Functional consequences of Kir2.1/Kir2.2 subunit heteromerization

Kir2 subunits form channels that underlie classical strongly inwardly rectifying potassium currents. While homomeric Kir2 channels display a number of distinct and physiologically important properties, the functional properties of heteromeric Kir2 assemblies, as well as the stoichiometries and the arrangements of Kir2 subunits in native channels, remain largely unknown. Therefore, we have implemented a concatemeric approach, whereby all four cloned Kir2 subunits were linked in tandem, in order to study the effects of Kir2.1 and Kir2.2 heteromerization on properties of the resulting channels. Kir2.2 subunits contributed stronger to single-channel conductance than Kir2.1 subunits, and channels containing two or more Kir2.2 subunits displayed conductances indistinguishable from that of a Kir2.2 homomeric channel. In contrast, single-channel kinetics was a more discriminating property. The open times were significantly shorter in Kir2.2 channels compared with Kir2.1 channels and decreased nearly proportionally to the number of Kir2.2 subunits in the heteromeric channel. Similarly, the sensitivity to block by barium also depended on the proportions of Kir2.1 to Kir2.2 subunits. Overall, the results showed that Kir2.1 and Kir2.2 subunits exert neither a dominant nor an anomalous effect on any of the properties of heteromeric channels. The data highlight opportunities and challenges of using differential properties of Kir2 channels in deciphering the subunit composition of native inwardly rectifying potassium currents.     

Chronic lentiviral expression of inwardly rectifying K+ channels (Kir2.1) reduces neuronal activity and downregulates voltage-gated potassium currents in hippocampus

Strongly inwardly rectifying K+ (Kir2) channels are endogenously expressed in rat brains and have recently been used as a tool to reduce the neuronal activity. But little is known about the role of Kir2 channels and the chronic effect of the reduced activity on the intrinsic excitability of neurons. Here we constructed a lentiviral vector that coexpressed Kir2.1 and GFP (LvKir2.1) and infected the vector to the hippocampal slice cultures. The LvKir2.1-infected CA1 neurons showed clear inwardly rectifying K+ currents for more than 15 days. The resting membrane potential was more negative by approximately 10 mV than those uninfected or infected with the lentiviral vector expressing GFP alone. The infection of LvKir2.1 reduced the voltage change in response to current injections and the amplitude of mEPSPs with a shunting effect. The LvKir2.1 infection significantly reduced the firings evoked by depolarizing currents in the CA1 neurons. The reduction of the firing was attributed to the hyperpolarized potential rather than to the shunting effect. These reductions were limited to modest current injections, suggesting that the overexpressed Kir2.1 plays the role of a noise-filter. Moreover, the chronic overexpression of Kir2.1 downregulated the expression of the delayed rectifier potassium current in a homeostatic manner, indicating a usefulness of this viral vector to study the activity-dependent neuronal development.     

内流性钾通道2.1蛋白在大鼠脊髓和小脑的分布

目的：内流性钾通道(Kir)2．1蛋白在大鼠脊髓和小脑的分布。方法：免疫荧光组织化学和免疫荧光双标记。结果：在脊髓灰质前角的运动神经元和白质的星形胶质细胞呈现Kir2．1免疫反应活性强阳性。少数少突胶质细胞呈现Kir2．1和GS共存。在小脑蒲肯野氏细胞及纤维呈Kir2．1免疫活性强阳性。双标记显示近蒲肯野氏细胞旁的少突胶质细胞的Kir2．1蛋白和GS蛋白共存。结论：脊髓和小脑灰质和白质中的Kir2．1表达不一，这种分布可能与保持细胞外的K 平衡、促进K 的内流维持细胞兴奋性有重要关系。     

Lack of the Kir4.1 channel subunit abolishes K+ buffering properties of astrocytes in the ventral respiratory group: impact on extracellular K+ regulation

