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PubMed2000

Localization of inward rectifier potassium channel Kir7.1 in the basolateral membrane of distal nephron and collecting duct.

Inward rectifier potassium channels (Kir) play an important role in the K(+) secretion from the kidney. Recently, a new subfamily of Kir, Kir7.1, has been cloned and shown to be present in the kidney as well as in the brain, choroid plexus, thyroid, and intestine. Its cellular and subcellular localization was examined along the renal tubule. Western blot from the kidney cortex showed a single band for Kir7.1 at 52 kD, which was also observed in microdissected segments from the thick ascending limb of Henle, distal convoluted tubule (DCT), connecting tubule, and cortical and medullary collecting ducts. Kir7.1 immunoreactivity was detected predominantly in the DCT, connecting tubule, and cortical collecting duct, with lesser expression in the thick ascending limb of Henle and in the medullary collecting duct. Kir7.1 was detected by electron microscopic immunocytochemistry on the basolateral membrane of the DCT and the principal cells of cortical collecting duct, but neither type A nor type B intercalated cells were stained. The message levels and immunoreactivity were decreased under low-K diet and reversed by low-K diet supplemented with 4% KCl. By the double-labeling immunogold method, both Kir7.1 and Na(+), K(+)-ATPase were independently located on the basolateral membrane. In conclusion, the novel Kir7.1 potassium channel is located predominantly in the basolateral membrane of the distal nephron and collecting duct where it could function together with Na(+), K(+)-ATPase and contribute to cell ion homeostasis and tubular K(+) secretion.

Animals↗

PubMed1998

P2Y receptor subtypes differentially couple to inwardly-rectifying potassium channels.

Subtypes of P2Y receptors are well characterized with respect to their agonist profile but little is known about differences in their intracellular signalling properties. When expressed in Xenopus oocytes, both P2Y2 and P2Y6 receptors effectively couple to endogenous Ca2+-dependent Cl--channels. However, only P2Y2 receptors increased currents mediated by inward-rectifier K+ channels of the Kir3.0 subfamily. This increase in Kir-current was sensitive to pertussis toxin, while activation of Ca2+-dependent Cl--channels was not. In contrast, suramin, a P2 receptor antagonist, inhibited activation of both channels. These observations suggest that, in contrast to P2Y6, P2Y2 receptors couple to two different classes of G proteins.

Adenosine Triphosphate↗

PubMed1996

Acute effects of thyroid hormone on inward rectifier potassium channel currents in guinea pig ventricular myocytes.

Thyroid hormones play an important role in cardiac electrophysiology. However, the regulation of cardiac ionic channels by thyroid hormones is still unclear. To evaluate the acute effect of 3,3',5-triiode-L-thyronine (T3) on inward rectifier potassium channel (IK1) action potentials, whole-cell IK1 currents and steady-state single IK1 currents were recorded in guinea pig ventricular myocytes. Acute exposure of cells to T3 resulted in shortening of the action potential durations. This effect was initiated at 5-15 min and reached a stable plateau at 25 min. The amplitude of steady-state whole-cell IK1 was significantly increased by T3 at 1 nM to 1 microM concentration rage and with ED50 12 nM/liter. T3 (1 microM) increased IK1 by 68 +/- 7% at -40 mV and 52 +/- 9% at -100 mV. Similar effects were observed with triiodothyroacetic acid, an analogue of T3 that does not stimulate DNA transcription. The single IK1 open probability (Po) was increased 7 +/- 1% by 1 nM T3 (n = 9, P < 0.05) and 42 +/- 6% by 1 microM T3 at -40 mV (n = 18, P < 0.0001). However, the channel unit amplitude, time constants of open and fast-closed time were not changed. T3 shortened interburst duration at each membrane potential but did not change the burst behavior. To elucidate detailed mechanism, we assumed a three-state model (C1 <==> C2 <==> O) and calculated each rate constant. T3 significantly increased the rate constant, k+1, for the transition from the C1 to the C2 state at RP -40 mV (2.84 +/- 0.56 to 7.28 +/- 1.23 sec-1, P < 0.01), RP -20 mV (3.63 +/- 0.95 to 10.17 +/- 2.60 sec-1, P < 0.05) and RP (6.73 +/- 1.20 to 21.94 +/- 4.49 sec-1, P < 0.01). However, the other rate constants were not affected. These results demonstrate that T3 enhances IK1 with the increment in Po, which mainly results from shortening of interburst duration without any changes in burst behavior. Hence, the shortening of interburst duration is due to acceleration of the transition from the C1 to the C2 state. The enhanced IK1 by T3 might be one of the causes for shortened action potential duration in hyperthyroidism.

