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PubMed2000

Homology modeling and molecular dynamics simulation studies of an inward rectifier potassium channel.

A homology model has been generated for the pore-forming domain of Kir6.2, a component of an ATP-sensitive K channel, based on the x-ray structure of the bacterial channel KcsA. Analysis of the lipid-exposed and pore-lining surfaces of the model reveals them to be compatible with the known features of membrane proteins and Kir channels, respectively. The Kir6.2 homology model was used as the starting point for nanosecond-duration molecular dynamics simulations in a solvated phospholipid bilayer. The overall drift from the model structure was comparable to that seen for KcsA in previous similar simulations. Preliminary analysis of the interactions of the Kir6.2 channel model with K(+) ions and water molecules during these simulations suggests that concerted single-file motion of K(+) ions and water through the selectivity filter occurs. This is similar to such motion observed in simulations of KcsA. This suggests that a single-filing mechanism is conserved between different K channel structures and may be robust to changes in simulation details. Comparison of Kir6.2 and KcsA suggests some degree of flexibility in the filter, thus complicating models of ion selectivity based upon a rigid filter.

Amino Acid Sequence↗
PubMed1997

Voltage-gated and inwardly rectifying potassium channels.

This lecture is dedicated to Max Delbrück and Seymour Benzer. Max Delbrück was our graduate advisor. He introduced us to a variety of biophysical problems, and taught us ways of thinking about these problems by example. Potassium channels was one of the topics included in his journal club in the early seventies; Max also carefully considered the feasibility of purifying potassium channels then. It was in Seymour Benzer's laboratory that we began to look for Drosophila mutants that affect synaptic transmission at the larval neuromuscular junction. Shaker was the first behavioural mutant we tested that gave a robust phenotype, a phenotype that could be mimicked by treating wild-type preparations with a potassium channel blocker. This mutant fly has led us to our subsequent molecular studies of potassium channels. Since we settled in the University of California, San Francisco, and began to study neural development as well as potassium channels, we have settled into the pattern of each attending meetings and presenting our studies on one of these two areas so as to avoid both being away from home and our children at the same time. In following this pattern, I will be presenting the studies of potassium channels as part of our long-term collaboration. In this talk I will first briefly take you through the path that led us to the molecular studies of potassium channels and then discuss the diversity and modulation of these potassium channels at the molecular and physiological level.

Animals↗
PubMed2005

Protein kinase A-dependent activation of inward rectifier potassium channels by adenosine in rabbit coronary smooth muscle cells.

We studied the effect of adenosine on the Ba(2+)-sensitive K(IR) channels in the smooth muscle cells isolated from the small-diameter (<100microm) coronary arteries of rabbit. Adenosine increased K(IR) currents in concentration-dependent manner (EC(50)=9.4+/-1.4microM, maximum increase of 153%). The adenosine-induced stimulation of K(IR) current was blocked by adenylyl cyclase inhibitor, SQ22536 and was mimicked by adenylyl cyclase activator, forskolin. The adenosine-induced increase of current was blocked by cyclic AMP-dependent protein kinase (PKA) inhibitors, KT 5720 and Rp-8-CPT-cAMPs. The adenosine-induced increase of K(IR) currents was blocked by an A(3)-selective antagonist MRS1334, while the antagonists of other subtypes (DPCPX for A(1), ZM241385 for A(2A), and alloxazine for A(2B)) were all ineffective. Furthermore, an A(3)-selective agonist, 2-Cl-IB-MECA induced increase of K(IR) currents. We also examined the effect of adenosine on coronary blood flow (CBF) rate by using the Langendorff-perfused heart. In the presence of glibenclamide to exclude the effects of ATP-sensitive K(+) (K(ATP)) channels, CBF was increased by adenosine (10microM), which was blocked by the addition of Ba(2+) (50microM). Above results suggest that adenosine increases K(IR) current via A(3) subtype through the activation of PKA in rabbit small-diameter coronary arterial smooth muscle cells.

Adenosine↗
PubMed1998

Expression of the Kir2.1 (inwardly rectifying potassium channel) gene in the human placenta and in cultured cytotrophoblast cells at different stages of differentiation.