Ongoing rhythmic neuronal activity in the ventral respiratory group (VRG) of the brain stem results in periodic changes of extracellular K+. To estimate the involvement of the weakly inwardly rectifying K+ channel Kir4.1 (KCNJ10) in extracellular K+ clearance, we examined its functional expression in astrocytes of the respiratory network. Kir4.1 was expressed in astroglial cells of the VRG, predominantly in fine astrocytic processes surrounding capillaries and in close proximity to VRG neurons. Kir4.1 expression was up-regulated during early postnatal development. The physiological role of astrocytic Kir4.1 was studied using mice with a null mutation in the Kir4.1 channel gene that were interbred with transgenic mice expressing the enhanced green fluorescent protein in their astrocytes. The membrane potential was depolarized in astrocytes of Kir4.1-/- mice, and Ba2+-sensitive inward K+ currents were diminished. Brain slices from Kir4.1-/- mice, containing the pre-B?tzinger complex, which generates a respiratory rhythm, did not show any obvious differences in rhythmic bursting activity compared with wild-type controls, indicating that the lack of Kir4.1 channels alone does not impair respiratory network activity. Extracellular K+ measurements revealed that Kir4.1 channels contribute to extracellular K+ regulation. Kir4.1 channels reduce baseline K+ levels, and they compensate for the K+ undershoot. Our data indicate that Kir4.1 channels 1) are expressed in perineuronal processes of astrocytes, 2) constitute the major part of the astrocytic Kir conductance, and 3) contribute to regulation of extracellular K+ in the respiratory network.     

Regional analysis of whole cell currents from hair cells of the turtle posterior crista

The turtle posterior crista is made up of two hemicristae, each consisting of a central zone containing type I and type II hair cells and a surrounding peripheral zone containing only type II hair cells and extending from the planum semilunatum to the nonsensory torus. Afferents from various regions of a hemicrista differ in their discharge properties. To see if afferent diversity is related to the basolateral currents of the hair cells innervated, we selectively harvested type I and II hair cells from the central zone and type II hair cells from two parts of the peripheral zone, one near the planum and the other near the torus. Voltage-dependent currents were studied with the whole cell, ruptured-patch method and characterized in voltage-clamp mode. We found regional differences in both outwardly and inwardly rectifying voltage-sensitive currents. As in birds and mammals, type I hair cells have a distinctive outwardly rectifying current (I(K,L)), which begins activating at more hyperpolarized voltages than do the outward currents of type II hair cells. Activation of I(K,L) is slow and sigmoidal. Maximal outward conductances are large. Outward currents in type II cells vary in their activation kinetics. Cells with fast kinetics are associated with small conductances and with partial inactivation during 200-ms depolarizing voltage steps. Almost all type II cells in the peripheral zone and many in the central zone have fast kinetics. Some type II cells in the central zone have large outward currents with slow kinetics and little inactivation. Although these currents resemble I(K,L), they can be distinguished from the latter both electrophysiologically and pharmacologically. There are two varieties of inwardly rectifying currents in type II hair cells: activation of I(K1) is rapid and monoexponential, whereas that of I(h) is slow and sigmoidal. Many type II cells either have both inward currents or only have I(K1); very few cells only have I(h). Inward currents are less conspicuous in type I cells. Type II cells near the torus have smaller outwardly rectifying currents and larger inwardly rectifying currents than those near the planum, but the differences are too small to account for variations in discharge properties of bouton afferents innervating the two regions of the peripheral zone. The large outward conductances seen in central cells, by lowering impedances, may contribute to the low rotational gains of some central-zone afferents.     

