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The KCNQI [K.sup.+] channel plays a key role in the regulation of several physiological functions, including cardiac excitability, cardiovascular tone, and body electrolyte homeostasis. The metabolic sensor AMP-activated protein kinase (AMPK) has been shown to regulate a growing number of ion transport proteins. To determine whether AMPK regulates KCNQ1, we studied the effects of AMPK activation on KCNQ1 currents in Xenopus laevis oocytes and collecting duct epithelial cells. AMPK activation decreased KCNQ1 currents and channel surface expression in X. laevis oocytes, but AMPK did not phosphorylate KCNQI in vitro, suggesting an indirect regulatory mechanism. As it has been recently shown that the ubiquitin-protein ligase Nedd4-2 inhibits KCNQI plasma membrane expression and that AMPK regulates epithelial [Na.sup.+] channels via Nedd4-2, we examined the role of Nedd4-2 in the AMPK-dependent regulation of KCNQl. Channel inhibition by AMPK was blocked in oocytes coexpressing either a dominant-negative or constitutively active Nedd4-2 mutant, or a Nedd4-2 interaction-deficient KCNQ1 mutant, suggesting that Nedd4-2 participates in the regulation of KCNQ1 by AMPK. KCNQ1 is expressed at the basolateral membrane in mouse polarized kidney cortical collecting duct ([mpkCCD.sub.c14]) cells and in rat kidney. Treatment with the AMPK activators AICAR (2 mM) or metformin (1 mM) reduced basolateral KCNQ1 currents in apically permeabilized polarized [mpkCCD.sub.c14] cells. Moreover, AICAR treatment of rat kidney slices ex vivo induced AMPK activation and intracellular redistribution of KCNQ1 from the basolateral membrane in collecting duct principal cells. AICAR treatment also induced increased ubiquitination of KCNQ1 immunoprecipitated from kidney slice homogenates. These results indicate that AMPK inhibits KCNQ 1 activity by promoting Nedd4-2-dependent channel ubiquitination and retrieval from the plasma membrane. KCNEI; ion channels; potassium transport doi: 10.1152/ajprenal.00423.2010

The voltage-gated potassium channel Kvl.3 has been recently identified as a molecular target that allows the selective pharmacological suppression of effector memory T cells ([T.sub.EM]) without affecting the function of naive T cells (TN) and central memory T cells ([T.sub.CM]). We found that Kvl.3 was expressed on glomeruli and some tubules in rats with anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). A flow cytometry analysis using kidney cells revealed that most of the [CD4.sup.+] T cells and some of the [CD8.sup.+] T cells had the [T.sub.EM] phenotype ([CD45RC.sup.-][CD62L.sup.-]). Double immunofluorescence staining using mononuclear cell suspensions isolated from anti-GBM GN kidney showed that Kvl.3 was expressed on T cells and some macrophages. We therefore investigated whether the Kvl.3 blocker Psora-4 can be used to treat anti-GBM GN. Rats that had been given an injection of rabbit anti-rat GBM antibody were also injected with Psora-4 or the vehicle intraperitoneally. Rats given Psora-4 showed less proteinuria and fewer crescentic glomeruli than rats given the vehicle. These results suggest that [T.sub.EM] and some macrophages expressing Kvl.3 channels play a critical role in the pathogenesis of crescentic GN and that Psora-4 will be useful for the treatment of rapidly progressive glomerulonephritis. WKY rats; crescentic glomerulonephritis; flow cytometric analysis doi: 10.1152/ajprenal.00374.2010

Loss or dysfunction of podocytes is a major cause of glomerular kidney disease. Several genetic forms of glomerular disease are caused by mutations in genes that encode structural elements of the slit diaphragm or the underlying cytoskeleton of podocyte foot processes. The recent discovery that gain-of-function mutations in [Ca.sup.2+]-permeable canonical transient receptor potential-6 channels (TRPC6) underlie a subset of familial forms of focal segmental glomerulosclerosis (FSGS) has focused attention on the basic cellular physiology of podocytes. Several recent studies have examined the role of [Ca.sup.2+] dynamics in normal podocyte function and their possible contributions to glomerular disease. This review summarizes the properties of TRPC6 and related channels, focusing on their permeation and gating properties, the nature of mutations associated with familial FSGS, and the role of TRPC channels in podocyte cell biology as well as in glomerular pathophysiology. TRPC6 interacts with several proteins in podocytes, including essential slit diaphragm proteins and mechanosensitive large-conductance [Ca.sup.2+]-activated [K.sup.+] channels. The signaling dynamics controlling ion channel function and localization in podocytes appear to be quite complex. cytoskeleton; glomerular disease; renal function; FSGS doi: 10.1152/ajprenal.00298.2010.

Sulfonylurea receptor-containing ATP-sensitive potassium ([K.sub.ATP]) channels have been implicated in cardioprotection, but the cell type and constitution of channels responsible for this protection have not been clear. Mice deleted for the first nucleotide binding region of sulfonylurea receptor 2 (SUR2) are referred to as SUR2 null since they lack full-length SUR2 and glibenclamide-responsive [K.sub.ATP] channels in cardiac, skeletal, and smooth muscle. As previously reported, SUR2 null mice develop electrocardiographic changes of ST segment elevation that were shown to correlate with coronary artery vasospasm. Here we restored expression of the cardiomyocyte SUR2-[K.sub.ATP] channel in SUR2 null mice by generating transgenic mice with ventricular cardiomyocyte-restricted expression of SUR2A. Introduction of the cardiomyocyte SUR2A transgene into the SUR2 null background restored functional cardiac [K.sub.ATP] channels. Hearts isolated from rescued mice, referred to as MLC2A, had significantly reduced infarct size (27 [+ or -] 3% of area at risk) compared with SUR2 null mice (36 [+ or -] 3% of area at risk). Compared with SUR2 null hearts, MLC2A hearts exhibited significantly improved cardiac function during the postischemia reperfusion period primarily because of preservation of low diastolic pressures. Additionally, restoration of cardiac SUR2-[K.sub.ATP] channels significantly reduced the degree and frequency of ST segment elevation episodes in MLC2A mice. Therefore, cardioprotective mechanisms both dependent and independent of SUR2-[K.sub.ATP] channels contribute to cardiac function. ATP-sensitive potassium channel; SUR2; SUR2A; ischemia; vasospasm doi: 10.1152/ajpheart.00084.2010.

Mechanosensitive large-conductance [Ca.sup.2+] or -activated [K.sup.+] channels encoded by the Slo1 gene ([BK.sub.Ca] channels) are expressed in podocytes. Here we show that [BK.sub.Ca] channels reciprocally coimmunoprecipitate with synaptopodin (Synpo) in mouse glomeruli, in mouse podocytes, and in a heterologous expression system (HEK293T cells) in which these proteins are transiently expressed. Synpo and Slo1 colocalize along the surface of the glomerular basement membrane in mouse glomeruli. Synpo interacts with [BK.sub.Ca] channels at COOH-terminal domains that overlap with an actin-binding domain on the channel molecule that is necessary for trafficking of [BK.sub.Ca] channels to the cell surface. Moreover, addition of exogenous [beta]-actin to mouse podocyte lysates reduces [BK.sub.Ca]-Synpo interactions. Coexpression of Synpo increases steady-state surface expression of [BK.sub.Ca] channels in HEK293T cells. However, Synpo does not affect the stability of cell surface [BK.sub.Ca] channels, suggesting a primary effect on the rate of forward trafficking, and Synpo coexpression does not affect [BK.sub.Ca] gating. Conversely, stable knockdown of Synpo expression in mouse podocyte cell lines reduces steady-state surface expression of [BK.sub.Ca] channels but does not affect total expression of [BK.sub.Ca] channels or their gating. The effects of Synpo on surface expression of [BK.sub.Ca] are blocked by inhibition of Rho signaling in HEK293T cells and in podocytes. Functional cell surface [BK.sub.Ca] channels in podocytes are also reduced by sustained (2 h) but not acute (15 min) depolymerization of actin with cytochalasin D. Synpo may regulate [BK.sub.Ca] channels through its effects on actin dynamics and by modulating interactions between [BK.sub.Ca] channels and regulatory proteins of the podocyte slit diaphragm. potassium channels; slit diaphragm; traffic; renal glomerulus; cytoskeleton doi: 10.1152/ajprenal.00206.2010.

