Suppressive effects of nonsteroidal anti-inflammatory drugs diclofenac sodium, salicylate and indomethacin on delayed rectifier K+-channel currents in murine thymocytes.

Lymphocytes predominantly express delayed rectifier K(+)-channels (Kv1.3) in their plasma membranes, and the channels play crucial roles in the lymphocyte activation and proliferation. Since nonsteroidal anti-inflammatory drugs (NSAIDs), the most commonly used analgesic and antipyretic drugs, exert immunomodulatory effects, they would affect the channel currents in lymphocytes. In the present study, employing the standard patch-clamp whole-cell recording technique, we examined the effects of diclofenac sodium, salicylate and indomethacin on the channel currents in murine thymocytes and the membrane capacitance. Diclofenac sodium and salicylate significantly suppressed the pulse-end currents of the channel. However, indomethacin suppressed both the peak and the pulse-end currents with a significant increase in the membrane capacitance. This study demonstrated for the first time that NSAIDs, such as diclofenac sodium, salicylate and indomethacin, exert inhibitory effects on thymocyte Kv1.3-channel currents. The slow inactivation pattern induced by indomethacin was thought to be associated with microscopic changes in the plasma membrane surface detected by the increase in the membrane capacitance.

Compensatory thrombopoietin production from the liver and bone marrow stimulates thrombopoiesis of living rat megakaryocytes in chronic renal failure.

BACKGROUND/AIMS: Decreased thrombopoiesis has been ascribed a role in the pathogenesis of uremic bleeding in chronic renal failure (CRF). However, serum thrombopoietin (TPO) levels are usually elevated in CRF patients, suggesting increased thrombopoiesis. The aim of this study was to determine the thrombopoietic activity in CRF. METHODS: Male Sprague-Dawley rats that underwent 5/6 nephrectomy were used as the model of CRF. Age-matched sham-operated rats were used as controls. Single megakaryocytes were isolated from the rat bone marrow, and their size distribution was examined. Megakaryocyte membrane invaginations were monitored by confocal imaging of di-8-ANEPPS staining, and patch clamp whole-cell recordings of membrane capacitance. TPO gene expression was assessed in various tissues. RESULTS: Circulating platelet counts and the number of large megakaryocytes were increased in the bone marrow of CRF rats. Massive di-8-ANEPPS staining and increased membrane capacitance in large megakaryocytes demonstrated increased membrane invaginations. Unaffected Kv1.3-channel currents per cell surface area demonstrated unaltered channel densities. TPO transcription was decreased in the renal cortex but increased in the liver and bone marrow of CRF rats. CONCLUSION: Increased thrombopoiesis in CRF was thought to be a reactive mechanism to platelet dysfunction. Increased TPO production from the liver and bone marrow compensated for decreased production from damaged kidneys.

Voltage-sensing phosphatase: actions and potentials.

Voltage sensors have been well studied in voltage-gated ion channels for neuronal excitation and muscle contraction. The recent discovery of a voltage-sensing phosphatase, VSP, has changed the idea that voltage sensors are unique to ion flux through membranes. Recent findings on mechanisms and potential applications of VSP are reviewed.

Asymmetry of Rb+ conduction emerged under bi-ionic conditions in epithelial maxi-K+ channels.

K(+) channels permit more than one ion within their conducting pathway at any given moment and show a saturating single-file behavior. The conduction of Rb(+) shows an unusual behavior, a so-called "Rb(+) anomaly," and it has been used to probe the mechanism of the ion conduction through K(+)-selective channels. Under the bi-ionic condition of K(+) and Rb(+), we carried out patch-clamp single-channel current measurements in MaxiK(+) channels from mouse submandibular acinar cells. Keeping only K(+) on one side of the membrane while varying fractional Rb(+) concentration on the opposite, we had a series of current-voltage relationships. It showed a characteristic inflection at which the ion conductance was divided into two components, one ascribed to pure K(+) conduction and the other to K(+) and Rb(+) bi-ionic conduction. By analyzing the latter, we depicted that (1) the bi-ionic conductance showed a characteristic reduction curve as the Rb(+) fractional concentration increased; (2) Rb(+) can bind the channel more tightly when it accesses from the outside than from the inside. Thus we conclude that such asymmetry of the Rb(+) binding determines the pattern of bi-ionic conductance reduction in K-selective channels.

