Yoshiyuki Horio

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.

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).

Clustering and enhanced activity of an inwardly rectifying potassium channel, Kir4.1, by an anchoring protein, PSD-95/SAP90.

An inwardly rectifying potassium channel predominantly expressed in glial cells, Kir4.1/KAB-2, has a sequence of Ser-Asn-Val in its carboxyl-terminal end, suggesting a possible interaction with an anchoring protein of the PSD-95 family. We examined the effects of PSD-95 on the distribution and function of Kir4.1 in a mammalian cell line. When Kir4.1 was expressed alone, the channel immunoreactivity was distributed homogeneously. In contrast, when co-expressed with PSD-95, prominent clustering of Kir4.1 in the cell membrane occurred. Kir4.1 was co-immunoprecipitated with PSD-95 in the co-expressed cells. Glutathione S-transferase-fusion protein of COOH terminus of Kir4.1 bound to PSD-95. These interactions disappeared when the Ser-Asn-Val motif was deleted. The magnitude of whole-cell Kir4.1 current was increased by 2-fold in cells co-expressing Kir4.1 and PSD-95 compared with cells expressing Kir4.1 alone. SAP97, another member of the PSD-95 family, showed similar effects on Kir4.1. Furthermore, we found that Kir4.1 as well as SAP97 distributed not diffusely but clustered in retinal glial cells. Therefore, PSD-95 family proteins may be a physiological regulator of the distribution and function of Kir4.1 in glial cells.

Immunolocalization of an inwardly rectifying K+ channel, KAB‐2 (Kir4.1), in the basolateral membrane of renal distal tubular epithelia

Immunolocalization of KAB−2 (Kir4.1), an inwardly ectifying K+ channel with a putative ATP‐binding domain, was examined in rat kidney where expression of KAB−2 mRNA was previously shown. Anti‐KAB−2 antibody was raised in rabbit and then affinity‐purified. An immunohistochemical study revealed that KAB‐2 immunoreactivity was detected specifically in the basolateral membrane of distal tubular epithelia. Therefore, KAB‐2 is the first K+ channel shown to be localized in the basolateral membrane of renal epithelia. The finding suggests that KAB‐2 may contribute to supplying K+ to the Na+‐K+ pump, which is abundant in the basolateral membrane of distal tubular epithelia, as well as to maintenance of the deep negative membrane potential of these cells.

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K^+ channel expressed in a mammalian cell line, HEK293T cells

Abstractu2002The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (KATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a Ki value of 160xa0nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATPi) equipotently in the presence and in the absence of intracellular Mg2+ (Mg2+i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATPi only in the presence of Mg2+i. Uridine 5’-diphosphate (UDP) antagonized the ATPi-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATPi-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac KATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the KATP 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.

SUR2 subtype (A and B)‐dependent differential activation of the cloned ATP‐sensitive K+ channels by pinacidil and nicorandil

The classical ATP sensitive K+ (KATP) channels are composed of a sulphonylurea receptor (SUR) and an inward rectifying K+ channel subunit (BIR/Kir6.2). They are the targets of vasorelaxant agents called K+ channel openers, such as pinacidil and nicorandil. In order to examine the tissue selectivity of pinacidil and nicorandil, in vitro, we compared the effects of these agents on cardiac type (SUR2A/Kir6.2) and vascular smooth muscle type (SUR2B/Kir6.2) of the KATP channels heterologously expressed in HEK293T cells, a human embryonic kidney cell line, by using the patch‐clamp method. In the cell‐attached recordings (145 mM K+ in the pipette), pinacidil and nicorandil activated a weakly inwardly‐rectifying, glibenclamide‐sensitive 80 pS K+ channel in both the transfected cells. In the whole‐cell configuration, pinacidil showed a similar potency in activating the SUR2B/Kir6.2 and SUR2A/Kir6.2 channels (EC50 of ∼2 and ∼10 μM, respectively). On the other hand, nicorandil activated the SUR2B/Kir6.2 channel >100 times more potently than the SUR2A/Kir6.2 (EC50 of ∼10 μM and >500 μM, respectively). Thus, nicorandil, but not pinacidil, preferentially activates the KATP channels containing SUR2B. Because SUR2A and SUR2B are diverse only in 42 amino acids at their C‐terminal ends, it is strongly suggested that this short part of SUR2B may play a critical role in the action of nicorandil on the vascular type classical KATP channel.

Somatostatin induces hyperpolarization in pancreatic islet α cells by activating a G protein-gated K+ channel

Somatostatin inhibits glucagon‐secretion from pancreatic α cells but its underlying mechanism is unknown. In mouse α cells, we found that somatostatin induced prominent hyperpolarization by activating a K+ channel, which was unaffected by tolbutamide but prevented by pre‐treating the cells with pertussis toxin. The K+ channel was activated by intracellular GTP (with somatostatin), GTPγS or Gβγ subunits. It was thus identified as a G protein‐gated K+ (KG) channel. RT‐PCR and immunohistochemical analyses suggested the KG channel to be composed of Kir3.2c and Kir3.4. This study identified a novel ionic mechanism involved in somatostatin‐inhibition of glucagon‐secretion from pancreatic α cells.

Intracellular nucleotide‐mediated gating of SUR/Kir6.0 complex potassium channels expressed in a mammalian cell line and its modification by pinacidil

1 We have examined the properties of intracellular nucleotide‐mediated gating of K+ channel constructs composed of the sulphonylurea receptor 2B and the inwardly rectifying K+ channel subunits Kir6.1 and Kir6.2 (SUR2B/Kir6.1 and SUR2B/Kir6.2 complex K+ channels) heterologously expressed in human embryonic kidney (HEK) 293T cells. In the cell‐attached form, both types of K+ channel were activated by pinacidil. 2 In inside‐out (IO) patches, the SUR2B/Kir6.2 channels opened spontaneously and were inhibited by intracellular ATP (ATPi). Pinacidil attenuated the ATPi‐mediated channel inhibition in a concentration‐dependent manner. In contrast, the SUR2B/Kir6.1 channels required intracellular nucleoside di‐ or tri‐, but not mono‐, phosphates for opening. The potency of adenine, guanine or uracil nucleotides to activate SUR2B/Kir6.1 channels was enhanced by pinacidil. 3 In the presence of pinacidil, adenine and guanine, but not uracil, nucleotides exhibited bell‐shaped concentration‐dependent activating effects on SUR2B/Kir6.1 channels. This was due to channel inhibition caused by adenine and guanine nucleotides, which was unaffected by pinacidil. 4 From power density spectrum analysis of SUR2B/Kir6.1 currents, channel activation could be described by the product of two gates, a nucleotide‐independent fast channel gate and a nucleotide‐dependent slow gate, which controlled the number of functional channels. Pinacidil specifically increased the potency of nucleotide action on the slow gate. 5 We conclude that Kir6.0 subunits play a crucial role in the nucleotide‐mediated gating of SUR/Kir6.0 complex K+ channels and may determine the molecular mode of pinacidil action.