PKA site mutations of ROMK2 channels shift the pH dependence to more alkaline values

Leipziger, Jens; MacGregor, Gordon G.; Cooper, Gordon J.; Xu, Jason; Hebert, Steven C.; Giebisch, Gerhard

doi:10.1152/ajprenal.2000.279.5.f919

Cited by 42 publications

(48 citation statements)

References 23 publications

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“…balance (13), a correlation attributed to the exquisite sensitivity of ROMK1 to cytosolic pH (37)(38)(39)(40). In face of the present observations, it is appealing to speculate that NHERF2 participates in the regulation of K ϩ excretion by acid base balance.…”

Section: Discussionmentioning

confidence: 59%

The Serum and Glucocorticoid-Inducible Kinase SGK1 and the Na+/H+ Exchange Regulating Factor NHERF2 Synergize to Stimulate the Renal Outer Medullary K+ Channel ROMK1

Yun¹,

Palmada

Embark

et al. 2002

Journal of the American Society of Nephrology

116

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Abstract. Mineralocorticoids stimulate Na ϩ reabsorption and K ϩ secretion in principal cells of connecting tubule and collecting duct. The involved ion channels are ENaC and ROMK1, respectively. In Xenopus oocytes, the serum and glucocorticoid-sensitive kinase SGK1 has been shown to increase ENaC activity by enhancing its abundance in the plasma membrane. With the same method, ROMK1 appeared to be insensitive to regulation by SGK1. On the other hand, ROMK1 has been shown to colocalize with NHERF2, a protein mediating targeting and trafficking of transport proteins into the cell membrane. The present study has been performed to test whether NHERF2 is required for regulation of ROMK1 by SGK1. Coexpression of neither NHERF2 nor SGK1 with ROMK1 increases ROMK1 activity. However, coexpression of NHERF2 and SGK1 together with ROMK1 markedly increases K ϩ channel activity. The combined effect of SGK1 and NHERF2 does not significantly alter the I/V relation of the channel but increases the abundance of the channel in the membrane and decreases the decay of channel activity after inhibition of vesicle insertion with brefeldin. Coexpression of NHERF2 and SGK1 does not modify cytosolic pH but leads to a slight shift of pK a of ROMK1 to more acidic values. In conclusion, NHERF2 and SGK1 interact to enhance ROMK1 activity in large part by enhancing the abundance of channel protein within the cell membrane. This interaction allows the integration of genomic regulation and activation of SGK1 and NHERF2 in the control of ROMK1 activity and renal K ϩ excretion.

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Section: Discussionmentioning

confidence: 59%

The Serum and Glucocorticoid-Inducible Kinase SGK1 and the Na+/H+ Exchange Regulating Factor NHERF2 Synergize to Stimulate the Renal Outer Medullary K+ Channel ROMK1

Yun¹,

Palmada

Embark

et al. 2002

Journal of the American Society of Nephrology

116

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“…Interestingly, single channel experiments revealed that the different PKA phospho-acceptors regulate the channel through different mechanisms (15). Phosphorylation of the two COOHterminal sites are required to maintain the channel in a high open probability state (15), controlling both pH-dependent gating (16) and phosphatidylinositol 4,5-biphosphate-dependent activation of the channel (17). Phosphorylation of the NH 2 -terminal site, on the other hand, has no effect on channel open probability.…”

Section: From the Department Of Physiology University Of Maryland Scmentioning

confidence: 99%

Cell Surface Expression of the ROMK (Kir 1.1) Channel Is Regulated by the Aldosterone-induced Kinase, SGK-1, and Protein Kinase A

Yoo¹,

Kim

Campo

et al. 2003

Journal of Biological Chemistry

159

146

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The Kir1.1 (ROMK) subtypes of inward rectifier K ؉ channels mediate potassium secretion and regulate sodium chloride reabsorption in the kidney. The density of ROMK channels on the cortical collecting duct apical membrane is exquisitely regulated in concert with physiological demands. Although protein kinase A-dependent phosphorylation of one of the three phospho-acceptors in Kir1.1, Ser-44, also a canonical serum-glucocorticoid-regulated kinase (SGK-1) phosphorylation site, controls the number of active channels, it is unknown whether this involves activating dormant channels already residing on the plasma membrane or recruiting new channels to the cell surface. Here we explore the mechanism and test whether SGK-1 phosphorylation of ROMK regulates cell surface expression. Removal of the phosphorylation site by point mutation (Kir1.1, S44A) dramatically attenuated the macroscopic current density in Xenopus oocytes. As measured by antibody binding of external epitope-tagged forms of Kir1.1, surface expression of Kir1.1 S44A was inhibited, paralleling the reduction in macroscopic current. In contrast, surface expression and macroscopic current density was augmented by a phosphorylation mimic mutation, Kir1.1 S44D. In vitro phosphorylation assays revealed that Ser-44 is a substrate of SGK-1 phosphorylation, and expression of SGK-1 with the wild type channel increased channel density to the same level as the phosphorylation mimic mutation. Moreover, the stimulatory effect of SGK-1 was completely abrogated by mutation of the phosphorylation site. In conclusion, SGK-1 phosphorylation of Kir1.1 drives expression on the plasmalemma. Because SGK-1 is an early aldosterone-induced gene, our results suggest a possible molecular mechanism for aldosterone-dependent regulation of the secretory potassium channel in the kidney.Extracellular potassium homeostasis, maintained by the regulation of renal potassium excretion, is dependent on the activity of weakly inward rectifying ''small conductance'' potassium channels (SK) 1 that are expressed on the apical membrane of epithelial cells in the distal nephron (1, 2). Encoded by the ROMK (Kir 1.1 or KCNJ1) gene (3, 4), these Kir channels are thought to be the major, but not exclusive (5, 6), route for potassium transport into the tubule lumen and constitute a final regulated component of the potassium secretory machinery of the kidney (7,8). Indeed, aldosterone, vasopressin, and other factors precisely regulate SK activity, controlling potassium excretion in accord with the demands of potassium balance. Because ROMK channels normally exhibit a very high open probability, near unity, physiologic augmentation of channel activity, as controlled by hormones and dietary potassium (9), is achieved largely by regulated changes in the number of active channels on the plasmalemma.Although the precise molecular mechanisms responsible for physiological augmentation of ROMK channel surface density have remained unclear, a growing body of evidence has pointed to an important role of protein kina...

