Yoshihisa Kurachi

Inwardly Rectifying Potassium Channels: Their Structure, Function, and Physiological Roles

Inwardly rectifying K(+) (Kir) channels allow K(+) to move more easily into rather than out of the cell. They have diverse physiological functions depending on their type and their location. There are seven Kir channel subfamilies that can be classified into four functional groups: classical Kir channels (Kir2.x) are constitutively active, G protein-gated Kir channels (Kir3.x) are regulated by G protein-coupled receptors, ATP-sensitive K(+) channels (Kir6.x) are tightly linked to cellular metabolism, and K(+) transport channels (Kir1.x, Kir4.x, Kir5.x, and Kir7.x). Inward rectification results from pore block by intracellular substances such as Mg(2+) and polyamines. Kir channel activity can be modulated by ions, phospholipids, and binding proteins. The basic building block of a Kir channel is made up of two transmembrane helices with cytoplasmic NH(2) and COOH termini and an extracellular loop which folds back to form the pore-lining ion selectivity filter. In vivo, functional Kir channels are composed of four such subunits which are either homo- or heterotetramers. Gene targeting and genetic analysis have linked Kir channel dysfunction to diverse pathologies. The crystal structure of different Kir channels is opening the way to understanding the structure-function relationships of this simple but diverse ion channel family.

A Novel Sulfonylurea Receptor Forms with BIR (Kir6.2) a Smooth Muscle Type ATP-sensitive K+ Channel

We have isolated a cDNA encoding a novel isoform of the sulfonylurea receptor from a mouse heart cDNA library. Coexpression of this isoform and BIR (Kir6.2) in a mammalian cell line elicited ATP-sensitive K+ (KATP) channel currents. The channel was effectively activated by both diazoxide and pinacidil, which is the feature of smooth muscle KATP channels. Sequence analysis indicated that this clone is a variant of cardiac type sulfonylurea receptor (SUR2). The 42 amino acid residues located in the carboxyl-terminal end of this novel sulfonylurea receptor is, however, divergent from that of SUR2 but highly homologous to that of the pancreatic one (SUR1). Therefore, this short part of the carboxyl terminus may be important for diazoxide activation of KATP channels. The reverse transcription-polymerase chain reaction analysis showed that mRNA of this clone was ubiquitously expressed in diverse tissues, including brain, heart, liver, urinary bladder, and skeletal muscle. These results suggest that this novel isoform of sulfonylurea receptor is a subunit reconstituting the smooth muscle KATP channel.

On the mechanism of activation of muscarinic K+ channels by adenosine in isolated atrial cells: involvement of GTP-binding proteins

The molecular mechanisms underlying activation of a K+ channel by adenosine (Ado) and acetylcholine (ACh) were examined in single atrial cells of guinea-pig. Whole cell clamp and patch clamp techniques were used to characterize the K+ channel. In the whole cell clamp conditions, Ado and ACh increased the K+ channel current in a dose-dependent manner. The maximum responses and the apparent dissociation constants were different for Ado and ACh activations of the current. Theophylline blocked activation of the K+ current by Ado, while atropine blocked ACh-activation, indicating that two different membrane receptors were involved. Measurements of the conductance and kinetic properties of both whole cell and single channel currents indicate that Ado and ACh regulate the same K+ channels. In “inside-out” patch conditions, GTP was required in the intracellular side of the membrane for activation of the K+ channel by agonists (present in the patch electrode). The A protomer of pertussis toxin inhibited the channel activation only when NAD was also present. Furthermore, GTP-γS, a non-hydrolyzable GTP analogue, gradually caused activation of the K+ channel in the absence of agonists. Therefore, it was concluded that Ado and m-ACh receptors link with the same population of K+ channels via GTP-binding proteins Ni and/or No in the atrial cell membrane.

Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Müller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains.

Postembedding immunogold labeling was used to examine the subcellular distribution of the inwardly rectifying K+ channel Kir4.1 in rat retinal Müller cells and to compare this with the distribution of the water channel aquaporin‐4 (AQP4). The quantitative analysis suggested that both molecules are enriched in those plasma membrane domains that face the vitreous body and blood vessels. In addition, Kir4.1, but not AQP4, was concentrated in the basal ∼300–400 nm of the Müller cell microvilli. These data indicate that AQP4 may mediate the water flux known to be associated with K+ siphoning in the retina. By its highly differentiated distribution of AQP4, the Müller cell may be able to direct the water flux to select extracellular compartments while protecting others (the subretinal space) from inappropriate volume changes. The identification of specialized membrane domains with high Kir4.1 expression provides a morphological correlate for the heterogeneous K+ conductance along the Müller cell surface. GLIA 26:47–54, 1999.

SULPHONYLUREA RECEPTOR 2B AND KIR6.1 FORM A SULPHONYLUREA-SENSITIVE BUT ATP-INSENSITIVE K+ CHANNEL

1. We analysed the K+ channel composed of the sulphonylurea receptor 2B (SUR2B) and an inwardly rectifying K+ channel subunit Kir6.1 coexpressed in a mammalian cell line, HEK293T, with the patch clamp technique. 2. In the cell‐attached configuration, K+ channel openers (pinacidil and nicorandil) activated approximately 33 pS K+ channels (approximately 145 mM external K+), which were inhibited by the sulphonylurea glibenclamide. 3. Although SUR2B forms an ATP‐sensitive K+ channel with Kir6.2, whose amino acid sequence is approximately 70% homologous with that of Kir6.1, the K+ channel composed of SUR2B and Kir6.1 surprisingly did not spontaneously open on patch excision in the absence of intracellular ATP. 4. In inside‐out patches, uridine diphosphate and guanosine diphosphate induced channel activity, which was inhibited by glibenclamide but not ATP. Intracellular ATP on its own activated the channels. K+ channel openers and intracellular nucleotides synergistically activated the channel. 5. Therefore, the K+ channel composed of SUR2B and Kir6.1 is not a classical ATP‐sensitive K+ channel but closely resembles the nucleotide diphosphate‐dependent K+ channel in vascular smooth muscle cells.

