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.     

Stretch-induced alterations of human Kir2.1 channel currents

The inward rectifier potassium channel, Kir2.1, contributes to the I(K1) current in cardiac myocytes and is closely associated with atrial fibrillation. Strong evidences have shown that atrial dilatation or stretch may result in atrial fibrillation. However, the role of Kir2.1 channels in the stretch-mediated atrial fibrillation is not clear. In this study, we constructed the recombinant plasmid of KCNJ2 that encodes the Kir2.1 channel and expressed it in CHO-K1 cells. We recorded I(K1) currents using the whole-cell patch clamping technique. Our data showed that I(K1) currents were significantly larger under stretch in the hypotonic solution than under non-stretch in the iso-osmotic solution, and the activation kinetics of the Kir2.1 channel were changed markedly by stretch as well. Thus, atrial stretch in human heart might result in excessive I(K1) currents, which is likely to increase the resting membrane potential and decrease the effective refractory period, to initiate and/or maintain atrial fibrillation.     

Heterogeneity of IK1 in the mouse heart

Previous studies have shown that cardiac inward rectifier potassium current (I(K1)) channels are heteromers of distinct Kir2 subunits and suggested that species- and tissue-dependent expression of these subunits may underlie variability of I(K1). In this study, we investigated the contribution of the slowly activating Kir2.3 subunit and free intracellular polyamines (PAs) to variability of I(K1) in the mouse heart. The kinetics of activation was measured in Kir2 concatemeric tetramers with known subunit stoichiometry. Inclusion of only one Kir2.3 subunit to a Kir2.1 channel led to an approximate threefold slowing of activation kinetics, with greater slowing on subsequent additions of Kir2.3 subunits. Activation kinetics of I(K1) in both ventricles and both atria was found to correspond to fast-activating Kir2.1/Kir2.2 channels, suggesting no major contribution of Kir2.3 subunits. In contrast, I(K1) displayed significant variation in both the current density and inward rectification, suggesting involvement of intracellular PAs. The total levels of PAs were similar across the mouse heart. Measurements of the free intracellular PAs in isolated myocytes, using transgenically expressed Kir2.1 channels as PA sensors, revealed "microheterogeneity" of I(K1) rectification as well as lower levels of free PAs in atrial myocytes compared with ventricular cells. These findings provide a quantitative explanation for the regional heterogeneity of I(K1).     

Distribution of the muscarinic K+ channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart.

The functionally important effects on the heart of ACh released from vagal nerves are principally mediated by the muscarinic K+ channel. The aim of this study was to determine the abundance and cellular location of the muscarinic K+ channel subunits Kir3.1 and Kir3.4 in different regions of heart. Western blotting showed a very low abundance of Kir3.1 in rat ventricle, although Kir3.1 was undetectable in guinea pig and ferret ventricle. Although immunofluorescence on tissue sections showed no labeling of Kir3.1 in rat, guinea pig, and ferret ventricle and Kir3.4 in rat ventricle, immunofluorescence on single ventricular cells from rat showed labeling in t-tubules of both Kir3.1 and Kir3.4. Kir3.1 was abundant in the atrium of the three species, as shown by Western blotting and immunofluorescence, and Kir3.4 was abundant in the atrium of rat, as shown by immunofluorescence. Immunofluorescence showed Kir3.1 expression in SA node from the three species and Kir3.4 expression in the SA node from rat. The muscarinic K+ channel is activated by ACh via the m2 muscarinic receptor and, in atrium and SA node from ferret, Kir3.1 labeling was co-localized with m2 muscarinic receptor labeling throughout the outer cell membrane.     