Kir2.1通道在牛视网膜胶质细胞中的分布和表达-mRNA和蛋白水平的研究

本研究目的为在 m RNA和蛋白质水平探讨 Kir2 .1通道在牛视网膜胶质细胞中的分布和表达。从神经视网膜和视网膜色素上皮分离 m RNA和蛋白质 ,以 RT-PCR,Northern blot,Western blot和免疫荧光组织化学法检测 Kir2 .1m RNA和 Kir2 .1蛋白的表达。结果表明 ,RT-PCR扩增的 Kir2 .1c DNA产物在神经视网膜中强表达 ,在视网膜色素上皮中弱表达 ;Northern斑点杂交的 Kir2 .1c DNA仅在神经视网膜表达 ;Western斑点杂交检测 Kir2 .1蛋白单带在神经视网膜中表达 ;免疫荧光组织化学显示 Kir2 .1蛋白在 Muller细胞中分布 ,并且主要分布在 Muller细胞与神经元相接触的细胞膜区域 ,与 Muller细胞的特异性标记蛋白质抗谷氨酸合成酶抗体双重标记显示其分布特性重叠。在视网膜色素上皮未检测到 Kir2 .1蛋白的免疫活性。上述结果表明 ,Kir2 .1分布在视网膜胶质细胞的 Muller细胞 ,这种分布特性可能与完成 Muller细胞功能 -保持细胞外 K+ 的平衡、促进 K+的内流有重要关系     

Characterization of acid-sensing ion channels in medium spiny neurons of mouse striatum

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases. They are highly expressed in the striatum, where medium spiny neurons (MSNs) are a major population. Given that the properties of ASICs in MSNs are unknown, in this study, we characterized ASICs in MSNs of the mouse striatum. A rapid drop in extracellular pH induced transient inward currents in all MSNs. The pH value for half-maximal activation was 6.25, close to that obtained in homomeric ASIC1a channels. Based on psalmotoxin 1 and zinc sensitivity, ASIC1a (70.5% of neurons) and heteromeric ASIC1a-2 channels (29.5% of neurons) appeared responsible for the acid-induced currents in MSNs. ASIC currents were diminished in MSNs from ASIC1, but not ASIC2, null mice. Furthermore, a drop in pH induced calcium influx by activating homomeric ASIC1a channels. Activation of ASICs increased the membrane excitability of MSNs and lowering extracellular Ca²⁺ potentiated ASIC currents. Our data suggest that the homomeric ASIC1a channel represents a majority of the ASIC isoform in MSNs. The potential function of ASICs in the striatum requires further investigation.     

An outwardly rectifying anion channel in human leukaemic K562 cells

In this study, an outwardly rectifying anion channel was characterized in the cell line K562 obtained from a chronic human leukaemia. Ion channel activity was recorded in the cell-detached (inside-out) configuration with standard patch-clamp technology. In most of the K562 cells studied, the channel exhibited low spontaneous activity, an outwardly rectifying current/voltage relationship and single-channel conductances of 19 pS and 40 pS for inwards and outwards currents respectively. The channel had a low permeability for gluconate with a relative permeability P(gluconate)/ P(Cl) of 0.14 and was blocked by glibenclamide (50 micro M) or diphenylamine-2-carboxylate (DPC, 1 mM) added to the cytoplasmic side of the patch. These results are characteristic of the outwardly rectifying Cl channel (ORCC) found in other types of cells.     

Outwardly rectifying chloride current in rabbit osteoclasts is activated by hyposmotic stimulation.

1. We characterized chloride currents in freshly isolated rabbit osteoclasts using whole-cell and single channel patch-clamp recording configurations. Depolarization activated an outwardly rectifying current in 40-50% of cells, distinct from the inwardly rectifying K+ current we have previously reported in osteoclasts. 2. The outwardly rectifying current persisted under conditions where all K+ currents were blocked. Furthermore, the outward current was reversibly inhibited by Cl- transport blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS); 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS); 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS); and niflumic acid. The blocked current had a reversal potential close to the predicted chloride equilibrium potential and was dependent on the chloride concentration gradient. 3. In those osteoclasts in which outwardly rectifying current was not initially apparent, exposure to hyposmotic extracellular solution resulted in its reversible activation. The induced current was due to Cl-, based on its reversal close to the chloride equilibrium potential and sensitivity to blockade by Cl- channel inhibitors. The hyposmotically induced current could be activated in Ca(2+)-free solutions containing 0.2 mM EGTA. 4. When studied in the current-clamp configuration, hyposmotic stimulation caused depolarization from -76 +/- 5 to -5 +/- 6 mV (mean +/- S.D., n = 7). 5. Unitary Cl- currents were recorded in the cell-attached patch configuration at positive potentials. Single channels had a slope conductance of 19 +/- 3 pS (n = 5). Reduction of the external [Cl-] shifted the current-voltage relationship in the positive direction, supporting the conclusion that these were Cl- currents. Like the whole-cell currents, single channel Cl- currents were activated by exposure of cells to hyposmotic bathing solution. 6. We conclude that rabbit osteoclasts express an outwardly rectifying Cl- current that can be activated by osmotic stress. Cl- channels may play a role in cell volume regulation and may also provide conductive pathways for dissipating the potential difference that arises from electrogenic proton transport during bone resorption.     