The aim of this study was to investigate whether the Kir2.1 gene is expressed by the human placenta throughout pregnancy and in cytotrophoblast cells at different stages of differentiation in culture. RNA was extracted from cytotrophoblast cells isolated from term placentas and maintained in culture for 18, 66 and 114 h and from first, second and third trimester placentas. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers, a cDNA product of 1.2 kb, as expected for Kir2.1 gene expression, was detected in all the RNA samples from cytotrophoblast cells and from placentas. The RT-PCR products were verified by sequencing and by detection of the expected transcript size for the Kir2.1 mRNA at 5.6-5.7 kb on Northern blots, using the 1.2 kb cDNA generated by RT-PCR. Northern blot quantification, using a control 28S rRNA probe, showed no significant difference in Kir2.1 mRNA expression between any of the three stages of cytotrophoblast cell differentiation studied (ANOVA; n = 3 RNA samples from each stage). These data demonstrate that the Kir2.1 gene is expressed by the human placenta and, specifically, by cytotrophoblast cells, at all stages of development and differentiation.

Cell Differentiation↗
PubMed1999

Cytoplasmic amino and carboxyl domains form a wide intracellular vestibule in an inwardly rectifying potassium channel.

We have studied the structural components and architecture of the intracellular vestibule of a strongly rectifying channel (Kir2.1) expressed in Xenopus oocytes. Putative vestibule-lining residues were identified by systematically examining covalent modification by sulfhydryl-specific reagents of cysteine residues engineered into two cytoplasmic regions. In a stretch of 33 amino acids in the amino terminus (from C54 to V86) and 22 amino acids in the carboxyl terminus (from R213 to S234), 15 and 11 residues, respectively, were found to be accessible to methanethiosulfonate ethylammonium (MTSEA) or methanethiosulfonate ethyltrimethylammonium (MTSET) and presumably project into the aqueous intracellular vestibule. The pattern of accessibility suggests that both stretches may adopt an extended loop structure. To explore the physical dimension of the intracellular vestibule, we covalently linked a constrained number (one to four) of positively charged moieties of different sizes to the E224 position and found that this vestibule region is sufficiently wide to accommodate four modifying groups with dimensions of 12 A x 10 A x 6 A. These results suggest that regions in both the amino and carboxyl domains of Kir2.1 channel form a long and wide intracellular vestibule that protrudes beyond the membrane into the cytoplasm.

Amino Acid Sequence↗
PubMed2005

Conduction through the inward rectifier potassium channel, Kir2.1, is increased by negatively charged extracellular residues.

Ion channel conductance can be influenced by electrostatic effects originating from fixed "surface" charges that are remote from the selectivity filter. To explore whether surface charges contribute to the conductance properties of Kir2.1 channels, unitary conductance was measured in cell-attached recordings of Chinese hamster ovary (CHO) cells transfected with Kir2.1 channels over a range of K+ activities (4.6-293.5 mM) using single-channel measurements as well as nonstationary fluctuation analysis for low K+ activities. K+ ion concentrations were shown to equilibrate across the cell membrane in our studies using the voltage-sensitive dye DiBAC4(5). The dependence of gamma on the K+ activity (a(K)) was fit well by a modified Langmuir binding isotherm, with a nonzero intercept as a(K) approaches 0 mM, suggesting electrostatic surface charge effects. Following the addition of 100 mM N-methyl-D-glucamine (NMG+), a nonpermeant, nonblocking cation or following pretreatment with 50 mM trimethyloxonium (TMO), a carboxylic acid esterifying agent, the gamma-a(K) relationship did not show nonzero intercepts, suggesting the presence of surface charges formed by glutamate or aspartate residues. Consistent with surface charges in Kir2.1 channels, the rates of current decay induced by Ba2+ block were slowed with the addition of NMG or TMO. Using a molecular model of Kir2.1 channels, three candidate negatively charged residues were identified near the extracellular mouth of the pore and mutated to cysteine (E125C, D152C, and E153C). E153C channels, but not E125C or D152C channels, showed hyperbolic gamma-a(K) relationships going through the origin. Moreover, the addition of MTSES to restore the negative charges in E53C channels reestablished wild-type conductance properties. Our results demonstrate that E153 contributes to the conductance properties of Kir2.1 channels by acting as a surface charge.

Animals↗
PubMed2005

Differential sensitivity of Kir2 inward-rectifier potassium channels to a mitochondrial uncoupler: identification of a regulatory site.