A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation

The inward rectifier K(+) channel Kir2.1 mediates the potassium I(K1) current in the heart. It is encoded by KCNJ2 gene that has been linked to Andersen's syndrome. Recently, strong evidences showed that Kir2.1 channels were associated with mouse atrial fibrillation (AF), therefore we hypothesized that KCNJ2 was associated with familial AF. Thirty Chinese AF kindreds were evaluated for mutations in KCNJ2 gene. A valine-to-isoleucine mutation at position 93 (V93I) of Kir2.1 was found in all affected members in one kindred. This valine and its flanking sequence is highly conserved in Kir2.1 proteins among different species. Functional analysis of the V93I mutant demonstrated a gain-of-function consequence on the Kir2.1 current. This effect is opposed to the loss-of-function effect of previously reported mutations in Andersen's syndrome. Kir2.1 V93I mutation may play a role in initiating and/or maintaining AF by increasing the activity of the inward rectifier K(+) channel.     

Kir2.3 knock-down decreases IK1 current in neonatal rat cardiomyocytes

Inward rectifier potassium Kir2.x channels mediate cardiac inward rectifier potassium currents (I(K1)). As a subunit of Kir2.x, the physiological role of Kir2.3 in native cardiomyocytes has not been reported. This study shows that Kir2.3 knock-down remarkably down-regulates Kir2.3 expression (Kir2.3 protein was reduced to 19.91+/-3.24% on the 2nd or 3rd day) and I(K1) current densities (at -120 mV, control vs. knock-down: -5.03+/-0.24 pA/pF, n=5 vs. -1.16+/-0.19 pA/pF, n=7, P<0.001) in neonatal rat cardiomyocytes. The data suggest that Kir2.3 plays a potentially important role in I(K1) currents in neonatal rat cardiomyocytes.     

The Interaction of Caveolin 3 Protein with the Potassium Inward Rectifier Channel Kir2.1: PHYSIOLOGY AND PATHOLOGY RELATED TO LONG QT SYNDROME 9 (LQT9)*

Mutations in CAV3 cause LQT syndrome 9 (LQT9). A previously reported LQT9 patient had prominent U waves on ECG, a feature that has been correlated with Kir2.1 loss of function. Our objective was to determine whether caveolin 3 (Cav3) associates with Kir2.1 and whether LQT9-associated CAV3 mutations affect the biophysical properties of Kir2.1. Kir2.1 current (I_K1) density was measured using the whole-cell voltage clamp technique. WT-Cav3 did not affect I_K1. However, F97C-Cav3 and T78M-Cav3 decreased I_K1 density significantly by ∼60%, and P104L-Cav3 decreased I_K1 density significantly by ∼30% at −60 mV. Immunostained rat heart cryosections and HEK293 cells cotransfected with Kir2.1 and WT-Cav3 both demonstrated colocalization of Kir2.1 and WT-Cav3 by confocal imaging. Cav3 coimmunoprecipitated with Kir2.1 in human ventricular myocytes and in heterologous expression systems. Additionally, FRET efficiency was highly specific, with a molecular distance of 5.6 ± 0.4 nm, indicating close protein location. Colocalization experiments found that Cav3 and Kir2.1 accumulated in the Golgi compartment. On-cell Western blot analysis showed decreased Kir2.1 cell surface expression by 60% when expressed with F97C-Cav3 and by 20% when expressed with P104L-Cav3 compared with WT-Cav3. This is the first report of an association between Cav3 and Kir2.1. The Cav3 mutations F97C-Cav3, P104L-Cav3, and T78M-Cav3 decreased I_K1 density significantly. This effect was related to a reduced cell surface expression of Kir2.1. Kir2.1 loss of function is additive to the increase described previously in late I_Na, prolonging repolarization and leading to arrhythmia generation in Cav3-mediated LQT9.     

Heteromeric assembly of inward rectifier channel subunit Kir2.1 with Kir3.1 and with Kir3.4

Heteromultimerization of different pore-forming subunits is known to contribute to the diversity of inward rectifier K⁺ channels. We examined if the subunits belonging to different subfamilies Kir2 and Kir3 can co-assemble to form heteromultimers in heterologous expression systems. We observed co-immunoprecipitation of Kir2.1 and Kir3.1 as well as Kir2.1 and Kir3.4 in HEK293T cells. Furthermore, analyses of subcellular localization using confocal microscopy revealed that co-expression of Kir2.1 promoted the cell surface localization of Kir3.1 and Kir3.4 in HEK293T cells. In electrophysiological experiments, co-expression of Kir2.1 with Kir3.1 and/or Kir3.4 in Xenopus oocytes and HEK293T cells did not yield currents with distinguishable features. However, co-expression of a dominant-negative Kir2.1 with the wild-type Kir3.1/3.4 decreased the Kir3.1/3.4 current amplitude in Xenopus oocytes. The results indicate that Kir2.1 is capable of forming heteromultimeric channels with Kir3.1 and with Kir3.4.     