International Union of Pharmacology. XLI. Compendium of Voltage-Gated Ion Channels: Potassium Channels

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

International Union of Pharmacology. LIV. Nomenclature and Molecular Relationships of Inwardly Rectifying Potassium Channels

Since the initial cDNA cloning of the first inward rectifiers Kir1.1 (ROMK1) and Kir2.1 (IRK1) in 1993, a succession of new members of this family have been identified, including the G protein-coupled Kir3 and the ATP-sensitive Kir6. These channels play an important physiological role in the

Muscarinic acetylcholine receptors

Muscarinic acetylcholine receptors mediate diverse physiological functions. At present, five receptor subtypes (M(1) - M(5)) have been identified. The odd-numbered receptors (M(1), M(3), and M(5)) are preferentially coupled to G(q/11) and activate phospholipase C, which initiates the phosphatidylinositol trisphosphate cascade leading to intracellular Ca(2+) mobilization and activation of protein kinase C. On the other hand, the even-numbered receptors (M(2) and M(4)) are coupled to G(i/o), and inhibit adenylyl cyclase activity. They also activate G protein-gated potassium channels, which leads to hyperpolarization of the plasma membrane in different excitable cells. Individual members of the family are expressed in an overlapping fashion in various tissues and cell types. Recent gene targeting approaches have unraveled the specific function of these muscarinic receptor subtypes, which were not able to be fully elucidated with pharmacological approaches because of the non-selective effects of the available ligands. Based on these findings, muscarinic receptors have been emerging as an important therapeutic target for various diseases, including dry mouth, incontinence and chronic obstructive pulmonary disease. Here we review the latest advances in the structural and functional characterization of muscarinic acetylcholine receptors and the pharmaceutical development of muscarinic receptor ligands.

Molecular and Cellular Diversity of Neuronal G-Protein-Gated Potassium Channels

Neuronal G-protein-gated potassium (GIRK) channels mediate the inhibitory effects of many neurotransmitters. Although the overlapping distribution of GIRK subunits suggests that channel composition varies in the CNS, little direct evidence supports the existence of structural or functional diversity in the neuronal GIRK channel repertoire. Here we show that the GIRK channels linked to GABAB receptors differed in two neuron populations. In the substantia nigra, GIRK2 was the principal subunit, and it was found primarily in dendrites of neurons in the substantia nigra pars compacta (SNc). Baclofen evoked prominent barium-sensitive outward current in dopamine neurons of the SNc from wild-type mice, but this current was completely absent in neurons from GIRK2 knock-out mice. In the hippocampus, all three neuronal GIRK subunits were detected. The loss of GIRK1 or GIRK2 was correlated with equivalent, dramatic reductions in baclofen-evoked current in CA1 neurons. Virtually all of the barium-sensitive component of the baclofen-evoked current was eliminated with the ablation of both GIRK2 and GIRK3, indicating that channels containing GIRK3 contribute to the postsynaptic inhibitory effect of GABAB receptor activation. The impact of GIRK subunit ablation on baclofen-evoked current was consistent with observations that GIRK1, GIRK2, and GABAB receptors were enriched in lipid rafts isolated from mouse brain, whereas GIRK3 was found primarily in higher-density membrane fractions. Altogether, our data show that different GIRK channel subtypes can couple to GABAB receptors in vivo. Furthermore, subunit composition appears to specify interactions between GIRK channels and organizational elements involved in channel distribution and efficient receptor coupling.

Molecular aspects of ATP-sensitive K+ channels in the cardiovascular system and K+ channel openers

ATP-sensitive K+ (K(ATP)) channels are inhibited by intracellular ATP (ATPi) and activated by intracellular nucleoside diphosphates and thus, provide a link between cellular metabolism and excitability. K(ATP) channels are widely distributed in various tissues and may be associated with diverse cellular functions. In the heart, the K(ATP) channel appears to be activated during ischemic or hypoxic conditions, and may be responsible for the increase of K+ efflux and shortening of the action potential duration. Therefore, opening of this channel may result in cardioprotective, as well as proarrhythmic, effects. These channels are clearly heterogeneous. The cardiac K(ATP) channel is the prototype of K(ATP) channels possessing approximately 80 pS of single-channel conductance in the presence of approximately 150 mM extracellular K+ and opens spontaneously in the absence of ATPi. A vascular K(ATP) channel called a nucleoside diphosphate-dependent K+ (K(NDP)) channel exhibits properties significantly different from those of the cardiac K(ATP) channel. The K(NDP) channel has the single-channel conductance of approximately 30-40 pS in the presence of approximately 150 mM extracellular K+, is closed in the absence of ATPi, and requires intracellular nucleoside di- or triphosphates, including ATPi to open. Nevertheless, K(ATP) and K(NDP) channels are both activated by K+ channel openers, including pinacidil and nicorandil, and inhibited by sulfonylurea derivatives such as glibenclamide. It recently was found that the cardiac K(ATP) channel is composed of a sulfonylurea receptor (SUR)2A and a two-transmembrane-type K+ channel subunit Kir6.2, while the vascular K(NDP) channel may be the complex of SUR2B and Kir6.1. By precisely comparing the functional properties of the SUR2A/Kir6.2 and the SUR2B/Kir6.1 channels, we shall show that the single-channel characteristics and pharmacological properties of SUR/Kir6.0 channels are determined by Kir and SUR subunits, respectively, while responses to intracellular nucleotides are determined by both SUR and Kir subunits.