The aim of this study was to gain insight into the mechanism by which members of the K(ir)2 subfamily are differentially sensitive to agents that inhibit mitochondrial function by identifying responsible site(s) in K(ir)2 proteins. K(ir)2 channels were expressed in Xenopus laevis oocytes and assayed by two-electrode voltage clamp and patch clamp. Incubation of oocytes in carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, inhibited K(ir)2.2 and K(ir)2.3, but not K(ir)2.1. Replacement of the first 44 amino acids of K(ir)2.2 the or of first 19 K(ir)2.3 with the first 45 of K(ir)2.1 did not affect the sensitivity of the channels to FCCP. In contrast, a larger substitution of K(ir)2.1 N-terminal sequence (1-78) into K(ir)2.2 or K(ir)2.3 produced channels that were resistant to FCCP. Sequence alignment between residues 46 and 78 (K(ir)2.1 numbering) revealed four residues that are the same in K(ir)2.2 and K(ir)2.3 but different in K(ir)2.1. Each of these four residues in the resistant chimera was converted back to the K(ir)2.2/K(ir)2.3 amino acid. Three of the mutants (D51N, I59A, and G65S) were not sensitive to FCCP, but the H53Q mutant was sensitive. K(ir)2.1-H53A and K(ir)2.1-H53E were also sensitive. In contrast, K(ir)2.1-H53R and K(ir)2.1-H53K were recovered during resistant. K(ir)2.2 and K(ir)2.3 currents perfusion of inside-out patches from FCCP-treated oocytes. FCCP was without effect on K(ir)2.2 and K(ir)2.3 when applied directly to inside-out patches. Together, these results suggest inhibition of K(ir)2.2 and K(ir)2.3 by a ligand that bears a positive charge and is produced by an intracellular action of FCCP.

Adenosine Triphosphate↗
PubMed1998

Differential distribution of inward rectifier potassium channel transcripts in human atrium versus ventricle.

BACKGROUND: The inward rectifier K+ current (IK1) plays an important role in governing cardiac electrical activity and is well known to have different properties in the atrium compared with the ventricle. Several inward rectifier K+ channel (IRK) subunits (hIRK, HH-IRK1, HIR, and TWIK-1) with different properties have been cloned from human tissues, but their relative expression in cardiac tissues has not been quantified. The present study was designed to define the relative levels of mRNA for various IRKs in human atrium and in failing and nonfailing ventricle. METHODS AND RESULTS: Competitive reverse transcription-polymerase chain reaction was used to quantify in human atrium and ventricle the mRNA levels of hIRK, HH-IRK1, HIR, and TWIK-1. The absence of important noncardiac contamination was confirmed by demonstrating a lack of detectable mRNA markers for neuronal (acetylcholine receptor) and vascular (maxi-K channel) tissue. mRNA of HIR was more abundant in normal atrium (7.1+/-1.3 amol/ microg total RNA) than ventricle (0.6+/-0.1 amol/ microg, P<0. 05), whereas TWIK-1 mRNA was more concentrated in ventricle (18. 1+/-4.3 amol/ microg) than atrium (1.4+/-0.3 amol/ microg, P<0.05). Concentrations of hIRK (42.7+/-6.7 amol/ microg in atrium vs 57. 1+/-9.2 amol/ microg in ventricle) and HH-IRK1 (2.0+/-0.5 amol/ microg in atrium vs 1.5+/-0.5 amol/ microg in ventricle) were comparable. No significant differences in IRK subunit transcript concentrations were found between normal and failing ventricles. CONCLUSIONS: mRNAs for all 4 IRKs are detected in human atrium and ventricle, but the mRNA copy number of a low-conductance subunit (HIR) is larger in atrium and the copy number of a weakly rectifying subunit (TWIK-1) is larger in ventricle. These differences in relative message levels may provide a potential molecular basis for different properties of IK1 in human atrium compared with ventricle.

Atrial Function↗
PubMed1994

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium.

UNLABELLED: Inward-rectifier K channel: using macroscopic voltage clamp and single-channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current-voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell-attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. CONCLUSIONS: under physiological ionic gradients, a 15-pS inward-rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.

Animals↗
PubMed1995

Cloning, localization, and functional expression of a human brain inward rectifier potassium channel (hIRK1).

