Akikazu Fujita

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.

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.

Cloning and Functional Expression of a Novel Cardiac Two-Pore Background K+ Channel (cTBAK-1)

We have cloned from a mouse heart cDNA library a novel K+ channel subunit that has two pore-forming domains and four transmembrane regions. Its amino acid sequence shares 25% identity with mouse TWIK-1, 22% with mouse TREK-1, and 33% with a putative K+ channel of Caenorhabditis elegans (C40C9). Strikingly abundant mRNA for this clone was expressed in the heart. The mRNA was also detected in kidney, brain, skin, testis, lung, skeletal muscle, small intestine, and stomach but not in liver, thymus, or spleen. Reverse transcription-polymerase chain reaction analyses of single cells showed that the mRNA of the clone was expressed in both atrial and ventricular myocytes per se. Xenopus oocytes injected with the cRNA of the clone expressed a Ba2+-sensitive K+-selective current with an almost linear steady-state current-voltage relationship. In cell-attached patches, the expressed channel exhibited short-lasting openings with a mean open time of approximately 2 milliseconds and a unitary conductance of approximately 16 pS (150 mmol/L [K+]o). The K+ current was insensitive to intracellular Na+ (50 mmol/L), Ca2+ (0.1 mmol/L), H+ (pH 6.4), and arachidonic acid (10 micromol/L) in inside-out patches. Thus, the current flowing through the channel may contribute to the cardiac cellular electrical activity as a linear background K+ conductance. Therefore, we designated the clone cTBAK (cardiac two-pore background K+ channel).

Specific localization of an inwardly rectifying K+ channel, Kir4.1, at the apical membrane of rat gastric parietal cells; its possible involvement in K+ recycling for the H+‐K+‐pump

Hydrochloric acid (HCl) is produced in parietal cells of gastric epithelium by a H+‐K+ pump. Protons are secreted into the gastric lumen in exchange for K+ by the action of the H+‐K+‐ATPase. Luminal K+ is essential for the operation of the pump and is thought to be supplied by unidentified K+ channels localized at the apical membrane of parietal cells. In this study, we showed that histamine‐ and carbachol‐induced acid secretion from isolated parietal cells monitored by intracellular accumulation of aminopyrine was depressed by Ba2+, an inhibitor of inwardly rectifying K+ channels. Among members of the inwardly rectifying K+ channel family, we found with reverse transcriptase‐polymerase chain reaction analyses that Kir4.1, Kir4.2 and Kir7.1 were expressed in rat gastric mucosa. With immunohistochemical analyses, Kir4.1 was found to be expressed in gastric parietal cells and localized specifically at their apical membrane. The current flowing through Kir4.1 channel expressed in HEK293T cells was not affected by reduction of extracellular pH from 7.4 to 3. These results suggest that Kir4.1 may be involved in the K+ recycling pathway in the apical membrane which is required for activation of the H+‐K+ pump in gastric parietal cells.

C-Terminal Tails of Sulfonylurea Receptors Control ADP-Induced Activation and Diazoxide Modulation of ATP-Sensitive K+ Channels

The ATP-sensitive K+ (KATP) channels are composed of the pore-forming K+ channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle–type KATP channels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-KATP channels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of KATP channels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on KATP channels containing different SURs.

Expression of an inwardly rectifying K+ channel, Kir5.1, in specific types of fibrocytes in the cochlear lateral wall suggests its functional importance in the establishment of endocochlear potential

Cochlear endolymph contains 150u2003mm K+ and has a highly positive potential of ≈u200a+80u2003mV. The specialized ionic composition and high potential in endolymph are essential for hearing and maintained by circulation of K+ from perilymph to endolymph through the cochlear lateral wall. Various types of K+ channel such as Kir4.1 and KCNQ1/KCNE1 are expressed in stria vascularis of the lateral wall and play essential roles in K+ circulation. In this study, we examined a distribution of another K+ channel, Kir5.1, and found it specifically expressed in the spiral ligament of the cochlear lateral wall. Specific immunoreactivity for Kir5.1 was detected in typeu2003II, IV and V fibrocytes of the ligament and spiral limbus, all of which are directly involved in K+ circulation. Kir5.1 was not found in either typeu2003I or III fibrocytes. Although Kir5.1 assembles with Kir4.1 to form a functional Kir channel in renal epithelia and retinal Müller cells, double‐immunolabelling revealed that they were expressed in distinct regions in the cochlea lateral wall, i.e. Kir4.1 only in stria vascularis vs. Kir5.1 in spiral ligament. During development, the expression of Kir5.1 subunits started significantly later than Kir4.1 and was correlated with the ‘rapid’ phase of the elevation of endocochlear potential (EP). Kir5.1 and Kir4.1 channel‐subunits may therefore play distinct functional roles in K+u2003circulation in the cochlear lateral wall.