Action Potentials↗

PubMed2001

Targeting of an A kinase-anchoring protein, AKAP79, to an inwardly rectifying potassium channel, Kir2.1.

Protein kinase A (PKA) is targeted to discrete subcellular locations close to its intended substrates through interaction with A kinase-anchoring proteins (AKAPs). Ion channels represent a diverse and important group of kinase substrates, and it has been shown that membrane targeting of PKA through association with AKAPs facilitates PKA-mediated phosphorylation and regulation of several classes of ion channel. Here, we investigate the effect of AKAP79, a membrane-associated multivalent-anchoring protein, upon the function and modulation of the strong inwardly rectifying potassium channel, Kir2.1. Functionally, the presence of AKAP79 enhanced the response of Kir2.1 to elevated intracellular cAMP, suggesting a requirement for a pool of PKA anchored close to the channel. Antibodies directed against a hemagglutinin epitope tag on Kir2.1 coimmunoprecipitated AKAP79, indicating that the two proteins exist together in a complex within intact cells. In support of this, glutathione S-transferase fusion proteins of both the intracellular N and C domains of Kir2.1 isolated AKAP79 from cell lysates, while glutathione S-transferase alone failed to interact with AKAP79. Together, these findings suggest that AKAP79 associates directly with the Kir2.1 ion channel and may serve to anchor kinase enzymes in close proximity to key channel phosphorylation sites.

A Kinase Anchor Proteins↗

PubMed2004

Laminin-induced aggregation of the inwardly rectifying potassium channel, Kir4.1, and the water-permeable channel, AQP4, via a dystroglycan-containing complex in astrocytes.

Dystroglycan (DG) is part of a multiprotein complex that links the extracellular matrix to the actin cytoskeleton of muscle fibers and that is involved in aggregating acetylcholine receptors at the neuromuscular junction. This complex is also expressed in regions of the central nervous system where it is localized to both neuronal and glial cells. DG and the inwardly rectifying potassium channels, Kir4.1, are concentrated at the interface of astroglia and small blood vessels. These channels are involved in siphoning potassium released into the extracellular space after neuronal excitation. This raises the possibility that DG may be involved in targeting Kir4.1 channels to specific domains of astroglia. To address this question, we used mixed hippocampal cultures to investigate the distribution of DG, syntrophin, dystrobrevin, and Kir4.1 channels, as well as aquaporin-permeable water channels, AQP4. These proteins exhibit a similar distribution pattern and form aggregates in astrocytes cultured on laminin. Both DG and syntrophin colocalize with Kir4.1 channel aggregates in astrocytes. Similarly, DG colocalizes with AQP4 channel aggregates. Quantitative studies show a significant increase of Kir4.1 and AQP4 channel aggregates in astrocytes cultured in the presence of laminin when compared with those in the absence of laminin. These findings show that laminin has a role in Kir4.1 and AQP4 channel aggregation and suggest that this may be mediated via a dystroglycan-containing complex. This study reveals a novel functional role for DG in brain including K+ buffering and water homeostasis.

Animals↗

PubMed1995

Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation.

To probe the protein environment of an ion channel, we have perturbed the structure of a transmembrane domain by substituting side chains with those of two different sizes by using site-specific mutagenesis. We have used Trp and Ala as a high- and a low-impact perturbation probe, respectively, to replace each of 18 consecutive residues within the putative second transmembrane segment, M2, of an inwardly rectifying potassium channel, ROMK1. Our rationale is that a change in the channel function as a consequence of these mutations at a particular position will reflect the structural environment of the altered side chain. Each position can then be assigned to one of three classes of environments, as grated by different levels of perturbation: very tolerant (channel functions with both Trp and Ala substitutions), tolerant (function preserved with Ala but not with Trp substitution), and intolerant (either Ala or Trp substitution destroys function). We identify the very tolerant environment as being lipid-facing, tolerant as protein-interior-facing, and intolerant as pore-facing. We observe a strikingly ordered pattern of perturbation of all three environmental classes. This result indicates that M2 is a straight alpha-helix.

Alanine↗

PubMed2003

Surface expression of inward rectifier potassium channels is controlled by selective Golgi export.