Activity-dependent regulation of inwardly rectifying potassium currents in non-myelinating Schwann cells in mice.

1. Voltage-gated potassium currents were recorded from freshly dissociated non-myelinating Schwann cells of sural and sympathetic nerves from 1- to 12-week-old mice using the whole-cell or a single channel variation of the patch-clamp technique. 2. All sural cells from 2-week-old mice showed inwardly rectifying potassium (Kir+) currents in whole-cell recordings. Kir+ currents were virtually undetectable in sural cells from mice more than 6 weeks old, which also showed depolarization of the resting membrane potential. On the other hand, the magnitude of Kir+ currents increased in cervical sympathetic trunk (CST) cells in parallel with an increase of cell capacitance 1-6 weeks after birth. The density of Kir+ currents in CST cells increased 1-4 weeks after birth and then stayed constant for up to 12 weeks. 3. The unitary conductance of a single Kir+ channel in CST cells was 30 pS 2-12 weeks after birth; this was recorded in a cell-attached configuration with 154 mM K+ in the pipette. The steady-state open channel probability of single Kir+ channels in CST cells decreased with membrane hyperpolarization, but was not markedly changed 2-12 weeks after birth. 4. Conduction block of CST for 5 days induced by local application of tetrodotoxin (TTX) resulted in a significant decrease in both the magnitude and the density of Kir+ currents in whole-cell recordings in CST cells rostral to the sites of TTX block. Similar changes of Kir+ currents in whole-cell recordings were observed in cells in the inferior postganglionic branch of a superior cervical ganglion after 5 days of TTX block of CST. 5. These results suggest that neuronal activity regulates the expression of functional Kir+ channels in non-myelinating Schwann cells in adult nerves. The activity-dependent regulation of the expression of glial potassium channels could play an important role in the regulation of the potassium microenvironment around active axons to maintain impulse conduction in unmyelinated fibres.     

Differential distribution of Kir4.1 in spinal cord astrocytes suggests regional differences in K+ homeostasis

Neuronal activity in the spinal cord results in extracellular potassium accumulation that is significantly higher in the dorsal horn than in the ventral horn. This is suggestive of differences in K(+) clearance, widely thought to involve diffusional K(+) uptake by astrocytes. We previously identified the inward rectifying K(+) channel Kir4.1 as the major K(+) conductance in spinal cord astrocytes in situ and hence hypothesized that different expression levels of Kir4.1 may account for the observed differences in potassium dynamics in spinal cord. Our results with immunohistochemical staining demonstrated highest Kir4.1 channel expression in the ventral horn and very low levels of Kir4.1 in the apex of the dorsal horn. Western blots from tissue of these two regions similarly confirmed much lower levels of Kir4.1 in the apex of the dorsal horn. Whole cell patch-clamp recordings from astrocytes in rat spinal cord slices also showed a difference in inwardly rectifying currents in these two regions. However, no statistical difference in either fast-inactivating (Ka) or delayed rectifying potassium currents (Kd) was observed, suggesting these differences were specific to Kir currents. Importantly, when astrocytes in each region were challenged with high [K(+)](o), astrocytes from the dorsal horn showed significantly smaller (60%) K(+) uptake currents than astrocytes from the ventral horn. Taken together, these data support the conclusion that regional differences in astrocytic expression of Kir4.1 channels result in marked changes in potassium clearance rates in these two regions of the spinal cord.     