Functional Kir7.1 channels localized at the root of apical processes in rat retinal pigment epithelium

1 The inwardly rectifying K+ channel current (IK(IR)) recorded from isolated retinal pigmented epithelial (RPE) cells showed poor dependence on external K+ ([K+]o) and low sensitivity to block by Ba2+. We examined the molecular identity and specific subcellular localization of the KIR channel in RPE cells. 2 The Kir7.1 channel current heterologously expressed in HEK293T cells (human embryonic kidney cell line) showed identical properties to those of the RPE IK(IR), i.e. poor dependence on [K+]o and low sensitivity to Ba2+ block. 3 Expression of Kir7.1 mRNA and protein was detected in RPE cells by RT‐PCR and immunoblot techniques, respectively. 4 Immunohistochemical studies including electron microscopy revealed that the Kir7.1 channel was localized specifically at the proximal roots of the apical processes of RPE cells, where Na+,K+‐ATPase immunoreactivity was also detected. 5 The middle‐distal portions of apical processes of RPE cells in the intact tissue exhibited immunoreactivity of Kir4.1, a common KIR channel. In the isolated RPE cells, however, Kir4.1 immunoreactivity was largely lost, while Kir7.1 immunoreactivity remained. 6 These data indicate that the only IK(IR) recorded in isolated RPE cells is derived from the functional Kir7.1 channel localized at the root of apical processes. Co‐localization with Na+,K+‐ATPase suggests that the Kir7.1 channel may provide the pathway for recycling of K+ to maintain pump activity and thus is essential for K+ handling in RPE cells.

Anchoring proteins confer G protein sensitivity to an inward-rectifier K(+) channel through the GK domain.

Anchoring proteins cluster receptors and ion channels at postsynaptic membranes in the brain. They also act as scaffolds for intracellular signaling molecules including synGAP and NO synthase. Here we report a new function for intracellular anchoring proteins: the regulation of synaptic ion channel function. A neuronal G protein‐gated inwardly rectifying K+ channel, Kir3.2c, can not be activated either by M2‐muscarinic receptor stimulation or by Gβγ overexpression. When coexpressed with SAP97, a member of the PSD/SAP anchoring protein family, the channel became sensitive to G protein stimulation. Although the C‐terminus of Kir3.2c bound to the second PDZ domain of SAP97, functional analyses revealed that the guanylate kinase (GK) domain of SAP97 is crucial for sensitization of the Kir3.2c channel to G protein stimulation. Furthermore, SAPAP1/GKAP, which binds specifically to the GK domain of membrane‐associated guanylate kinases, prevented the SAP97‐induced sensitization. The function of a synaptic ion channel can therefore be controlled by a network of various intracellular proteins.

Molecular cloning and characterization of a novel splicing variant of the Kir3.2 subunit predominantly expressed in mouse testis

1 One of the features of weaver mutant mice is male infertility, which suggests that Kir3.2, a G‐protein‐gated inwardly rectifying K+ channel subunit, may be involved in spermatogenesis. Therefore, we have characterized the Kir3.2 isoform in mouse testis using immunological, molecular biological and electrophysiological techniques. 2 Testicular membrane contained a protein that was recognized by the antibody specific to the C‐terminus of Kir3.2c (aG2C‐3). Its molecular mass was ≈45 kDa, which was smaller than that of Kir3.2c (≈48 kDa). The immunoprecipitant obtained from testis with aG2C‐3 contained a single band of the 45 kDa protein, which could not be detected by the antibody to the N‐terminus common to the known Kir3.2 isoforms (aG2N‐2). 3 A novel alternative splicing variant of Kir3.2, designated Kir3.2d, was isolated from a mouse testis cDNA library. The cDNA had an open reading frame encoding 407 amino acids, whose molecular mass was calculated to be ≈45 kDa. Kir3.2d was 18 amino acids shorter than Kir3.2c at its N‐terminal end, which was the only difference between the two clones. The 18 amino acid region possesses the epitope for aG2N‐2. 4 In heterologous expression systems of both Xenopus oocytes and mammalian cells (HEK 293T), Kir3.2d either alone or with Kir3.1 exhibited G‐protein‐gated inwardly rectifying K+ channel activity. 5 Prominent Kir3.2d immunoreactivity in the testis was detected exclusively in the acrosomal vesicles of spermatids, while Kir3.1 immunoreactivity was diffuse in the spermatogonia and spermatocytes. These results indicate the possibility that the testicular variant of Kir3.2, Kir3.2d, may assemble to form a homomultimeric G‐protein‐gated K+ channel and be involved in the development of the acrosome during spermiogenesis.

PSD-95 mediates formation of a functional homomeric Kir5.1 channel in the brain.

Homomeric assembly of Kir5.1, an inward-rectifying K+ channel subunit, is believed to be nonfunctional, although the subunit exists abundantly in the brain. We show that HEK293T cells cotransfected with Kir5.1 and PSD-95 exhibit a Ba(2+)-sensitive inward-rectifying K+ current. Kir5.1 coexpressed with PSD-95 located on the plasma membrane in a clustered manner, while the Kir5.1 subunit expressed alone distributed mostly in cytoplasm, probably due to rapid internalization. The binding of Kir5.1 with PSD-95 was prevented by protein kinase A (PKA)-mediated phosphorylation of its carboxyl terminus. The currents flowing through Kir5.1/PSD-95 were suppressed promptly and reversibly by PKA activation. Because the Kir5.1/PSD-95 complex was detected in the brain, this functional brain K+ channel is potentially a novel physiological target of PKA-mediated signaling.