Large-conductance, calcium-activated potassium channels (BK) are expressed in principal cells (PC) and intercalated cells (IC) in mammalian nephrons as BK-[alpha]/[beta]1 and BK-[alpha]/[beta]4, respectively. IC, which protrude into the lumens of tubules, express substantially more BK than PC despite lacking sufficient Na-K-ATPase to support K secretion. We previously showed in mice that IC exhibit size reduction when experiencing high distal flows induced by a high-K diet. We therefore tested the hypothesis that BK-[alpha]/[beta]4 are regulators of IC volume via a shear stress ([tau])-induced, calcium-dependent mechanism, resulting in a reduction in intracellular K content. We determined by Western blot and immunocytochemical analysis that C11-Madin-Darby canine kidney cells contained a predominance of BK-[alpha]/[beta]4. To determine the role of BK-[alpha]/[beta]4 in [tau]-induced volume reduction, we exposed C11 cells to [tau] and measured K efflux by flame photometry and cell volume by calcein staining, which changes inversely to cell volume. With 10 dynes/[cm.sup.2], calcein intensity significantly increased 39% and monovalent cationic content decreased significantly by 37% compared with static conditions. Furthermore, the shear-induced K loss from C11 was abolished by the reduction of extracellular calcium, addition of 5 mM TEA, or BK-[beta]4 small interfering (si) RNA, but not by addition of nontarget siRNA. These results show that BK-[alpha]/[beta]4 plays a role in shear-induced K loss from IC, suggesting that BK-[alpha]/[beta]4 regulate IC volume during high-flow conditions. Furthermore, these results support the use of C11 cells as in vitro models for studying BK-related functions in IC of the kidney. potassium; volume regulation; mechanotransduction; intercalated cells; parallel plate flow chamber; calcein doi: 10.1152/ajprenal.00222.2010.

Human ether-a-go-go-related gene (HERG) potassium channels play an important role in cardiac action potential repolarization, and HERG dysfunction can cause cardiac arrhythmias. However, recent evidence suggests a role for HERG in the proliferation and progression of multiple types of cancers, making it an attractive target for cancer therapy. Ceramide is an important second messenger of the sphingolipid family, which due to its proapoptotic properties has shown promising results in animal models as an anticancer agent. Yet the acute effects of ceramide on HERG potassium channels are not known. In the present study we examined the effects of cell-permeable [C.sub.6]-ceramide on HERG potassium channels stably expressed in HEK293 cells. [C.sub.6]-ceramide (10 [micro]M) reversibly inhibited HERG channel current ([I.sub.HERG]) by 36 [+ or -] 5%. Kinetically, ceramide induced a significant hyperpolarizing shift in the current-voltage relationship ([DELTA][V.sub.1/2] = -8 [+ or -] 0.5 mV) and increased the deactivation rate (43 [+ or -] 3% for [[tau].sub.fast] and 51 [+ or -] 3% for [[tau].sub.low]). Mechanistically, ceramide recruited HERG channels within caveolin-enriched lipid rafts. Cholesterol depletion and repletion experiments and mathematical modeling studies confirmed that inhibition and gating effects are mediated by separate mechanisms. The ceramide-induced hyperpolarizing gating shift (raft mediated) could offset the impact of inhibition (raft independent) during cardiac action potential repolarization, so together they may nullify any negative impact on cardiac rhythm. Our results provide new insights into the effects of [C.sub.6]-ceramide on HERG channels and suggest that [C.sub.6]-ceramide can be a promising therapeutic for cancers that overexpress HERG. human ether-a-go-go-related gene; activation kinetics; deactivation kinetics; cholesterol; inhibition doi: 10.1152/ajpcell.00462.2009.

Large-conductance voltage- and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstruction-induced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 ([MLC.sub.20]) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel [alpha]- and [beta]-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BK[beta] mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK [beta]-subunit was greater than that of the BK [alpha]-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK [beta]-subunit was employed to study the effect of BK depletion on [MLC.sub.20] phosphorylation. siRNA-mediated BK channel reduction was associated with an increased [MLC.sub.20] phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of [MLC.sub.20] phosphorylation. benign prostatic hyperplasia; MLC phosphorylation doi: 10.1152/ajprenal.00595.2009.

In ether-a-go-go (eag) [K.sup.+] channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are ~4 [Angstrom] apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular [Mg.sup.2+]. In I287D+F324D, [Mg.sup.2+] significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that [Mg.sup.2+] binds to the voltage sensor in the resting conformation. Supporting this conclusion, [Mg.sup.2+] shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for [Mg.sup.2+] binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation. doi/ 10.1085/jgp.200910320

Zhao H, Sprunger LK, Simasko SM. Expression of transient receptor potential channels and two-pore potassium channels in subtypes of vagal afferent neurons in rat. Am J Physiol Gastrointest Liver Physiol 298: G212-G221, 2010. First published December 3, 2009; doi: 10.1152/ajpgi.00396.2009.--Vagal afferent neurons relay important information regarding the control of the gastrointestinal system. However, the ionic mechanisms that underlie vagal activation induced by sensory inputs are not completely understood. We postulate that transient receptor potential (TRP) channels and/or two-pore potassium (K2p) channels are targets for activating vagal afferents. In this study we explored the distribution of these channels in vagal afferents by quantitative PCR after a capsaicin treatment to eliminate capsaicinsensitive neurons, and by single-cell PCR measurements in vagal afferent neurons cultured after retrograde labeling from the stomach or duodenum. We found that TRPC1/3/5/6, TRPV1-4, TRPM8, TRPA1, TWIK2, TRAAK, TREK1, and TASK1/2 were all present in rat nodose ganglia. Both lesion results and single-cell PCR results suggested that TRPA1 and TRPCI were preferentially expressed in neurons that were either capsaicin sensitive or TRPV1 positive. Expression of TRPM8 varied dynamically after various manipulations, which perhaps explains the disparate results obtained by different investigators. Last, we also examined ion channel distribution with the A-type CCK receptor (CCK-[R.sub.A]) and found there was a significant preference for neurons that express TRAAK to also express CCK-[R.sub.A], especially in gut-innervating neurons. These findings, combined with findings from prior studies, demonstrated that background conductances such as TRPC1, TRPA1, and TRAAK are indeed differentially distributed in the nodose ganglia, and not only do they segregate with specific markers, but the degree of overlap is also dependent on the innervation target. nodose; vagal afferent; TRP; K2p; CCK-[R.sub.A] doi: 10.1152/ajpgi.00396.2009