We have cloned a novel human brain inward rectifier K+ channel (hIRK1), which shares approximately 60% amino acid identity with another human inward rectifier (hIRK2) but 98% identity with the mouse IRK1. The hIRK1 mRNA is expressed in several human tissues: skeletal muscle > placenta > heart > brain > lung > kidney. In human brain, the hIRK1 mRNA is uniformly distributed (except for a higher level in the corpus callosum, which contains white matter and glial cells), whereas the hIRK2 mRNA is expressed in major regions of the basal ganglia and limbic system. Xenopus oocytes injected with hIRK1 cRNA expressed an inwardly rectifying K+ current that was blocked by extracellular Ba2+. The hIRK1 channel carried a significant outward current when membrane potential was more positive than the K+ equilibrium potential (EK) and therefore had an "N-shape" current-voltage relation, resembling that of the native cardiac IRK channel. The resting membrane potential was near EK in oocytes expressing hIRK1, but was approximately -40 mV in H2O-injected or non-injected oocytes. The ability of hIRK1 to set the resting membrane potential depended on the outward current. Single-channel conductance of hIRK1 was 32 pS measured with 150 mM KCl in the patch pipette, significantly higher than 23 pS measured for mouse IRK1 and approximately 10 pS for hIRK2.

Amino Acid Sequence↗
PubMed2002

Cloning, expression, and localization of a rat hepatocyte inwardly rectifying potassium channel.

Bile formation involves anion accumulation within the apical lumen of hepatocytes. Potassium flux through hepatocellular basolateral membrane channels may provide the counterion for apical anion efflux. Here we cloned a molecular candidate for maintaining charge balance during bile secretion. Two transcripts resembling the Kir4.2 subclass of inwardly rectifying potassium channels were found. The longer deduced isoform (4.2a) has 30 additional NH(3)-terminal amino acids, which identifies this as a new isoform. The short-form isoform shared 86-91% identity with the mouse, human, and guinea pig channels. Whole cell currents of either rat isoform expressed in HEK293T cells demonstrated time independence and inward rectification. Antibodies against a COOH-terminal fragment recognized bands between 40 and 45 kDa and at 90 kDa and recognized a high molecular mass band around 200 kDa in overexpressing HEK cells. Immunohistology of liver tissue shows hepatocellular plasma membrane localization. In hepatocyte couplets, Kir4.2 was predominantly localized to the basolateral membrane. Results demonstrate expression of a new Kir4.2 isoform in the rat hepatocyte whose functional properties are compatible with a role in maintaining electrical integrity of bile-generating hepatocytes.

Animals↗
PubMed2001

Identification of G protein-coupled, inward rectifier potassium channel gene products from the rat anterior pituitary gland.

Dopamine (DA) is a physiological regulator of PRL secretion, exerting tonic inhibitory control. DA activates an inward rectifier K(+) (IRK) channel in rat lactotropes, causing membrane hyperpolarization and inhibition of Ca(2+)-dependent action potentials. Both the activation of this effector K(+) channel and the inhibition of PRL release are mediated by D(2)-type receptor activation and pertussis toxin- sensitive G proteins. To study the molecular basis of this physiologically relevant channel, a homology-based PCR approach was employed to identify members of the IRK channel family expressed in the anterior pituitary gland. Nondegenerate primers corresponding to regions specific for IRK channels known to be G protein activated (GIRKs; gene subfamily Kir 3.0) were synthesized and used in the PCR with reverse transcribed female rat anterior pituitary messenger RNA as the template. PCR products of predicted sizes for Kir 3.1, 3.2, and 3.4 were consistently observed by ethidium bromide staining after 16 amplification cycles. The identities of the products were confirmed by subcloning and sequencing. Expression of each of these gene products in anterior pituitary was confirmed by Northern blot analysis. Functional analysis of the GIRK proteins was performed in the heterologous expression system, Xenopus laevis oocytes. Macroscopic K(+) currents were examined in oocytes injected with different combinations of Kir 3.0 complementary RNA (cRNA) and G protein subunit (beta(1)gamma(2)) cRNA. The current-voltage relationships demonstrated strong inward rectification for each individual and pairwise combination of GIRK channel subunits. Oocytes coinjected with any pair of GIRK subunit cRNA exhibited significantly larger inward K(+) currents than oocytes injected with only one GIRK channel subtype. Ligand-dependent activation of only one of the GIRK combinations (GIRK1 and GIRK4) was observed when channel subunits were coexpressed with the D(2) receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent K(d) similar to that for DA binding in anterior pituitary tissue. GIRK1 and GIRK4 proteins were coimmunoprecipitated from anterior pituitary lysates, confirming the presence of native GIRK1/GIRK4 oligomers in this tissue. These data indicate that GIRK1 and GIRK4 are excellent candidate subunits for the D(2)-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.

Animals↗
PubMed1994

Endothelin-1 inhibits inward rectifier potassium channels and activates nonspecific cation channels in cultured endothelial cells.