Traffic of integral membrane proteins along the secretory pathway is not simply a default process but can be selective. Such selectivity is achieved by sequence information within the cargo protein that recruits coat protein complexes to drive the formation of transport vesicles. A number of sequence motifs have been identified in the cytoplasmic domains of ion channels that regulate early trafficking events between the endoplasmic reticulum and the Golgi complex. Here, we demonstrate that the following trafficking step from the Golgi compartment to the plasma membrane can also be selective. The N-terminal domain of the inward rectifier potassium channel Kir2.1 contains specific sequence information that is necessary for its efficient export from the Golgi complex. Lack of this information results in accumulation of the protein within the Golgi and a significant decrease in cell surface expression. As similar results were obtained for the N terminus of another Kir channel subfamily member, Kir4.1, which could functionally substitute for the Kir2.1 N terminus, we propose a more general role of the identified N-terminal domains for post-Golgi trafficking of Kir channels.

Amino Acid Sequence↗

PubMed2004

Differential subunit composition of the G protein-activated inward-rectifier potassium channel during cardiac development.

Parasympathetic slowing of the heart rate is predominantly mediated by acetylcholine-dependent activation of the G protein-gated potassium (K+) channel (IK,ACh). This channel is composed of 2 inward-rectifier K+ (Kir) channel subunits, Kir3.1 and Kir3.4, that display distinct functional properties. Here we show that subunit composition of IK,ACh changes during embryonic development. At early stages, IK,ACh is primarily formed by Kir3.1, while in late embryonic and adult cells, Kir3.4 is the predominant subunit. This change in subunit composition results in reduced rectification of IK,ACh, allowing for marked K+ currents over the whole physiological voltage range. As a consequence, IK,ACh is able to generate the membrane hyperpolarization that underlies the strong negative chronotropy occurring in late- but not early-stage atrial cardiomyocytes upon application of muscarinic agonists. Both strong negative chronotropy and membrane hyperpolarization can be induced in early-stage cardiomyocytes by viral overexpression of the mildly rectifying Kir3.4 subunit. Thus, a switch in subunit composition is used to adopt IK,ACh to its functional role in adult cardiomyocytes.

Acetylcholine↗

PubMed1999

Corticosteroids alter G protein inwardly rectifying potassium channels protein levels in hippocampal subfields.

Corticosterone or cortisol, stress hormones in rat and human, respectively, alter neurotransmitter receptor-mediated responses in the brain. Corticosterone could alter these responses by modifying any component of the receptor-effector pathway. Many of these receptors are linked to guanine nucleotide regulatory proteins (G proteins) which, in turn, can activate second messenger systems and/or ion channels, such as G protein inwardly rectifying potassium channels (GIRK). The aim of these experiments was to determine whether corticosterone treatment altered the levels of GIRK proteins in rat hippocampus. Corticosterone treatment selectively altered the levels of GIRK1 and GIRK2 (measured on immunoblots) depending on the subfield of the hippocampus examined. These data lend credence to the hypothesis that corticosterone differentially alters neurotransmitter receptor-mediated responses dependent on the brain area.

Adrenalectomy↗

PubMed2004

Inward rectifier potassium channels in the basilar artery during chronic alcohol consumption.

BACKGROUND: The goals of this study were to determine whether chronic alcohol consumption alters potassium channel-mediated reactivity in the basilar artery and to determine a potential mechanism that might account for the effects of alcohol on the basilar artery. METHODS: Sprague-Dawley rats were fed liquid diets with or without alcohol for 2 to 3 months. We measured diameter of the basilar artery in response to potassium channel inhibitors and activators. Protein level of inward rectifier potassium channel subunit Kir2.1 in the basilar artery was determined by Western blot. RESULTS: Topical application of glibenclamide (1 and 10 microM) significantly constricted the basilar artery at high dose; iberiotoxin (10 and 100 nM), 4-AP (0.1 and 1 mM), and BaCl2 (1 and 10 microM) produced dose-related constriction in both non-alcohol-fed and alcohol-fed rats. However, the magnitude of constriction in response to BaCl2 was significantly less in alcohol-fed rats compared with non-alcohol-fed rats. Topical application of KCl (1 and 3 mM), cromakalim (0.1 and 0.3 microM), and NS1619 (10 and 30 microM) induced dose-related dilation in non-alcohol-fed and alcohol-fed rats. However, the magnitude of vasodilation in response to KCl was significantly less in alcohol-fed rats compared with non-alcohol-fed rats. In addition, Kir2.1 protein level in the basilar artery was significantly reduced in alcohol-fed compared with non-alcohol-fed rats. CONCLUSIONS: These findings suggest that chronic alcohol consumption reduces expression of inward rectifier potassium channels and inhibits KIR channel-mediated dilation in the basilar artery.