The follicular phase of the ovine ovarian cycle demonstrates parallel increases in ovarian estrogens and uterine blood flow (UBF). Although estrogen and nitric oxide contribute to the rise in UBF, the signaling pathway remains unclear. We examined the relationship between the rise in UBF during the ovarian cycle of nonpregnant sheep and changes in the uterine vascular cGMP-dependent pathway and large-conductance Ca2+-activated K+ channels (BKca). Nonpregnant ewes (n = 19) were synchronized to either follicular or luteal phase using a vaginal progesterone-releasing device (CIDR), followed by intramuscular [PGF2.sub.[alpha]], CIDR removal, and treatment with pregnant mare serum gonadotropin. UBF was measured with flow probes before tissue collection, and second-generation uterine artery segments were collected from nine follicular and seven luteal phase ewes. The poreforming [alpha]- and regulatory [beta]-subunits that constitute the [BK.sub.Ca], soluble guanylyl cyclase (sGC), and cGMP-dependent protein kinase G (cPKG) isoforms ([cPKG.sub.1[alpha]] and [cPKG.sub.1[beta]]) were measured by Western analysis and cGMP levels by RIA. BKca subunits were localized by immunohistochemistry. UBF rose >3-fold (P < 0.04) in follicular phase ewes, paralleling a 2.3-fold rise in smooth muscle cGMP and 32% increase in [cPKG.sub.1[alpha]], (P < 0.05). sGC, [cPKG.sub.1[beta]], and the [BK.sub.Ca] [alpha]-subunit were unchanged. Notably, expression of 131- and [32-regulatory subunits rose 51 and 79% (P -< 0.05), respectively. Increases in endogenous ovarian estrogens in follicular-phase ewes result in increases in UBF associated with upregulation of the cGMP- and cPKG-dependent pathway and increased vascular [BK.sub.Ca] [beta]/[alpha]-subunit stoichiometry, suggesting enhanced [BK.sub.Ca] activation contributes to the follicular phase rise in UBF. estrogen; uterine blood flow; protein kinase G; guanylyl cyclase doi: 10.1152/ajpendo.00375.2009

Valenda-Cruz G, Shabala L, Delgado-Enciso I, Shabala S, Bonales-Alatorre E, Pottosin H, Dobrovinskaya OR. [K.sub.bg] and Kv1.3 channels mediate potassium efflux in the early phase of apoptosis in Jurkat T lymphocytes. Am J Physiol Cell Physiol 297: C1544-C1553, 2009. First published September 30, 2009; doi: 10.1152/ajpcell.00064.2009.--Microelectrode ion flux estimation (MIFE) and patch-clamp techniques were combined for nonmvasive [K.sup.+] flux measwements and recording of activities of the dominant [K.sup.+] channels in the early phases of apoptosis in Jurkat cells. Staurosporine (STS, 1 [micro]M) evoked rapid (peaking around 15 min) wansient [K.sup.+] efflux, which then gradually decreased. This transient [K.sup.+] efflux occurred concurrently with the transient increase of the [K.sup.+] background ([K.sub.bg]) TWIK-related spinal cord [K.sup.+] channel-like current density, followed by a drastic decrease and concomitant membrane depolarization. The Kvl.3 current density remained almost constant. Kv1.3 activation was not altered by STS, whereas the inactivation was shifted to more positive potentials. Contribution of [K.sub.bg] and Kv1.3 channels to the transient and posttransient STS-induced [K.sup.+] efflux components, respectively, was confirmed by the effects of bupivacaine, predominantly blocking [K.sub.bg] current, and the Kv1.3-specific blocker margatoxin. Channel-mediated [K.sup.+] efflux provoked a substantial cellular shrinkage and affected the activation of caspases. staurosporine; Jurkat cells; [K.sup.+] background current doi: 10.1152/ajpcell.00064.2009

Kim EY, Chiu YH, Dryer SE. Neph1 regulates steady-state surfaces expression of Slo1 [Ca.sup.2+]-activated [K.sup.+] channels: different effects in embryonic neurons and podocytes. Am J Physiol Cell Physiol 297; C1379-C1388, 2009. First published September 30, 2009; doi: 10.1152/.ajpcell.00354.2009.--Large-conductance [Ca.sup.2+]-activated [K.sup.+] (B[K.sub.ca]) channels encoded by the Slo1 gene are often components of large multiprotein complexes in excitable and nonexcitable cells. Here we show that Slo1 proteins interact with Neph1, a member of the immunoglobulin superfamily expressed in slit diaphragm domains of podocytes and in vertebrate and invertebrate nervous systems. This interaction was established by reciprocal coimmunoprecipitation of endogenous proteins from differentiated cells of a podocyte cell line, from parasympathetic neurons of the embryonic chick ciliary ganglion, and from HEK293T cells heterologously expressing both proteins. Neph1 can interact with all three extreme COOH-terminal variants of Slo1 ([Slo1.sub.VEDEC], [Slo1.sub.QEERL], and [Slo1.sub.EMVYR]) as ascertained by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation. Neph1 is partially colocalized in intracellular compartments with endogenous Slo1 in podocytes and ciliary ganglion neurons. Coexpression in HEK293T cells of Nepbl with any of the Slo1 extreme COOH-terminal splice variants suppresses their steady-state expression on the cell surface, as assessed by cell surface biotinylation assays, confocal microscopy, and whole cell recordings. Consistent with this, small interfering RNA (siRNA) knockdown of endogenous Neph1 in embryonic day 10 ciliary ganglion neurons causes an increase in steady-state surface expression of Slo1 and an increase in whole cell [Ca.sup.2+]-dependent [K.sup.+] current. Surprisingly, a comparable Neph1 knockdown in podocytes causes a decrease in surface expression of Slo1 and a decrease in whole cell B[K.sub.Ca] currents. In podocytes, Neph1 siRNA also caused a decrease in nephrin, even though the Neph1 siRNA had no sequence homology with nephrin. However, we could not detect nephrin in ciliary ganglion neurons. potassium channels; slit diaphragm; traffic; renal glomerulus; parasympathetic neurons doi: 10.1152/ajpcell.00354.2009

Henney NC, Li B, Elford C, Reviriego P, Campbell AK, Wann KT, Evans BA. A large-conductance (BK) potassium channel sub-type affects both growth and mineralization of human osteoblasts. Am J Physiol Cell Physiol 297: Cc1397-C1408, 2009. First published September 23, 2009; doi: 10.1152/ajpcell.00311.2009.--The pharmacology of the large-conductance [K.sup.+] (BK) channel in human osteoblasts is not well defined, and its role in bone is speculative. Here we assess BK channel properties in MG63 cells and primary human osteoblasts and determine whether pharmacological modulation affects cell function. We used RT-PCR and patch-clamp methods to determine the expression of BK channel subunits and cell number assays in the absence and presence of BK channel modulators. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3, and KCNMB4 subunits. The BK channel was voltage dependent, with a mean unitary conductance of 228.8 pS (n = 10) in cell-attached patches (140 mM [K.sup.+]/140 mM [K.sup.+]) and a conductance of 142.5 pS (n = 16) in excised outside-out and 155 pS (n = 6) in inside-out patches in 3 mM [K.sup.+]/140 mM [K.sup.+]. The selectivity ratio (ratio of [K.sup.+] to [Na.sup.+] permeability) was 15:1. The channel was blocked by tetraethylammonium (TEA, 0.3 mM), iberiotoxin (5-60 nM), tetrandrine (5-30 [micro]M), and paxilline (10 [micro]M) and activated by isopimaric acid (20 [micro]M). BK channel modulators affected MG63 cell numbers: TEA and tetrandrine significantly increased cell numbers at low concentrations (3 mM and 3 [micro]M, respectively) and reduced cell numbers at higher concentrations (>10 mM and >10 [micro]M, respectively). Neither iheriotoxin (20-300 nM) nor slotoxin (300 nM) affected cell numbers. The increase in cell numbers by TEA was blocked by isopimafic acid. TEA (0.1-3.0 mM) significantly increased mineralization in primary osteoblasts. In conclusion, the BK channel has a distinctive pharmacology and is thus a target for therapeutic strategies aimed at modulating osteoblast proliferation and function. MG63 cells; bone; potassium current; tetrandrine doi: 10.1152/ajpcell.00311.2009