Cholesterol sensitivity and lipid raft targeting of Kir2.1 channels

This study investigates how changes in the level of cellular cholesterol affect inwardly rectifying K+ channels belonging to a family of strong rectifiers (Kir2). In an earlier study we showed that an increase in cellular cholesterol suppresses endogenous K+ current in vascular endothelial cells, presumably due to effects on underlying Kir2.1 channels. Here we show that, indeed, cholesterol increase strongly suppressed whole-cell Kir2.1 current when the channels were expressed in a null cell line. However, cholesterol level had no effect on the unitary conductance and only little effect on the open probability of the channels. Moreover, no cholesterol effect was observed either on the total level of Kir2.1 protein or on its surface expression. We suggest, therefore, that cholesterol modulates not the total number of Kir2.1 channels in the plasma membrane but rather the transition of the channels between active and silent states. Comparing the effects of cholesterol on members of the Kir2.x family shows that Kir2.1 and Kir2.2 have similar high sensitivity to cholesterol, Kir2.3 is much less sensitive, and Kir2.4 has an intermediate sensitivity. Finally, we show that Kir2.x channels partition virtually exclusively into Triton-insoluble membrane fractions indicating that the channels are targeted into cholesterol-rich lipid rafts.     

Characterisation of Kir2.0 proteins in the rat cerebellum and hippocampus by polyclonal antibodies

Rabbit polyclonal antibodies were raised to rat Kir2.0 (Kir2.1, Kir2.2 and Kir2.3) inwardly rectifying potassium ion channel proteins. The antibody specificities were confirmed by immunoprecipitation of [³⁵S]-methionine-labelled in vitro translated channel proteins and western blotting. Immunohistochemistry revealed a different patterns of expression of Kir2.0 subfamily proteins in the rat hind-brain (cerebellum and medulla) and fore-brain (hippocampus). Notably, only Kir2.2 protein was detected in the cerebellum and medulla, Kir2.1, Kir2.2 and Kir2.3 proteins were expressed in the hippocampus and immunostaining was not limited to neuronal cell types. Anti-Kir2.1 (fore-brain only) and anti-Kir2.2 (fore- and hind-brain) antibodies showed positive staining in macroglia, endothelia, ependyma and vascular smooth muscle cells. In contrast, anti-Kir2.3 (fore-brain only) immunostaining was limited to neurons, macroglia and vascular smooth muscle. These results indicate that specific regions within the rat fore- and hind-brain have differential distributions of inwardly rectifying potassium ion channel proteins. Accepted: 12 October 1999     

Losartan prevents stretch-induced electrical remodeling in cultured atrial neonatal myocytes