Alcohol Drinking↗

PubMed2007

Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes.

Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.

Animals↗

PubMed1999

Channelopathies of inwardly rectifying potassium channels.

Mutations in genes encoding ion channels have increasingly been identified to cause disease conditions collectively termed channelopathies. Recognizing the molecular basis of an ion channel disease has provided new opportunities for screening, early diagnosis, and therapy of such conditions. This synopsis provides an overview of progress in the identification of molecular defects in inwardly rectifying potassium (Kir) channels. Structurally and functionally distinct from other channel families, Kir channels are ubiquitously expressed and serve functions as diverse as regulation of resting membrane potential, maintenance of K(+) homeostasis, control of heart rate, and hormone secretion. In humans, persistent hyperinsulinemic hypoglycemia of infancy, a disorder affecting the function of pancreatic beta cells, and Bartter's syndrome, characterized by hypokalemic alkalosis, hypercalciuria, increased serum aldosterone, and plasma renin activity, are the two major diseases linked so far to mutations in a Kir channel or associated protein. In addition, the weaver phenotype, a neurological disorder in mice, has also been associated with mutations in a Kir channel subtype. Further genetic linkage analysis and full understanding of the consequence that a defect in a Kir channel would have on disease pathogenesis are among the priorities in this emerging field of molecular medicine.

Animals↗

PubMed2002

G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase.

A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.

Adenylyl Cyclases↗

PubMed2000

Permeation properties of inward-rectifier potassium channels and their molecular determinants.

The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K(+) channels ROMK2 (Kir1.1b), IRK1 (Kir2.1), and their chimeras using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode with different permeant cations in the pipette. For inward K(+) conduction, replacing the extracellular loop of ROMK2 with that of IRK1 increased single-channel conductance by 25 pS (from 39 to 63 pS), whereas replacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance by 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH(2) terminus or transmembrane domains, suggesting that the two domains form two resistors in series. The larger conductance of the extracellular loop of IRK1 was attributable to a single amino acid difference (Thr versus Val) at the 3P position, three residues in front of the GYG motif. Permeability sequences for the conducted ions were similar for the two channels: Tl(+) > K(+) > Rb(+) > NH(4)(+). The ion selectivity sequence for ROMK2 based on conductance ratios was NH(4)(+) (1.6) > K(+) (1) > Tl(+) (0.5) > Rb(+) (0.4). For IRK1, the sequence was K(+) (1) > Tl(+) (0.8) > NH(4)(+) (0.6) >> Rb(+) (0.1). The difference in the NH(4)(+)/ K(+) conductance (1.6) and permeability (0.09) ratios can be explained if NH(4)(+) binds with lower affinity than K(+) to sites within the pore. The relatively low conductances of NH(4)(+) and Rb(+) through IRK1 were again attributable to the 3P position within the P region. Site-directed mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred the relatively high Tl(+) conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectivity properties of the pore.

Algorithms↗

PubMed2002

mRNA expression alterations of inward rectifier potassium channels in rat brain with cholinergic impairment.

Potassium channel dysfunction has been indicated in Alzheimer's disease. In the present study, spatial memory, the activity of cortical acetylcholinesterase (AChE) and the expressions of inward rectifier potassium channels (Kir) were measured after ibotenic acid lesions of nucleus basalis magnocellularis (nbm) in rats. Expressions of Kir (Kir2.1, Kir3.1, Kir6.1 and Kir6.2) at mRNA level were assessed using reverse transcription-polymerase chain reaction. At 28 days after ibotenic acid injection, the spatial memory of rats was significantly impaired accompanied by a 32% reduction of cortical AChE activity. Furthermore, the expression of Kir6.2 was increased by 79.3% in cortex, and that of Kir6.1 was increased by 172.1% in hippocampus, while no obvious changes in the mRNA expression of Kir2.1 and Kir3.1 were detected. This study indicated that the expression of adenosine triphosphate-sensitive potassium channel was area and channel subtype specifically increased following nbm lesions.

Acetylcholine↗

PubMed1995

beta-Adrenergic modulation of the inwardly rectifying potassium channel in isolated human ventricular myocytes. Alteration in channel response to beta-adrenergic stimulation in failing human hearts.