We examined the impact of coexpressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, synapse-associated protein (SAP) 97, and determined that coexpression of these proteins caused an approximately twofold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channel conductance, or channel open probability. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3, leading to prominent colocalization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was ~13 pS, whereas coexpression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16, 29, and 42 pS. These changes occurred without altering channel open probability, current rectification properties, or pH sensitivity. Thus association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kit2.3 COOH-terminal domain and altering channel conformation. inward rectifier; postsynaptic density protein domain; channelosome; membrane cytoskeleton doi:10.1152/ajpheart.00638.2008

Voltage-gated potassium ([K.sub.v]) channels in vascular smooth muscle cells (VSMC) are critical regulators of membrane potential and vascular tone. These channels exert a hyperpolarizing influence to counteract the depolarizing effects of intraluminal pressure and vasoconstrictors. However, the contribution of [K.sub.v] channel activity to the functional regulation of cerebral (parenchymal) arterioles within the brain is not known. Thus [K.sub.v] channel properties in parenchymal arteriolar SMCs were characterized. Isolated, pressurized parenchymal arterioles and arterioles in cortical brain slices exhibited robust constriction in the presence of the [K.sub.v] channel inhibitor 4-aminopyridine (4-AP). 4-AP also decreased the amplitude of [K.sub.v] currents recorded from SMCs. The steady-state activation and inactivation properties of [K.sub.v] currents suggested that these channels are composed of [K.sub.v]1.2 and 1.5 subunits, which was confirmed by RT-PCR. [K.sub.v] channels can be regulated by extracellular glucose, which may be involved in the functional hyperemic response in the brain. Thus the effects of glucose on [K.sub.v] channel activity and arteriolar function were investigated. Elevation of glucose from 4 to 14 mM significantly decreased the peak [K.sub.v] current amplitude and constricted arterioles. Arteriolar constriction was prevented by inhibition of protein kinase C (PKC), consistent with previous studies showing enhanced PKC activity in the presence of elevated glucose. In cortical brain slices, the dilation generated by neuronal activity induced by electrical field stimulation was decreased by 54% in 14 mM glucose when compared with the dilation in 4 mM glucose. In anesthetized mice the whisker stimulation-induced increase in local cerebral blood flow was also significantly decreased in 14 mM glucose, and this effect was similarly prevented by PKC inhibition. These findings point to a critical role for [K.sub.v] channels in the regulation of intracerebral arteriolar function and suggest that changes in perivascular glucose levels could directly alter vascular diameter resulting in a modulation of local cerebral blood flow. cerebral blood flow; protein kinase C; vascular smooth muscle cells

Hypoxia activates chemoreceptor cells of the carotid body (CB) promoting an increase in their normoxic release of neurotransmitters. Catecholamine (CA) release rate parallels the intensity of hypoxia. Coupling of hypoxia to CA release requires cell depolarization, produced by inhibition of [O.sub.2]-regulated [K.sup.+] channels, and [Ca.sup.2+] entering the cells via voltage-operated channels. In rat chemoreceptor cells hypoxia inhibits large-conductance, calcium-sensitive K channels (maxiK) and a two-pore domain weakly inward rectifying [K.sup.+] channel (TWIK)-like acid-sensitive [K.sup.+] channel (TASK)-like channel, but the significance of maxiK is controversial. A proposal envisions maxiK contributing to set the membrane potential ([E.sub.m]) and the hypoxic response, but the proposal is denied by authors finding that maxiK inhibition does not depolarize chemoreceptor cells or alters intracellular [Ca.sup.2+] concentration or CA release in normoxia or hypoxia. We found that maxiK channel blockers (tetraethylammonium and iberiotoxin) did not modify CA release in rat chemoreceptor cells, in either normoxia or hypoxia, and iberiotoxin did not alter the [Ca.sup.2+] transients elicited by hypoxia. On the contrary, both maxiK blockers increased the responses elicited by dinitrophenol, a stimulus we demonstrate does not affect maxiK channels in isolated patches of rat chemoreceptor cells. We conclude that in rat chemoreceptor cells maxiK channels do not contribute to the genesis of the [E.sub.m], and that their full inhibition by hypoxia, preclude further inhibition by maxiK channel blockers. We suggest that full inhibition of this channel is required to generate the spiking behavior of the cells in acute hypoxia. hypoxia; catecholamine; iberiotoxin; intracellular calcium

Human ether a go-go related gene (hERG) potassium channels play a central role in cardiac repolarization where channel closing (deactivation) regulates current density during action potentials. Consequently, mutations in hERG that perturb deactivation are linked to long QT syndrome (LQTS), a catastrophic cardiac arrhythmia. Interactions between an N-terminal domain and the pore-forming 'core' of the channel were proposed to regulate deactivation, however, despite its central importance the mechanistic basis for deactivation is unclear. Here, to more directly examine the mechanism for regulation of deactivation, we genetically fused N-terminal domains to fluorescent proteins and tested channel function with electrophysiology and protein interactions with Forster resonance energy transfer (FRET) spectroscopy. Truncation of hERG N-terminal regions markedly sped deactivation, and here we report that reapplication of gene fragments encoding N-terminal residues 1-135 (the 'eag domain') was sufficient to restore regulation of deactivation. We show that fluorophore-tagged eag domains and N-truncated channels were in close proximity at the plasma membrane as determined with FRET. The eag domains with Y43A or R56Q (aLQTS locus) mutations showed less regulation of deactivation and less FRET, whereas eag domains restored regulation of deactivation gating to full-length Y43A or R56Q channels and showed FRET. This study demonstrates that direct, noncovalent interactions between the eag domain and the channel core were sufficient to regulate deactivation gating, that an LQTS mutation perturbed physical interactions between the eag domain and the channel, and that small molecules such as the eag domain represent a novel method for restoring function to channels with disease-causing mutations. eag domain | FRET | LQTS

To sustain cardiac muscle contractility relatively independent of temperature, some fish species are capable of temporarily altering excitation-contraction coupling processes to meet the demands of their environment. The Pacific bluefin tuna, Thunnus orientalis, is a partially endothermic fish that inhabits a wide range of thermal niches. The present study examined the effects of temperature and thermal acclimation on sarcolemmal [K.sup.+] currents and their role in action potential (AP) generation in bluefin tuna cardiomyocytes. Atrial and ventricular myocytes were enzymatically isolated from cold (14[degrees]C)- and warm (24[degrees]C)-acclimated bluefin tuna. APs and current-voltage relations of [K.sup.+] channels were measured using the whole cell current and voltage clamp techniques, respectively. Data were collected either at the cardiomyocytes' respective acclimation temperature of 14 or 24[degrees]C or at a common test temperature of 19[degrees]C (to reveal the effects of acclimation). AP duration (APD) was prolonged in cold-acclimated (CA) cardiomyocytes tested at 14[degrees]C compared with 19[degrees]C and in warm-acclimated (WA) cardiomyocytes tested at 19[degrees]C compared with 24[degrees]C. This effect was mirrored by a decrease in the density of the delayed-rectifier current ([I.sub.Kr]), whereas the density of the background inward-rectifier current ([I.sub.K1]) was unchanged. When CA and WA cardiomyocytes were tested at a common temperature of 19[degrees]C, no significant effects of temperature acclimation on AP shape or duration were observed, whereas [I.sub.Kr] density was markedly increased in CA cardiomyocytes. [I.sub.K1] density was unaffected in CA ventricular myocytes but was significantly reduced in CA atrial myocytes, resulting in a depolarization of atrial resting membrane potential. Our results indicate the bluefin AP is relatively short compared with other teleosts, which may allow the bluefin heart to function at cold temperatures without the necessity for thermal compensation of APD. thermal acclimation; bluefin tuna; cardiac; potassium ion channels; action potential