A predominant inward rectifier and a small outward potassium current were obtained in whole-cell patch-clamp recordings from cultured bovine pulmonary arterial endothelial cells. Application of endothelial-1 (ET-1; 10-100 nmol/l) inhibited the inward rectifier. Washout with bath solution did not recover the current decreased by ET-1. In cell-attached studies, ET-1 (1 nmol/l) inhibited single-channel activity of the inward rectifier and in some patches enhanced activity of the outward potassium current without change of conductance. A non-specific cation current which is permeable to calcium was identified in cell-attached patches in cultured human umbilical vein endothelial cells. ET-1 (1 nmol/l) increased activity of the nonspecific cation channel. ET-1 may increase calcium influx into endothelial cells and promote synthase and release of endothelium-derived factors.

Animals↗
PubMed2002

Structural basis of inward rectifying potassium channel gating.

The last 10 years have seen rapid advances in the understanding of potassium channel function. Since the first inward rectifying (Kir) channels were cloned in 1994, the structural basis of channel function has been significantly elucidated, and determination of the crystal structure of a bacterial K channel (KcsA) in 1998 provided an atomic resolution of the permeation pathway. This review considers recent experimental studies aimed at uncovering the structural basis of Kir channel activity, and the applicability of comparative models based on KcsA to illuminate Kir channel pore structure and opening and closing processes.

Humans↗
PubMed1994

Cloning and expression of a family of inward rectifier potassium channels.

Five new members of the two-transmembrane domain potassium channel family have been identified from rat brain, heart and skeletal muscle. The channel mRNAs are differentially expressed and found in both the central nervous system and periphery. Expression of two of these channels in Xenopus oocytes gave rise to inwardly rectifying potassium currents which were voltage-dependently blocked by barium and cesium. Voltage command pulses negative to Ek evoked inward currents which rapidly reached a peak amplitude and relaxed to a steady-state level. The quantity of current relaxation differed in the two channels and was increased at more negative potentials. The degree of current rectification was also different for the two channels. The results demonstrate the existence of a large and widely expressed family of inward rectifier potassium channel subunits with distinct tissue distributions and functional properties.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of a novel human inwardly rectifying potassium channel predominantly expressed in small intestine.

A new member of the two transmembrane domain potassium (K+) channel family was identified and isolated from a human brain cDNA library. The cDNA clone contains an open reading frame which encodes a 360 amino acid sequence with a characteristic P domain flanked by two hydrophobic regions representing the membrane spanning segments. The closest homologue of this gene product is the inwardly rectifying potassium channel subunit, Kir1.2 (identity approximately 42%). Northern blot analysis of human tissues with a selective cDNA probe for this new K+ subunit showed a single major transcript of 3.4 kb predominantly expressed at high levels in small intestine, with lower levels in stomach, kidney and brain. The main regions of expression in the central nervous system were medulla, hippocampus and corpus callosum. cRNA-injected oocytes and transiently transfected HEK293 cells expressed a K+ conductance which displays an inward rectification. This conductance is blocked by cesium and barium but is insensitive to tolbutamide and diazoxide even upon co-transfection of this novel subunit with the plasmid encoding the sulfonylurea receptor SUR1. Taken together, these results demonstrate that we have isolated and characterized a novel K+ channel subunit belonging to the inwardly rectifying K+ (Kir) channel family to which, upon homology classification, we have given the nomenclature Kir7.1.

Amino Acid Sequence↗
PubMed1996

Molecular determinants for activation and inactivation of HERG, a human inward rectifier potassium channel.

1. The human eag-related potassium channel, HERG, gives rise to inwardly rectifying K+ currents when expressed in Xenopus oocytes. 2. The apparent inward rectification is caused by rapid inactivation. In extracellular Cs+ solutions, large outward currents can be recorded having an inactivation time constant at 0 mV of about 50 ms with an e-fold change every 37 mV. 3. HERG channel inactivation is not caused by an amino-terminal ball structure, as a deletion of the cytoplasmic amino terminus (HERG delta 2-373) did not eliminate inactivation. However, channel deactivation was accelerated about 12-fold at -80 mV. 4. Mutation of S631 to A, the homologous residue of eag channels, in the outer mouth of the HERG pore completely abolished channel inactivation. 5. Activity of HERG channels depended on extracellular cations, which are effective for channel activation, in the order Cs+ > K+ > > Li+ > Na+. The point mutation S631A strongly reduced this channel regulation. 6. By analogy to functional aspects of cloned voltage-gated potassium channels, rectification of HERG, as well as its kinetic properties during the course of an action potential, are presumably governed by a mechanism reminiscent of C-type inactivation.

Alanine↗