The beta-adrenergic modulation of the inwardly-rectifying K+ channel (IK1) was examined in isolated human ventricular myocytes using patch-clamp techniques. Isoproterenol (ISO) reversibly depolarized the resting membrane potential and prolonged the action potential duration. Under the whole-cell C1- -free condition, ISO applied via the bath solution reversibly inhibited macroscopic IdK1. The reversal potential of the ISO-sensitive current was shifted by approximately 60 mV per 10-fold change in the external K+ concentration and was sensitive to Ba2+. The ISO-induced inhibition of IK1 was mimicked by forskolin and dibutyrl cAMP, and was prevented by including a cAMP-dependent protein kinase (PKA) inhibitor (PKI) in the pipette solution. In single-channel recordings from cell-attached patches, bath applied ISO could suppress IK1 channels by decreasing open state probability. Bath application of the purified catalytic sub-unit of PKA to inside-out patches also inhibited IK1 and the inhibition could be antagonized by alkaline phosphatase. When beta-adrenergic modulation of IK1 was compared between ventricular myocytes isolated from the failing and the nonfailing heart, channel response to ISO and PKA was significantly reduced in myocytes from the failing heart. Although ISO inhibited IK1 in a concentration-dependent fashion in both groups, a half-maximal concentration was greater in failing (0.12 microM) than in nonfailing hearts (0.023 microM). These results suggest that IK1 in human ventricular myocytes can be inhibited by a PKA-mediated phosphorylation and the modulation is significantly reduced in ventricular myocytes from the failing heart compared to the nonfailing heart.

Adrenergic beta-Agonists↗

PubMed1994

Molecular cloning and functional expression of a novel brain-specific inward rectifier potassium channel.

We have cloned a novel brain-specific inward rectifier K+ channel from a mouse brain cDNA library and designated it MB-IRK3. The mouse brain cDNA library was screened using a fragment of the mouse macrophage inward rectifier K+ channel (IRK1) cDNA as a probe. The amino acid sequence of MB-IRK3 shares 61% and 64% identity to MB-IRK1 and RB-IRK2, respectively. Xenopus oocytes injected with cRNA derived from this clone expressed a potassium current which showed inward-rectifying channel characteristics similar to MB-IRK1 and RB-IRK2 currents, but distinct from ROMK1 or GIRK1 current. However, the single channel conductance of MB-IRK3 was approximately 10 pS with 140 mM extracellular K+, which was distinct from that of MB-IRK1 (20 pS). MB-IRK3 mRNA expressed specifically in the forebrain, which clearly differed from MB-IRK1 and RB-IRK2 mRNAs. These results indicate that members of the IRK family with distinct electrophysiological properties express differentially and may play heterogenous functional roles in brain functions.

Amino Acid Sequence↗

PubMed1995

Identification of strong modifications in cation selectivity in an Arabidopsis inward rectifying potassium channel by mutant selection in yeast.

The Arabidopsis thaliana cDNA, KAT1, encodes a hyperpolarization-activated K+ channel. In the present study, we utilized a combination of random site-directed mutagenesis, genetic screening in a potassium uptake-deficient yeast strain, and electrophysiological analysis in Xenopus oocytes to identify strong modifications in cation selectivity of the inward rectifying K+ channel KAT1. Threonine at position 256 was replaced by 11 other amino acid residues. Six of these mutated KAT1 cDNAs complemented a K+ uptake-deficient yeast strain at low concentrations of potassium. Among these, two mutants (T256D and T256G) showed a sensitivity of yeast growth toward high ammonium concentrations and a dramatic increase in current amplitudes of rubidium and ammonium ions relative to K+ by 39-72-fold. These single site mutations gave rise to Rb+- and NH4(+)-selective channels with Rb+ and NH4+ currents that were approximately 10-13-fold greater in amplitude than K+ currents, whereas the NH4+ to K+ current amplitude ratio of wild type KAT1 was 0.28. This strong conversion in cation specificity without loss of general selectivity exceeds those reported for other mutations in the pore domain of voltage-dependent K+ channels. Yeast growth was greatly impaired by sodium in two other mutants at this site (T256E and T256Q), which were blocked by millimolar sodium (K1/2 = 1.1 mM for T256E), although the wild type channel was not blocked by 110 mM sodium. Interestingly, the ability of yeast to grow in the presence of toxic cations correlated to biophysical properties of KAT1 mutants, illustrating the potential for qualitative K+ channel mutant selection in yeast. These data suggest that the size of the side chain of the amino acid at position 256 in KAT1 is important for enabling cation permeation and that this site plays a crucial role in determining the cation selectivity of hyperpolarization-activated potassium channels.

Amino Acid Sequence↗