Atrial fibrillation (AF) is the most frequent arrhythmia found in clinical practice. In recent studies, a decrease in the development or recurrence of AF was found in hypertensive patients treated with angiotensin-converting enzyme inhibitors or angiotensin receptor-blocking agents. Hypertension is related to an increased wall tension in the atria, resulting in increased stretch of the individual myocyte, which is one of the major stimuli for the remodeling process. In the present study, we used a model of cultured atrial neonatal rat cardiomyocytes under conditions of stretch to provide insight into the mechanisms of the preventive effect of the angiotensin receptor-blocking agent losartan against AF on a molecular level. Stretch significantly increased protein-to-DNA ratio and atrial natriuretic factor mRNA expression, indicating hypertrophy. Expression of genes encoding for the inward rectifier K(+) current (I(K1)), Kir 2.1, and Kir 2.3, as well as the gene encoding for the ultrarapid delayed rectifier K(+) current (I(Kur)), Kv 1.5, was significantly increased. In contrast, mRNA expression of Kv 4.2 was significantly reduced in stretched myocytes. Alterations of gene expression correlated with the corresponding current densities: I(K1) and I(Kur) densities were significantly increased in stretched myocytes, whereas transient outward K(+) current (I(to)) density was reduced. These alterations resulted in a significant abbreviation of the action potential duration. Losartan (1 microM) prevented stretch-induced increases in the protein-to-DNA ratio and atrial natriuretic peptide mRNA expression in stretched myocytes. Concomitantly, losartan attenuated stretch-induced alterations in I(K1), I(Kur), and I(to) density and gene expression. This prevented the stretch-induced abbreviation of action potential duration. Prevention of stretch-induced electrical remodeling might contribute to the clinical effects of losartan against AF.     

Expression of Kir2.1 channels in astrocytes under pathophysiological conditions

Astrocyte ion channels participate in ionic homeostasis in the brain. Inward rectifying potassium channels (Kir channels) in astrocytes have been particularly implicated in K(+) homeostasis because of their high open probability at resting potential and their increased conductance at high concentrations of extracellular K(+). We examined the expression of the Kir2.1 subunit, one of the Kir channel subunits, in the mouse brain by immunohistochemistry. Kir2.1 channels were widely distributed throughout the brain, with high expression in the olfactory bulb and the cerebellum. Interestingly, they were abundantly expressed in astrocytes of the olfactory bulb, while astrocytes in other brain regions including the hippocampus did not show any detectable expression. However, Kir2.1 channel-expressing cells were dramatically increased in the hippocampus by kainic acid-induced seizure and the cells were glial fibrillary acidic protein (GFAP)-positive, which confirms that astrocytes in the hippocampus express Kir2.1 channels under pathological conditions. Our results imply that Kir2.1 channels in astrocyte may be involved in buffering K(+) against accumulated extracellular K(+) caused by neuronal hyperexcitability under phathophysiological conditions.     

Pregnenolone Sulfate Potentiates the Inwardly Rectifying K+ Channel Kir2.3

Background

Neurosteroids have various physiological and neuropsychopharmacological effects. In addition to the genomic effects of steroids, some neurosteroids modulate several neurotransmitter receptors and channels, such as N-methyl-D-aspartate receptors, γ-aminobutyric acid type A (GABA_A) receptors, and σ₁ receptors, and voltage-gated Ca²⁺ and K⁺ channels. However, the molecular mechanisms underlying the various effects of neurosteroids have not yet been sufficiently clarified. In the nervous system, inwardly rectifying K⁺ (Kir) channels also play important roles in the control of resting membrane potential, cellular excitability and K⁺ homeostasis. Among constitutively active Kir2 channels in a major Kir subfamily, Kir2.3 channels are expressed predominantly in the forebrain, a brain area related to cognition, memory, emotion, and neuropsychiatric disorders.

Methodology/Principal Findings

The present study examined the effects of various neurosteroids on Kir2.3 channels using the Xenopus oocyte expression assay. In oocytes injected with Kir2.3 mRNA, only pregnenolone sulfate (PREGS), among nine neurosteroids tested, reversibly potentiated Kir2.3 currents. The potentiation effect was concentration-dependent in the micromolar range, and the current-voltage relationship showed inward rectification. However, the potentiation effect of PREGS was not observed when PREGS was applied intracellularly and was not affected by extracellular pH conditions. Furthermore, although Kir1.1, Kir2.1, Kir2.2, and Kir3 channels were insensitive to PREGS, in oocytes injected with Kir2.1/Kir2.3 or Kir2.2/Kir2.3 mRNA, but not Kir2.1/Kir2.2 mRNA, PREGS potentiated Kir currents. These potentiation properties in the concentration-response relationships were less potent than for Kir2.3 channels, suggesting action of PREGS on Kir2.3-containing Kir2 heteromeric channels.