[gamma]-Glutamyl transpeptidase ([gamma]-GT) is a key enzyme in GSH metabolism that regulates intracellular GSH levels in response to extracellular GSH ([GSH.sub.o]). The objective of this study was to identify the role of [gamma]-GT in reversing pathogenic [K.sup.+] channel remodeling in the diseased heart. Chronic ventricular dysfunction was induced in rats by myocardial infarction (MI), and studies were done after 6-8 wk. Biochemical assays of tissue extracts from post-MI hearts revealed significant increases in [gamma]-GT activity in left ventricle (47%) and septum (28%) compared with sham hearts, which paralleled increases in protein abundance and mRNA. Voltage-clamp studies of isolated left ventricular myocytes from post-MI hearts showed that downregulation of transient outward [K.sup.+] current ([I.sub.to]) was reversed after 4-5 h by 10 mmol/l [GSH.sub.o] or N-acetylcysteine ([NAC.sub.o]), and that the effect of [GSH.sub.o] but not [NAC.sub.o] was blocked by the [gamma]-GT inhibitors, acivicin or S-hexyl-GSH. Inhibition of [gamma]-glutamylcysteine synthetase by buthionine sulfoximine did not prevent upregulation of [I.sub.to] by [GSH.sub.o], suggesting that intracellular synthesis of GSH was not directly involved. However, pretreatment of post-MI myocytes with an SOD mimetic [manganese (III) tetrapyridylporphyrin] and catalase completely blocked recovery of [I.sub.to] by [GSH.sub.o]. Confocal microscopy using the fluorogenic dye 2',7'-dichlorodihydrofluorescein diacetate confirmed that [GSH.sub.o] increased reactive oxygen species (ROS) generation by post-MI myocytes and to a lesser extent in myocytes from sham hearts. Furthermore, [GSH.sub.o]-mediated upregulation of [I.sub.to] was blocked by inhibitors of tyrosine kinase (genistein, lavendustin A, and AG1024) and thioredoxin reductase (auranofin and 13-cis-retinoic acid). These data suggest that [GSH.sub.o] elicits [gamma]-GT- and ROS-dependent transactivation of tyrosine kinase signaling that upregulates [K.sup.+] channel activity or expression via redox-mediated mechanisms. The signaling events stimulated by [gamma]-GT catalysis of [GSH.sub.o] may be a therapeutic target to reverse pathogenic electrical remodeling of the failing heart. glutathione; voltage-dependent [K.sup.+] channel; thioredoxin; transient outward current

Acute changes in cAMP and protein kinase A (PKA) signaling can regulate ion channel protein activities such as gating. Effects on channels due to chronic PKA signaling, as in stress or disease states, are less understood. We examined the effects of prolonged PKA activity on the human ether-a-go-go-related gene (HERG) [K.sup.+] channel in stably transfected human embryonic kidney (HEK)293 cells. Sustained elevation of cAMP by either chlorophenylthiol (CPT)-cAMP or forskolin increased the HERG channel protein abundance two- to fourfold within 24 h, with measurable difference as early as 4 h. The cAMP-induced augmentation was not due to changes in transcription and was specific for HERG compared with other cardiac [K.sup.+] channels (Kv1.4, Kv1.5, Kit2.1, and KvLQT1). PKA activity was necessary for the effect on HERG protein and did not involve other cAMP signaling pathways. Direct PKA phosphorylation of the HERG protein was responsible for the cAMP-induced augmentation. Enhanced abundance of HERG protein was detected in endoplasmic reticulum-enriched, Golgi, and plasma membrane without significant changes in trafficking rates or patterns. An increase in the [K.sup.+] current density carried by the HERG channel was also observed, but with a delay, suggesting that traffic to the surface is rate-limiting traffic. Acceleration of the HERG protein synthesis rate was the primary factor in the cAMP/PKA effect with lesser effects on protein stability. These results provide evidence for a novel mechanism whereby phosphorylation of a nascent protein dictates its rate of synthesis, resetting its steady-state abundance. adenosine 3',5'-cyclic monophosphate; human ether-a-go-go-related gene; potassium channel; protein kinase A

Organized neuronal firing is crucial for cortical processing and is disrupted in schizophrenia. Using rapid amplification of 5&apos; complementary DNA ends in human brain, we identified a primate-specific isoform (3.1) of the ether-a-go-go-related [K.sup.+] channel KCNH2 that modulates neuronal firing. KCNH2-3.1 messenger RNA levels are comparable to full-length KCNH2 (lA) levels in brain but three orders of magnitude lower in heart. In hippocampus from individuals with schizophrenia, KCNH2-3.1 expression is 2.5-fold greater than KCNH2-lA expression. A meta-analysis of five clinical data sets (367 families, 1,158 unrelated cases and 1,704 controls) shows association of single nucleotide polymorphisms in KCNH2 with schizophrenia. Risk-associated alleles predict lower intelligence quotient scores and speed of cognitive processing, altered memory-linked functional magnetic resonance imaging signals and increased KCNH2-3.1 mRNA levels in postmortem hippocampus. KCNH2-3.1 lacks a domain that is crucial for slow channel deactivation. Overexpression of KCNH2-3.1 in primary cortical neurons induces a rapidly deactivating [K.sup.+] current and a high-frequency, nonadapting firing pattern. These results identify a previously undescribed KCNH2 channel isoform involved in cortical physiology, cognition and psychosis, providing a potential new therapeutic drug target., A major challenge in modern medicine is to understand cellular and molecular mechanisms underlying common mental illnesses such as schizophrenia, which involve complicated genetic and environmental determinantsl. Prevailing etiological hypotheses [...]

Human ether-a-go-go-related gene (HERG) potassium channel acts as a delayed rectifier in cardiac myocytes and is an important target for both pro-and antiarrhythmic drugs. Many drugs have been pulled from the market for unintended HERG block causing arrhythmias. Conversely, recent evidence has shown that HERG plays a role in cell proliferation and is overexpressed both in multiple tumor cell lines and in primary tumor cells, which makes HERG an attractive target for cancer treatment. Therefore, a drug that can block HERG but that does not induce cardiac arrhythmias would have great therapeutic potential. Roscovitine is a cyclin-dependent kinase (CDK) inhibitor that is in phase II clinical trials as an anticancer agent. In the present study we show that R-roscovitine blocks HERG potassium current (human embryonic kidney-293 cells stably expressing HERG) at clinically relevant concentrations. The block ([IC.sub.50] = 27 [micro]M) was rapid ([tau] = 20 ms) and reversible ([tau] = 25 ms) and increased with channel activation, which supports an open channel mechanism. Kinetic study of wild-type and inactivation mutant HERG channels supported block of activated channels by roscovitine with relatively little effect on either closed or inactivated channels. A HERG gating model reproduced all roscovitine effects. Our model of open channel block by roscovitine may offer an explanation of the lack of arrhythmias in clinical trials using roscovitine, which suggests the utility of a dual CDK/HERG channel block as an adjuvant cancer therapy. S620T mutant; open channel block; terfenadine; human ether-a-go-go-related gene model

Primary hypomagnesemia is a heterogeneous group of disorders characterized by renal or intestinal magnesium ([Mg.sup.2+]) wasting, resulting in tetany, cardiac arrhythmias, and seizures. The kidney plays an essential role in maintaining blood [Mg.sup.2+] levels, with a prominent function for the [Mg.sup.2+]-transporting channel transient receptor potential cation channel, subfamily M, member 6 (TRPM6) in the distal convoluted tubule (DCT). In the DCT, [Mg.sup.2+] reabsorption is an active transport process primarily driven by the negative potential across the luminal membrane. Here, we studied a family with isolated autosomal dominant hypomagnesemia and used a positional cloning approach to identify an N255D mutation in KCNA1, a gene encoding the voltage-gated potassium ([K.sup.+]) channel Kv1.1. Kv1.1 was found to be expressed in the kidney, where it colocalized with TRPM6 along the luminal membrane of the DCT. Upon overexpression in a human kidney cell line, patch clamp analysis revealed that the KCNA1 N255D mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. These data suggest that Kv1.1 is a renal [K.sup.+] channel that establishes a favorable luminal membrane potential in DCT cells to control TRPM6-mediated [Mg.sup.2+] reabsorption., Introduction Occurrence of hypomagnesemia (serum [Mg.sup.2+] levels below 0.70 mmol/l) in the general population has been estimated to be around 2%, while hospitalized patients are more prone to develop hypomagnesemia [...]