ATP-sensitive potassium channels: structures, functions, and pathophysiology

ATP-sensitive potassium channels (KATP channels) play important roles in various tissues by coupling cell metabolic status to electrical activity. Recently, molecular biological and electrophysiological techniques have revealed the molecular basis of the KATP channels to be a complex of the Kir6.0 subunit, a member of the inwardly rectifying K+ channel subfamily Kir6.0, and the sulfonylurea receptor (SUR) subunit, a member of ATP-binding cassette (ABC) superfamily; the functional diversity of the various KATP channels is being determined by a combination of the Kir6.0 subunit (Kir6.1 or Kir6.2) and the SUR subunit (SUR1 or SUR2) comprising it. Recent studies of the KATP channels have suggested mechanisms of KATP channel regulation and pathophysiology and also a new model in which ABC proteins regulate the functional expression of ion channels.     

Effect of Extracellular Cations on the Inward Rectifying K+ Channels Kir2.1 and Kir3.1/Kir3.4

The effects of Ba2+, Mg2+, Ca2+ and Na+ as blocking ions were investigated in 90 and 10 mM extracellular K+ solutions on the cloned inward rectifying K+ channel Kir2.1 expressed in Xenopus oocytes. Some data were also obtained using another inward rectifying K+ channel Kir3.1/Kir3.4. The addition of Ba2+ caused a concentration-, voltage- and time-dependent block of both channels. Decreasing the extracellular K+ concentration augmented the block. The data suggest that Ba2+ blocks the channels by binding to a site within the channel pore and that the electrical binding distance, delta, of the site is significantly different for Kir2.1 and Kir3. 1/Kir3.4 (0.38 and 0.22, respectively). Mg2+ and Ca2+ caused an instantaneous concentration- and voltage-dependent block of both channels. With Kir2.1, decreasing the K+ concentration augmented the block. The voltage dependence of the block was less than that of Ba2+ ([delta], 0.1), indicating a more superficial binding site for these ions within the channel pore. The affinity of the channels for Mg2+ and Ca2+ was 1000-fold lower than that for Ba2+. Addition of Na+ resulted in a concentration-, voltage- and time-dependent block of Kir2.1, similar to that observed with Ba2+. The competition between the blocking cations (for Kir2.1: Ba2+, Mg2+, Ca2+; for Kir3. 1/Kir3.4: Ba2+) and extracellular K+ suggests that the binding sites for the blocking cations may be sites to which K+ binds as part of the normal passage of K+ through the channels. It is possible that under normal physiological conditions naturally occurring extracellular cations may partly block the two inward rectifying K+ channels.     

Multiple neuropeptide Y receptors regulate K+ and Ca2+ channels in acutely isolated neurons from the rat arcuate nucleus

We examined the effects of neuropeptide Y (NPY) and related peptides on Ca2+ and K+ currents in acutely isolated neurons from the arcuate nucleus of the rat. NPY analogues that activated all of the known NPY receptors (Y1-Y5), produced voltage-dependent inhibition of Ca2+ currents and activation of inwardly rectifying K+ currents in arcuate neurons. Both of these effects could occur simultaneously in the same cells. In some cells, activation of Y4 NPY receptors also caused oscillations in [Ca2+]i. NPY hyperpolarized arcuate neurons through the activation of a K+ conductance and increased the spike threshold. Molecular biological studies indicated that arcuate neurons possessed all of the previously cloned NPY receptor types (Y1, Y2, Y4, and Y5). Thus activation of multiple types NPY receptors on arcuate neurons can regulate both Ca2+ and K+ conductances leading to a reduction in neuronal excitability and a suppression of neurotransmitter release.     