Conclusions/Significance

The present results suggest that PREGS acts as a positive modulator of Kir2.3 channels. Kir2.3 channel potentiation may provide novel insights into the various effects of PREGS.     

Suppression of Kir2.3 activity by protein kinase C phosphorylation of the channel protein at threonine 53

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

Identification of gamma-aminobutyric acid receptor-interacting factor 1 (TRAK2) as a trafficking factor for the K+ channel Kir2.1

To identify proteins that regulate potassium channel activity and expression, we performed functional screening of mammalian cDNA libraries in yeast that express the mammalian K(+) channel Kir2.1. Growth of Kir2.1-expressing yeast in media with low K(+) concentration is a function of K(+) uptake via Kir2.1 channels. Therefore, the host strain was transformed with a human cDNA library, and cDNA clones that rescued growth at low K(+) concentration were selected. One of these clones was identical to the protein of unknown function isolated previously as gamma-aminobutyric acid receptor-interacting factor 1 (GRIF-1) (Beck, M., Brickley, K., Wilkinson, H., Sharma, S., Smith, M., Chazot, P., Pollard, S., and Stephenson, F. (2002) J. Biol. Chem. 277, 30079-30090). GRIF-1 specifically enhanced Kir2.1-dependent growth in yeast and Kir2.1-mediated (86)Rb(+) efflux in HEK293 cells. Quantitative microscopy and flow cytometry analysis of immunolabeled surface Kir2.1 channel showed that GRIF-1 significantly increased the number of Kir2.1 channels in the plasma membrane of COS and HEK293 cells. Physical interaction of Kir2.1 channel and GRIF-1 was demonstrated by co-immunoprecipitation from HEK293 lysates and yeast two-hybrid assay. In vivo association of Kir2.1 and GRIF-1 was demonstrated by co-immunoprecipitation from brain lysate. Yeast two-hybrid assays showed that an N-terminal region of GRIF-1 interacts with a C-terminal region of Kir2.1. These results indicate that GRIF-1 binds to Kir2.1 and facilitates trafficking of this channel to the cell surface.     

Regulation of Kir2.1 channels by the Rho-GTPase, Rac1

Mutations in Kir2.1 inwardly rectifying potassium channels are associated with Andersen syndrome, a disease characterized by potentially fatal cardiac arrhythmias. While several Andersen-associated mutations affect membrane expression, the cytoplasmic signals that regulate Kir2.1 trafficking are poorly understood. Here, we investigated whether the Rho-family of small GTPases regulates trafficking of Kir2.1 channels expressed in HEK-293 cells. Treatment with Clostridium difficile toxin B, an inhibitor of Rho-family GTPases, or co-expression of the dominant-negative mutant of Rac1 (Rac1(DN)) increased Kir2.1 channels approximately 2-fold. However, the dominant-negative forms of other Rho-family GTPases, RhoA or Cdc42, did not alter Kir2.1 currents, suggesting a selective effect of Rac1 on Kir2.1 channels. Single-channel properties (gamma, tau(o), tau(c)) and total protein levels of Kir2.1 were unchanged with co-expression of Rac1(DN); however, studies using TIRF microscopy and CFP-tagged Kir2.1 revealed increased channel surface expression. Immunohistochemical detection of extracellularly tagged HA-Kir2.1 channels showed that Rac1(DN) reduced channel internalization when co-expressed. Finally, the dominant-negative mutant of dynamin, which interferes with endocytosis, occluded the Rac1(DN)-induced potentiation of Kir2.1 currents. These data suggest that inhibition of Rac1 increases Kir2.1 surface expression by interfering with endocytosis, likely via a dynamin-dependent pathway. Surprisingly, Rac1(DN) did not alter Kir2.2 current density or internalization, suggesting subunit specific modulation of Kir2.1 channels. Consistent with this, construction of Kir2.1/2.2 chimeras implicated the C-terminal domain of Kir2.1 in mediating the potentiating effect of Rac1(DN). This novel pathway for regulating surface expression of cardiac Kir2.1 channels could have implications for normal and diseased cardiac states.