Analysis of Mendelian [Mg.sup.2+] wasting disorders helps us to unravel the mechanisms of [Mg.sup.2+] homeostasis. In this issue of the JCI, Glaudemans and colleagues show that mutations in voltage-gated [K.sup.+] channel subtype 1.1 (Kv1.1) cause autosomal dominant hypomagnesemia in humans (see the related article beginning on page 936). Interestingly, other mutations in the same protein cause the neurological disease episodic ataxia type 1. The authors show, using cells with heterologous expression of the wild-type and mutant channels, that the mutant channel is dysfunctional and speculate that [Mg.sup.2+] wasting results from changes in apical membrane voltage along the nephron. Mechanisms by which the apical voltage is generated and how Kv1.1 fits within this context are discussed herein., Rare Mendelian diseases are windows into both physiology and pathogenesis. Examples include the rare [Mg.sup.2+] wasting disorders that form the basis for most of our current understanding of renal [Mg.sup.2+] [...]

The cardiac-delayed rectifier [K.sup.+] current ([I.sub.KS]) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensor domains and the pore, and auxiliary KCNE1 (E1) subunits, required for the characteristic [I.sub.KS] voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1-TM) relative to the Q1 TM helices (S1-S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1-S6 and E1-TM to Cys, coexpressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide-bond formation. Cys-flanking E1-TM readily formed disulfides with Cys-flanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 on different subunits of Q1. The salient functional effects of selected cross-links were as follows. A disulfide from E1 K41C to S1 I145C strongly slowed deactivation, and one from E1 L42C to S6 V324C eliminated deactivation. Given that E1-TM is between S1 and S6 and that K41C and L42C are likely to point approximately oppositely, these two cross-links are likely to favor similar axial rotations of E1-TM. In the opposite orientation, a disulfide from E1 K41C to S6 V324C slightly slowed activation, and one from E1 L42C to S1 I145C slightly speeded deactivation. Thus, the first E1 orientation strongly favors the open state, while the approximately opposite orientation favors the closed state. arrhythmias | cardiac repolarization | electrophysiology | atrial fibrillation | S1

Potassium channels at the cardiomyocyte surface must eventually be internalized and degraded, and changes in cardiac potassium channel expression are known to occur during myocardial disease. It is not known which trafficking pathways are involved in the control of cardiac potassium channel surface expression, and it is not clear whether all cardiac potassium channels follow a common pathway or many pathways. In the present study we have surveyed the role of retrograde microtubule-dependent transport in modulating the surface expression of several cardiac potassium channels in ventricular myocytes and heterologous cells. The disruption of microtubule transport in rat ventricular myocytes with nocodazole resulted in significant changes in potassium currents. A-type currents were enhanced 1.6-fold at +90 mV, rising from control densities of 20.9 [+ or -] 2.8 to 34.0 [+ or -] 5.4 pA/pF in the nocodazole-treated cells, whereas inward rectifier currents were reduced by one-third, perhaps due to a higher nocodazole sensitivity of Kir channel forward trafficking. These changes in potassium currents were associated with a significant decrease in action potential duration. When expressed in heterologous human embryonic kidney (HEK-293) cells, surface expression of Kv4.2, known to substantially underlie A-type currents in rat myocytes, was increased by nocodazole, by the dynein inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, and by p50 overexpression, which specifically interferes with dynein motor function. Peak current density was 360 [+ or -] 61.0 pA/pF in control cells and 658 [+ or -] 94.5 pA/pF in cells overexpressing p50. The expression levels of Kv2.1, Kv3.1, human ether-a-go-go-related gene, and Kir2.1 were similarly increased by p50 overexpression in this system. Thus the regulation of potassium channel expression involves a common dynein-dependent process operating similarly on the various channels. voltage-gated potassium channel; inward rectifier; microtubules; p50/ dynamitin

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive [K.sup.+] ([K.sub.ATP]) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic [beta] cells. These [beta]-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from [beta]-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of [K.sub.ATP] channels in pancreatic [beta] cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of [K.sub.ATP] channels, closed [K.sub.ATP] channels, elevated intracellular calcium levels, and stimulated insulin release in [beta]-V59M [beta] cells, indicating that events downstream of [K.sub.ATP] channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic [beta] cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. [beta]-V59M islets also displayed a reduced percentage of [beta] cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of [beta]-V59M mice. Their cause requires further investigation., Introduction Neonatal diabetes (ND) is a rare disease that affects about 1 in 200,000 live births (1). It is defined as diabetes that manifests within the first 6 months of [...]

Benign familial neonatal convulsion (BNFC) is a neurological disorder caused by mutations in the potassium channel genes KCNQ2 and KCNQ3, which are thought to contribute to the medium afterhyperpolarization (mAHP). Despite their importance in normal brain function, it is unknown whether they invariably function as heteromeric complexes. Here, we examined the contribution of KCNQ3 and KCNQ2 in mediating the apamin-insensitive mAHP current (ImAHP) in hippocampus. The ImAHP was not impaired in CA1 pyramidal neurons from mice genetically deficient for either KCNQ3 or KCNQ2 but was reduced [approximately or equal to] 50% in dentate granule cells. While recording from KCNQ-deficient mice, we observed that the calcium-activated slow afterhyperpolarization current (IsAHP) was also reduced in dentate granule cells, suggesting that KCNQ channels might also contribute to this potassium current whose molecular identity is unknown. Further pharmacological and molecular experiments manipulating KCNQ channels provided evidence in support of this possibility. Together our data suggest that multiple KCNQ subunit compositions can mediate the ImAHP, and that the very same subunits may also contribute to the IsAHP. We also present data suggesting that the neuronal calcium sensor protein hippocalcin may allow for these dual signaling processes. Epilepsy | KCNQ | M-current | Retigabine | sAHP

The regulation of M-type (KCNQ [Kv7]) [K.sup.+] channels by phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]) has perhaps the best correspondence to physiological signaling, but the site of action and structural motif of [PIP.sub.2] on these channels have not been established. Using single-channel recordings of chimeras of Kv7.3 and 7.4 channels with highly differential [PIP.sub.2] sensitivities, we localized a carboxy-terminal inter-helix linker as the primary site of [PIP.sub.2] action. Point mutants within this linker in Kv7.2 and Kv7.3 identified a conserved cluster of basic residues that interact with the lipid using electrostatic and hydrogen bonds. Homology modeling of this putative [PIP.sub.2]-binding linker in Kv7.2 and Kv7.3 using the solved structure of Kir2.1 and Kir3.1 channels as templates predicts a structure of Kv7.2 and 7.3 very similar to the Kit channels, and to the seven-[beta]-sheet barrel motif common to other [PIP.sub.2]-binding domains. Phosphoinositide-docking simulations predict affinities and interaction energies in accord with the experimental data, and furthermore indicate that the precise identity of residues in the interacting pocket alter channel-[PIP.sub.2] interactions not only by altering electrostatic energies, but also by allosterically shifting the structure of the lipid-binding surface. The results are likely to shed light on the general structural mechanisms of phosphoinositide regulation of ion channels.

