SeSAME syndrome caused mistrafficking of the mutant channels and inhibited their expression on the basolateral surface of tubular cells. These findings suggest mislocalization of the Kir4.1 channels contributes to renal salt wasting.     

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.     

Expression and Coexpression of CO<Subscript>2</Subscript>-sensitive Kir Channels in Brainstem Neurons of Rats

Several inward rectifier K⁺ (Kir) channels are inhibited by hypercapnic acidosis and may be involved in CO₂ central chemoreception. Among them are Kir1.1, Kir2.3, and Kir4.1. The Kir4.1 is expressed predominantly in the brainstem. Although its CO₂ sensitivity is low, coexpression of Kir4.1 with Kir5.1 in Xenopus oocytes greatly enhances the CO₂/pH sensitivities of the heteromeric channels. If these Kir channels play a part in the central CO₂ chemosensitivity, they should be expressed in neurons of brainstem cardio-respiratory nuclei. To test this hypothesis, we performed in-situ hybridization experiments in which the expression of Kir1.1, Kir2.3, Kir4.1 and Kir5.1, and coexpression of Kir4.1 and Kir5.1 were studied in brainstem neurons using non-radioactive riboprobes. We found that mRNAs of these Kir channels were present in several brainstem nuclei, especially those involved in cardio-respiratory controls. Strong labeling was observed in the locus coeruleus, ventralateral medulla, parabrachial-Kölliker-Fuse nuclei, solitary tract nucleus, and area postrema. Strong expression was also seen in several cranial motor nuclei, including the nucleus of ambiguus, hypoglossal nucleus, facial nucleus and dorsal vagus motor nucleus. In general, the expression of Kir5.1 and Kir4.1 was much more prominent than that of Kir1.1 and Kir2.3 in all the nuclei. Evidence for the coexpression of Kir4.1 and Kir5.1 was found in a good number of neurons in these nuclei. The expression and coexpression of these CO₂/pH-sensitive Kir channels suggest that they are likely to contribute to CO₂ chemosensitivity of the brainstem neurons.     

Inwardly Rectifying K<Superscript>+</Superscript> Currents in Cultured Oligodendrocytes from Rat Optic Nerve are Insensitive to pH

Inwardly rectifying K⁺ (Kir) channel expression signals at an advanced stage of maturation during oligodendroglial differentiation. Knocking down their expression halts the generation of myelin and produces severe abnormalities in the central nervous system. Kir4.1 is the main subunit involved in the tetrameric structure of Kir channels in glial cells; however, the precise composition of Kir channels expressed in oligodendrocytes (OLs) remains partially unknown, as participation of other subunits has been proposed. Kir channels are sensitive to H⁺; thus, intracellular acidification produces Kir current inhibition. Since Kir subunits have differential sensitivity to H⁺, we studied the effect of intracellular acidification on Kir currents expressed in cultured OLs derived from optic nerves of 12-day-old rats. Unexpectedly, Kir currents in OLs (2–4 DIV) did not change within the pH range of 8.0–5.0, as observed when using standard whole-cell voltage-clamp recording or when preserving cytoplasmic components with the perforated patch-clamp technique. In contrast, low pH inhibited astrocyte Kir currents, which was consistent with the involvement of the Kir4.1 subunit. The H⁺-insensitivity expressed in OL Kir channels was not intrinsic because Kir cloning showed no difference in the sequence reported for the Kir4.1, Kir2.1, or Kir5.1 subunits. Moreover, when Kir channels were heterologously expressed in Xenopus oocytes they behaved as expected in their general properties and sensitivity to H⁺. It is therefore concluded that Kir channel H⁺-sensitivity in OLs is modulated through an extrinsic mechanism, probably by association with a modulatory component or by posttranslational modifications.     

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti

Inward-rectifying K⁺ (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K⁺ currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na⁺. Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl⁺ > K⁺ > Rb⁺ > NH₄⁺) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K⁺.