A voltage-sensing phosphatase, Ci-VSP, which shares sequence identity with PTEN, dephosphorylates phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol lipids play diverse physiological roles, and their concentrations are tightly regulated by various kinases and phosphatases. The enzymatic activity of Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP), recently identified as a member of the PTEN (phosphatase and tensin homolog deleted on chromosome 10) family of phosphatidylinositol phosphatases, is regulated by its own voltage-sensor domain in a voltage-dependent manner. However, a detailed mechanism of Ci-VSP regulation and its substrate specificity remain unknown. Here we determined the in vitro substrate specificity of Ci-VSP by measuring the phosphoinositide phosphatase activity of the Ci-VSP cytoplasmic phosphatase domain. Despite the high degree of identity shared between the active sites of PTEN and Ci-VSP, Ci-VSP dephosphorylates not only the PTEN substrate, phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], but also, unlike PTEN, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Enzymatic action on PI(4,5)P2 removes the phosphate at position 5 of the inositol ring, resulting in the production of phosphatidylinositol 4-phosphate [PI(4)P]. The active site Cys-X(5)-Arg (CX(5)R) sequence of Ci-VSP differs with that of PTEN only at amino acid 365 where a glycine residue in Ci-VSP is replaced by an alanine in PTEN. Ci-VSP with a G365A mutation no longer dephosphorylates PI(4,5)P2 and is not capable of inducing depolarization-dependent rundown of a PI(4,5)P2-dependent potassium channel. These results indicate that Ci-VSP is a PI(3,4,5)P3/PI(4,5)P2 phosphatase that uniquely functions in the voltage-dependent regulation of ion channels through regulation of PI(4,5)P2 levels.

Depolarization activates the phosphoinositide phosphatase Ci-VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2.

Voltage-evoked signals play critical roles in neural activities, muscle contraction and exocytosis. Ciona voltage-sensor containing phosphatase (Ci-VSP) consists of the transmembrane voltage sensor domain (VSD) and a cytoplasmic domain of phosphoinositide phosphatase, homologous to phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Previous experiments utilizing potassium channels as the sensor for phosphoinositides have demonstrated that phosphatase activities of Ci-VSP are voltage dependent. However, it still remained unclear whether enzyme activity is activated by depolarization or hyperpolarization. Further, a large gap in voltage dependency was found between the charge movement of the VSD and potassium channel-reporting phosphatase activities. In this study, voltage-dependent dynamics of phosphoinositides mediated by Ci-VSP were examined by confocal imaging and electrical measurements in Xenopus oocytes. Imaging of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) using green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains from phospholipase C delta subunit (PLC-delta) showed that PtdIns(4,5)P(2) concentration is reduced during depolarization. In the presence of Ci-VSP, IRK1 channels with higher sensitivity to phosphoinositide than GIRK2 channels decreased their magnitude during depolarization over 0 mV, indicating that the PtdIns(4,5)P(2) level is reduced upon depolarization. KCNQ2/3 channels coexpressed with Ci-VSP exhibited voltage-dependent decay of the outward current that became sharper with higher depolarization in a voltage range up to 100 mV. These results indicate that Ci-VSP has an activity that depletes PtdIns(4,5)P(2) unlike PTEN and that depolarization-activated voltage sensor movement is translated into activation of phosphatase activity.

Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes.

Ion fluxes through membrane ion channels play crucial roles both in neuronal signaling and the homeostatic control of body electrolytes. Despite our knowledge about the respective ion channels, just how diversification of ion channel genes underlies adaptation of animals to the physical environment remains unknown. Here we systematically survey up to 160 putative ion channel genes in the genome of Ciona intestinalis and compare them with corresponding gene sets from the genomes of the nematode Chaenorhabditis elegans, the fruit fly Drosophila melanogaster, and the more closely related genomes of vertebrates. Ciona has a set of so-called "prototype" genes for ion channels regulating neuronal excitability, or for neurotransmitter receptors, suggesting that genes responsible for neuronal signaling in mammals appear to have diversified mainly via gene duplications of the more restricted members of ancestral genomes before the ascidian/vertebrate divergence. Most genes responsible for modulation of neuronal excitability and pain sensation are absent from the ascidian genome, suggesting that these genes arose after the divergence of urochordates. In contrast, the divergent genes encoding connexins, transient receptor potential-related channels and chloride channels, channels involved rather in homeostatic control, indicate gene duplication events unique to the ascidian lineage. Because several invertebrate-unique channel genes exist in Ciona genome, the crown group of extant vertebrates not only acquired novel channel genes via gene/genome duplications but also discarded some ancient genes that have persisted in invertebrates. Such genome-wide information of ion channel genes in basal chordates enables us to begin correlating the innovation and remodeling of genes with the adaptation of more recent chordates to their physical environment.

Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor.

Changes in membrane potential affect ion channels and transporters, which then alter intracellular chemical conditions. Other signalling pathways coupled to membrane potential have been suggested but their underlying mechanisms are unknown. Here we describe a novel protein from the ascidian Ciona intestinalis that has a transmembrane voltage-sensing domain homologous to the S1-S4 segments of voltage-gated channels and a cytoplasmic domain similar to phosphatase and tensin homologue. This protein, named C. intestinalis voltage-sensor-containing phosphatase (Ci-VSP), displays channel-like 'gating' currents and directly translates changes in membrane potential into the turnover of phosphoinositides. The activity of the phosphoinositide phosphatase in Ci-VSP is tuned within a physiological range of membrane potential. Immunocytochemical studies show that Ci-VSP is expressed in Ciona sperm tail membranes, indicating a possible role in sperm function or morphology. Our data demonstrate that voltage sensing can function beyond channel proteins and thus more ubiquitously than previously realized.

Identification of a site involved in the block by extracellular Mg(2+) and Ba(2+) as well as permeation of K(+) in the Kir2.1 K(+) channel.

The inward rectifier potassium channel Kir2.1 is more sensitive to the weakly voltage-dependent block by extracellular Mg(2+) (Mg2+o) than Kir2.2 and Kir2.3. We identified Glu125 in an extracellular loop before the pore region of Kir2.1 as a site responsible for this sensitivity to M2+o block, based on the observations that the Glu125Gln (E125Q) mutation strongly decreased the sensitivity, while a mutation to Glu at the corresponding sites of Kir2.2 and 2.3 led to an increase. The negative charge proved to be crucial since the Glu125Asp (E125D) mutant showed similar properties to the wild type (WT). A similar weakly voltage-dependent block was also caused by extracellular Ca(2+) and La(3+) in Kir2.1 WT but not in the E125Q mutant. The sensitivity to block by extracellular Ba(2+) (Ba2+o) was also decreased in the E125Q mutant, although the voltage dependency of half-inhibition concentration was not changed, as reported previously. We additionally observed that the speed of Ba2+o block and recovery was decelerated by the presence of Mg2+o in WT, but not in the E125Q mutant. The sensitivity to the block by Mg2+o was increased by lowering extracellular K(+) (K+o), suggesting a competitive interaction of Mg2+o and K+o. The single-channel conductance of the WT in 140 mM K(+) was 39.6 pS (0 mM Mg2+o) and 11.5 pS (10 mM), while that of the E125Q mutant was 26.0 pS (0 mM) and 19.6 pS (10 mM). These results demonstrate that Mg(2+) competes with K(+) permeation in the WT and that E125 is required for efficient K(+) permeation in the absence of Mg2+o. We conclude that E125 in an extracellular loop of Kir2.1 is a site which facilitates K(+) permeation and entry of Ba(2+) toward a deeper plugging site, and that Mg2+o competes with K+o and Ba2+o at this site.

Alternative splicing of RGS8 gene determines inhibitory function of receptor type-specific Gq signaling.

The regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling. RGS8 is a brain-specific RGS protein of 180 aa. Here we identified a short isoform of RGS8, RGS8S, that arises by alternative splicing. RGS8S cDNA encodes a N terminus of 7 aa instead of amino acids 1-9 of RGS8 and 10-180 of RGS8. The subcellular distribution of RGS8 and RGS8S did not differ significantly in transfected cells. RGS8S accelerated, not as efficiently as RGS8, the turning on and off of Gi/o-mediated modulation of G protein-gated inwardly rectifying K(+) channels in Xenopus oocytes. We next examined the effects of RGS8 and RGS8S on Gq-mediated signaling. RGS8 decreased the amplitude of the response upon activation of m1 muscarinic or substance P receptors, but did not remarkably inhibit signaling from m3 muscarinic receptors. In contrast, RGS8S showed much less inhibition of the response of either of these Gq-coupled receptors. By quantitative analysis of the inhibitory effect and the protein expression level, we confirmed that the difference of inhibitory effect is caused by both the qualitative difference between RGS8 and RGS8S and the quantitative difference of the protein expression level. We also confirmed that the receptor-type specificity of inhibition is not caused by the difference of the expression level of the receptors. In summary, we showed that 9 aa in the N terminus of RGS8 contribute to the function to inhibit Gq-coupled signaling in a receptor type-specific manner and that the regulatory function of RGS8S is especially diminished on Gq-coupled responses.