Several analyses are conducted to explain the symptoms, causes and effects of the maternally transmitted birk barel dysmorphism syndrome of mental retardation, namely hypotonia. The disorder is shown to be caused by a mutation in the genomically imprinted potassium channel KCNK9 by exhibiting a dual function of a homodimer, as well as of a heterodimer.

We previously reported an enhanced tonic dilator impact of ATP-sensitive [K.sup.+] channels in afferent arterioles of rats with streptozotocin (STZ)-induced diabetes. The present study explored the hypothesis that other types of [K.sup.+] channel also contribute to afferent arteriolar dilation in STZ rats. The in vitro blood-perfused juxtamedullary nephron technique was utilized to quantify afferent arteriolar lumen diameter responses to [K.sup.+] channel blockers: 0.1-3.0 mM 4-aminopyridine (4-AP; Kv channels), 10-100 [micro]M barium (Kin channels), 1-100 nM tertiapin-Q (TPQ; Kirl. 1 and Kir3.x subfamilies of [K.sub.IR] channels), 100 nM apamin (SKca channels), and 1 mM tetraethylammonium (TEA; BKca channels). In kidneys from normal rats, 4-AP, TEA, and [Ba.sup.2+] reduced afferent diameter by 23 [+ or -] 3, 8 [+ or -] 4, and 18 [+ or -] 2%, respectively, at the highest concentrations employed. Neither TPQ nor apamin significantly altered afferent diameter. In arterioles from STZ rats, a constrictor response to TPQ (22 [+ or -] 4% decrease in diameter) emerged, and the response to [Ba.sup.2+] was exaggerated (28 [+ or -] 5% decrease in diameter). Responses to the other [K.sup.+] channel blockers were similar to those observed in normal rats. Moreover, exposure to either TPQ or [Ba.sup.2+] reversed the afferent arteriolar dilation characteristic of STZ rats. Acute surgical papillectomy did not alter the response to TPQ in arterioles from normal or STZ rats. We conclude that 1) [K.sub.v], [K.sub.IR], and [BK.sub.ca] channels tonically influence normal afferent arteriolar tone, 2) [K.sub.IR] channels (including Kir1.1 and/or Kir3.x) contribute to the afferent arteriolar dilation during diabetes, and 3) the dilator impact of Kir1. 1/Kir3.x channels during diabetes is independent of solute delivery to the macula densa. tertiapin-Q; barium; TEA; apamin; 4-aminopyridine

Voltagegated [K.sup.+] (Kv) channels are important in the regulation of pulmonary vascular function having both physiological and pathophysiological implications. The pulmonary vasculature is essential for reoxygenation of the blood, supplying oxygen for cellular respiration. Mitochondria have been proposed as the major oxygen-sensing organelles in the pulmonary vasculature. Using electrophysiological techniques and immunofluorescence, an interaction of the mitochondria with Kv channels was investigated. Inhibitors, blocking the mitochondrial electron transport chain at different complexes, were shown to have a dual effect on Kv currents in freshly isolated rat pulmonary arterial smooth muscle cells (PASMCs). These dual effects comprised an enhancement of Kv current in a negative potential range (manifested as a 5- to 14-mV shift in the Kv activation to more negative membrane voltages) with a decrease in current amplitude at positive potentials. Such effects were most prominent as a result of inhibition of Complex III by antimycin A. Investigation of the mechanism of antimycin A-mediated effects on Kv channel currents ([I.sub.Kv]) revealed the presence of a mitochondria-mediated [Mg.sup.2+] and ATP-dependent regulation of Kv channels in PASMCs, which exists in addition to that currently proposed to be caused by changes in intracellular reactive oxygen species. rat; Kv channel currents; antimycin A; magnesium ions; ATP; Kv channel activation

G protein-gated inwardly rectifying potassium (GIRK/Kir3) channels mediate the inhibitory effects of many neurotransmitters on excitable cells. Four Girk genes have been identified (Girk1-4). Whereas GIRK4 is associated with the cardiac GIRK channel, Girk4 expression has been detected in a few neuron populations. Here, we used a transgenic mouse expressing enhanced green fluorescent protein (EGFP) under the control of the Girk4 gene promoter to clarify the expression pattern of Girk4 in the brain. Although small subsets of EGFP-positive neurons were evident in some areas, prominent labeling was seen in the hypothalamus. EGFP expression was most pronounced in the ventromedial, paraventricuiar, and arcuate nuclei, neuron populations implicated in energy homeostasis. Consistent with a contribution of GIRK4-containing channels to energy balance, Girk4 knockout (-/-) mice were predisposed to late-onset obesity. By 9 months, [Girk4.sup.-l-] mice were [approximately equal to] 25% heavier than wild-type controls, a difference attributed to greater body fat. Before the development of overweight, [Girk4.sup.-/-] mice exhibited a tendency toward greater food intake and an increased propensity to work for food in an operant task. [Girk4.sup.-/-] mice also exhibited reduced net energy expenditure, despite displaying elevated resting heart rates and core body temperatures. These data implicate G1RK4-containing channels in signaling crucial to energy homeostasis and body weight. body weight | energy balance | hypothalamus | Kir3

[K.sup.+] channels in the basolateral membrane of mouse cortical collecting duct (CCD) principal cells were identified with patch-clamp technique, real-time PCR, and immunohistochemistry. In cell-attached membrane patches, three [K.sup.+] channels with conductances of ~75, 40, and 20 pS were observed, but the [K.sup.+] channel with the intermediate conductance (40 pS) predominated. In inside-out membrane patches exposed to an [Mg.sup.2+]-free medium, the current-voltage relationship of the intermediate-conductance channel was linear with a conductance of 38 pS. Addition of 1.3 mM internal [Mg.sup.2+] had no influence on the inward conductance ([G.sub.in] = 35 pS) but reduced outward conductance ([G.sub.out]) to 13 pS, yielding a [G.sub.in]/[G.sub.out] of 3.2. The polycation spermine (6 x [10.sup.-7] M) reduced its activity on inside-out membrane patches by 50% at a clamp potential of 60 mV. Channel activity was also dependent on intracellular pH ([pH.sub.i]: a sigmoid relationship between [pH.sub.i] and channel normalized current ([NP.sub.o]) was observed with a pK of 7.24 and a Hill coefficient of 1.7. By real-time PCR on CCD extracts, inwardly rectifying [K.sup.+] (Kir)4.1 and Kir5.1, but not Kir4.2, mRNAs were detected. Kir4.1 and Kir5.1 proteins cellularly colocalized with aquaporin 2 (AQP2), a specific marker of CCD principal cells, while AQP2-negative cells (i.e., intercalated cells) showed no staining. Dietary [K.sup.+] had no influence on the properties of the intermediate-conductance channel, but a [Na.sup.+]-depleted diet increased its open probability by ~25%. We conclude that the Kir4.1/Kir5.1 channel is a major component of the [K.sup.+] conductance in the basolateral membrane of mouse CCD principal cells. kidney; patch clamp