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“…Activity of the Kir1.1 channel is directly regulated by intracellular pH, protein kinase A, protein kinase C, phosphatidylinositol-4,5-biphosphate, and WNK4 (serine-threonine kinase 4 with no lysine) (8 -11). Inhibition of protein kinase A activity or phosphatidylinositol-4,5-biphosphate production shifts the channel sensitivity toward more alkaline pH levels (12,13).…”

Section: Kir11 (Romk1) Is a Member Of The Inward Rectifier Kmentioning

confidence: 99%

Subunit Stoichiometry of the Kir1.1 Channel in Proton-dependent Gating

Wang

et al. 2005

Journal of Biological Chemistry

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Kir1.1 channel regulates membrane potential and K؉ secretion in renal tubular cells. This channel is gated by intracellular protons, in which a lysine residue (Lys 80 ) plays a critical role. Mutation of the Lys 80 to a methionine (K80M) disrupts pH-dependent channel gating. To understand how an individual subunit in a tetrameric channel is involved in pH-dependent channel gating, we performed these studies by introducing K80M-disrupted subunits to tandem tetrameric channels. The pH sensitivity was studied in whole-cell voltage clamp and inside-out patches. Homomeric tetramers of the wild-type (wt) and K80M-disrupted channels showed a pH sensitivity almost identical to that of their monomeric counterparts. In heteromeric tetramers and dimers, pH sensitivity was a function of the number of wt subunits. Recruitment of the first single wt subunit shifts the pK a greatly, whereas additions of any extra wt subunit had smaller effects. Single-channel analysis revealed that the tetrameric channel with two or more wt subunits showed one substate conductance at ϳ40% of the full conductance, suggesting that four subunits act as two pairs. However, three and four substates of conductance were seen in the tetrameric wt-3K80M and 4K80M channels. Acidic pH increased long-time closures when there were two or more wt subunits. Disruption of more than two subunits led to flicking activity with appearance of a new opening event and loss of the long period of closures. Interestingly, the channel with two wt subunits at diagonal and adjacent configurations showed the same pH sensitivity, substate conductance, and long-time closure. These results thus suggest that one functional subunit is sufficient to act in the pH-dependent gating of the Kir1.1 channel, the channel sensitivity to pH increases with additional subunits, the full pH sensitivity requires contributions of all four subunits, and two subunits may be coordinated in functional dimers of either trans or cis configuration.Kir1.1 (ROMK1) is a member of the inward rectifier K ϩ (Kir) channel family. A functional channel consists of four Kir subunits, and each Kir subunit has two membrane-spanning helices and a pore-forming sequence (1, 2). The Kir1.1 channel was originally cloned from the outer medulla of the kidney and later found in several other tissues (3, 4). The channel regulates K ϩ recycling in the thick ascending limb of Henle and K ϩ secretion in the cortical collecting duct (4, 5). Dysfunction of the Kir1.1 channel is one of the major causes for Bartter syndrome, which is characterized by decreased salt transport in the thick ascending limb of Henle (5). Kir1.1 surface expression and trafficking rely on protein kinase A and protein kinase C (6, 7). Activity of the Kir1.1 channel is directly regulated by intracellular pH, protein kinase A, protein kinase C, phosphatidylinositol-4,5-biphosphate, and WNK4 (serine-threonine kinase 4 with no lysine) (8 -11). Inhibition of protein kinase A activity or phosphatidylinositol-4,5-biphosphate production shifts the channel sen...

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PKA site mutations of ROMK2 channels shift the pH dependence to more alkaline values

Cited by 42 publications

References 23 publications

The Serum and Glucocorticoid-Inducible Kinase SGK1 and the Na+/H+ Exchange Regulating Factor NHERF2 Synergize to Stimulate the Renal Outer Medullary K+ Channel ROMK1

The Serum and Glucocorticoid-Inducible Kinase SGK1 and the Na+/H+ Exchange Regulating Factor NHERF2 Synergize to Stimulate the Renal Outer Medullary K+ Channel ROMK1

Cell Surface Expression of the ROMK (Kir 1.1) Channel Is Regulated by the Aldosterone-induced Kinase, SGK-1, and Protein Kinase A

Subunit Stoichiometry of the Kir1.1 Channel in Proton-dependent Gating

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