Electrophysiological and molecular characterization of the inward rectifier in juxtaglomerular cells from rat kidney

Renin, the key element of the renin–angiotensin–aldosterone system, is mainly produced by and stored in the juxtaglomerular cells in the kidney. These cells are situated in the media of the afferent arteriole close to the vessel pole and can transform into smooth muscle cells and vice versa. In this study, the electrophysiological properties and the molecular identity of the K⁺ channels responsible for the resting membrane potential (∼−60 mV) of the juxtaglomerular cells were examined. In order to increase the number of juxtaglomerular cells, afferent arterioles from NaCl-depleted rats were used, and > 90% of the afferent arterioles were renin positive at the distal end of the arteriole. Whole-cell and cell-attached single-channel patch-clamp experiments showed that juxtaglomerular cells are endowed with a strongly inwardly rectifying K⁺ channel (Kir). The channel was highly sensitive to inhibition by Ba²⁺ (inhibition constant 37 μ m at 0 mV), but relatively insensitive to Cs⁺ and, with 142 m m K⁺ in the pipette, had a single-channel conductance of 31.5 pS. Immunocytochemical studies showed the presence of Kir2.1 but no signal for Kir2.2 in the media of the afferent arteriole. In PCR analyses using isolated juxtaglomerular cells, the mRNA for Kir2.1 and Kir2.2 was detected. Collectively, the results show that Kir2.1 is the dominant component of the channel. The current carried by these channels plays a decisive role in setting the membrane potential of juxtaglomerular cells.     

Phasic and tonic attenuation of EPSPs by inward rectifier K+ channels in rat hippocampal pyramidal cells

We made whole-cell recordings from CA1 pyramidal cells of hippocampal slices in combination with brief dendritic glutamate pulses to study the role of constitutive inwardly rectifying K⁺ channels (IRK, Kir2.0) and G-protein-activated inwardly rectifying K⁺ channels (GIRK, Kir3.0) in the processing of excitatory inputs. Phasic activation of GIRK channels by baclofen (20 μ m ) produced a reversible reduction of glutamate-evoked postsynaptic potentials (GPSPs), our equivalent of EPSPs, by about one-third. Conversely, tertiapin (30 n m ), a selective inhibitor of GIRK channels, and Ba²⁺ (200 μ m ), a non-selective blocker of inwardly rectifying K⁺ channels, enhanced GPSPs and, in voltage-clamp experiments, reduced the underlying K⁺ conductances, indicating a functionally significant background GIRK conductance, in addition to constitutive IRK channel activity. When examined after suppression of endogenous adenosinergic inhibition, using either adenosine deaminase or the selective A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, tertiapin failed to influence either the GPSPs or the inwardly rectifying K⁺ conductance. Voltage-clamp recordings from acutely isolated CA1 pyramidal cells not exposed to ambient adenosine exhibited no response to tertiapin, whereas Ba²⁺ was still capable of reducing hyperpolarizing inward rectification. Our data indicate that in hippocampal pyramidal cells, two components of the inwardly rectifying K⁺ conductance can be identified, which together exert a tonic modulation of excitatory synaptic input: one arises from constitutive putative IRK channels, the other is mediated by the background activity of GIRK channels that results from the tonic activation of A1 receptors by ambient adenosine.     

Outward currents through the inwardly rectifying potassium channel of guinea-pig ventricular cells

Currents through the inwardly rectifying K channel were studied under whole-cell clamp of collagenase-treated single ventricular cells of guinea-pigs. The inwardly rectifying K channel was fully activated by hyperpolarizing the membrane from the equilibrium potential for K+ (EK) by 30-40 mV. Following depolarization above EK, a decaying outward current was elicited. Prolongation of the hyperpolarizing prepulse increased the amplitude of the decaying outward current, with a time course similar to the increase of the inward current during the prepulse. Time-dependent changes in both outward and inward currents could be fitted with a single exponential function and were attributed to deactivation and activation of the inwardly rectifying K channel. The instantaneous current-voltage relation was almost linear, indicating that the conductance of the channel is ohmic and that the rectification of the steady-state current was due to the kinetic properties of the inwardly rectifying K channel. The activation kinetics of the channel was measured at different concentrations of K+ in both the external and internal solutions. The time constant and the steady-state activation were not a function of the absolute membrane potential value, but were dependent on the driving force.