Bartter's syndrome represents a group of hereditary salt- and water-losing renal tubulopathies caused by loss-of-function mutations in proteins mediating or regulating salt transport in the thick ascending limb (TAL) of Henle's loop. Mutations in the ROMK channel cause type II antenatal Bartter's syndrome that presents with maternal polyhydramnios and postnatal life-threatening volume depletion. We have developed a colony of Romk null mice showing a Bartter-like phenotype and with increased survival to adulthood, suggesting the activation of compensatory mechanisms. To test the hypothesis that upregulation of [Na.sup.+]-transporting proteins in segments distal to the TAL contributes to compensation, we studied expression of salt-transporting proteins in ROMK-deficient ([Romk.sup.-/-]) mice. Plasma aldosterone was 40% higher and urinary [PGE.sub.2] excretion was 1.5-fold higher in [Romk.sup.-/-] compared with wild-type littermates. Semiquantitative immunoblotting of kidney homogenates revealed decreased abundances of proximal tubule [Na.sup.+]/[H.sup.+] exchanger (NHE3) and [Na.sup.+]-[P.sub.i] cotransporter (NaPi-IIa) and TAL-specific [Na.sup.+]-[K.sup.+]-2[Cl.sup.-]-cotransporter (NKCC2/BSC1) in [Romk.sup.-/-] mice, while the distal convoluted tubule (DCT)-specific [Na.sup.+]-[Cl.sup.-] cotransporter (NCC/TSC) was markedly increased. The abundance of the [alpha]-,[beta]-, and [gamma]-subunits of the epithelial [Na.sup.+] channel (ENaC) was slightly increased, although only differences for [gamma]-ENaC reached statistical significance. Morphometry revealed a fourfold increase in the fractional volume of DCT but not of connecting tubule (CNT) and collecting duct (CCD). Consistently, CNT and CD of [Romk.sup.-/-] mice revealed no apparent increase in the luminal abundance of the ENaC compared with those of wild-type mice. These data suggest that the loss of ROMK-dependent [Na.sup.+] absorption in the TAL is compensated predominately by upregulation of [Na.sup.+] transport in downstream DCT cells. These adaptive changes in [Romk.sup.-/-] mice may help to limit renal [Na.sup.+] loss, and thereby, contribute to survival of these mice. thick ascending limb of Henle's loop; distal convoluted tubule; thiazide-sensitive NaCl cotransporter; renal outer medulla [K.sup.+] channel; hyperprostaglandin E syndrome

KCNQ1 and KCNE1 (Q1 and El) associate to form the slow delayed rectifier [I.sub.Ks] channels in the heart. A short stretch of eight amino acids at the extracellular end of S1 in Q1 (positions 140-147) harbors six arrhythmia-associated mutations. Some of these mutations affect the Q1 channel function only when coexpressed with E1, suggesting that this Q1 region may engage in the interaction with E1 critical for the [I.sub.Ks] channel function. Identifying the Q1/E1 contact points here may provide new insights into how the [I.sub.Ks] channel operates. We focus on Q1 position 145 and E1 positions 40-43. Replacing all native cysteine (Cys) in Q1 and introducing Cys into the above Q1 and E1 positions do not significantly perturb the Q1 channel function or Q1/E1 interactions. Immunoblot experiments on COS-7 cells reveal that Q1 145C can form disulfide bonds with E1 40C and 41C, but not E1 42C or 43C. Correspondingly, voltage clamp experiments in oocytes reveal that Q1 145C coexpressed with E1 40C or E1 41C manifests unique gating behavior and DTT sensitivity. Our data suggest that E1 40C and 41C come close to Q1 145C in the activated and resting states, respectively, to allow disulfide bond formation. These data and those in the literature lead us to propose a structural model for the Q1/E1 channel complex, in which E1 is located between S1, S4, and S6 of three separate Q1 subunits. We propose that E1 is not a passive partner of the Q1 channel, but instead can engage in molecular motions during [I.sub.Ks] gating.

Cell migration is an important physiological process among others controlled by ion channel activity. Calcium-activated potassium channels ([K.sub.Ca]3.1) are required for optimal cell migration. Previously, we identified single human (h) [K.sub.Ca]3.1 channel proteins in the plasma membrane by means of quantum dot (QD) labeling. In the present study, we tracked single-channel proteins during migration to classify their dynamics in the plasma membrane of MDCK-F cells. Single [hK.sub.Ca]3.1 channels were visualized with QD- or Alexa488-conjugated antibodies and tracked at the basal cell membrane using time-lapse total internal reflection fluorescence (TIRF) microscopy. Analysis of the trajectories allowed the classification of channel dynamics. Channel tracks were compared with those of free QD-conjugated antibodies. The size of the label has a pronounced effect on [hK.sub.Ca]3.1 channel diffusion. QD-labeled channels have a (sub)diffusion coefficient [D.sub.QDbound] = 0.067 [micro][m.sup.2]/[s.sup.alpha], whereas that of Alexa488-1abeled channels is [D.sub.Alexa] = 0.139 [micro][m.sup.2]/s. Free QD-conjugated antibodies move much faster: [D.sub.QDfree] = 2.163 [micro][m.sup.2]/[s.sup.alpha] Plotting the mean squared distances (msd) covered by [hK.sub.Ca]3.1 channels as a function of time points to the mode of diffusion. Alexa488-1abeled channels diffuse normally, whereas the QD-label renders [hK.sub.Ca]3.1 channel diffusion anomalous. Free QDlabeled antibodies also diffuse anomalously. Hence, QDs slow down diffusion of [hK.sub.Ca]3.1 channels and change the mode of diffusion. These results, referring to the role of label size and properties of the extracellular environment, suggest that the pericellular glycocalyx has an important impact on labels used for single molecule tracking. Thus tracking fluorescent particles within the glycocalyx opens up a possibility to characterize the pericellular nanoenvironment. calcium-activated potassium channel 3.1, quantum dot; single molecule tracking; cell migration

The long QT syndrome genes human ether-a-go-go-related gene (HERG1) and voltage-gated [K.sup.+] channel, KQT-like subfamily, member 1, gene (KCNQ1), encoding [K.sup.+] channels critical to the repolarization rate and repolarization reserve in cardiac cells, and thereby the likelihood of arrhythmias, are both composed of two isoforms: HERG1a and HERG1b and KCNQ1a and KCNQ1b, respectively. Expression of these genes is dynamic, depending on the differentiation status and disease states. We identified their core promoter regions and transcription start sites. Our data suggest that HERG1a and HERG1b, and KCNQ1a and KCNQ1b, represent independent transcripts instead of being alternatively spliced variants of the same gene, for they each have their own transcription start sites and their own promoter regions. We obtained data pointing to the potential role of stimulating protein 1 (Sp1) in the transactivation of these genes. We compared expression profiling of these genes across a variety of human tissues. Consistent with the general lack of cis elements for cardiac-specific transcription factors and the presence of multiple sites for ubiquitous Sp1 sites in the core promoter regions of HERG1a/HERG1b and KCNQ1a/KCNQ1b genes, the transcripts demonstrated widespread distribution across a variety of human tissues. We further revealed that the mRNA levels of all HERG1 and KCNQ1 isoforms were asymmetrically distributed within the heart, being more abundant in the right atria and ventricles relative to the left atria and ventricles. These findings open up an opportunity for studying interventricular gradients of slow and rapid delayed rectifier [K.sup.+] current and of cardiac repolarization as well. Our study might help us understand the molecular mechanisms for arrhythmias since heterogeneity of ion channel activities is an important substrate for arrhythmogenesis. potassium channels; promoter; transcription; human ether-a-go-go-related gene; voltage-gated potassium channel, KQT-like subfamily, member 1; stimulating protein 1

Shaker-type [K.sup.+] channels in plants display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, an Arabidopsis [K.sup.+] channel (SKOR) and a tomato [K.sup.+] channel (LKT1) share high amino acid sequence similarity and identical domain structures; however, SKOR conducts outward [K.sup.+] current and is activated by positive membrane potentials (depolarization), whereas LKT1 conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the 'opposite' voltage-sensing properties of SKOR and LKT1 remains unknown. Using a screening procedure combined with random mutagenesis, we identified in the SKOR channel single amino acid mutations that converted an outwardconducting channel into an inward-conducting channel. Further domain-swapping and random mutagenesis produced similar results, suggesting functional interactions between several regions of SKOR protein that lead to specific voltage-sensing properties. Dramatic changes in rectifying properties can be caused by single amino acid mutations, providing evidence that the inward and outward channels in the Shaker family from plants may derive from the same ancestor. Arabidopsis | ion channels | voltage-gating | DNA shuffling | mutagenesis