Identification of a titratable lysine residue that determines sensitivity of kidney potassium channels (ROMK) to intracellular pH.

Fakler, Bernd; Schultz, Jobst‐Hendrik; Yang, Ji-jie; Schulte, Uwe; Brändle, Uwe; Zenner, Hans‐Peter; Jan, L. Y.; Ruppersberg, J. P.

doi:10.1002/j.1460-2075.1996.tb00784.x

Cited by 165 publications

(215 citation statements)

References 39 publications

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“…2 A). Similar observations for ROMK1 and ROMK2 have been reported by others (8,10). The half-maximal inhibitory pH values, pH 0.5 , and Hill coefficients of the isoforms were not significantly different (Fig.…”

Section: Resultssupporting

confidence: 89%

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Subunit–subunit interactions are critical for proton sensitivity of ROMK: Evidence in support of an intermolecular gating mechanism

MacGregor

Dong

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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, and the gating of this channel is highly sensitive to changes in cytosolic pH. Although charge-charge interactions have been implicated in pH sensing by this K channel tetramer, the molecular mechanism linking pH sensing and the gating of ion channels is poorly understood. The x-ray crystal structure KirBac1.1, a prokaryotic ortholog of ROMK, has suggested that channel gating involves intermolecular interactions of the N-and C-terminal domains of adjacent subunits. Here we studied channel gating behavior to changes in pH using giant patch clamping of Xenopus laevis oocytes expressing WT or mutant ROMK, and we present evidence that no single charged residue provides the pH sensor. Instead, we show that N-C-and C-Cterminal subunit-subunit interactions form salt bridges, which function to stabilize ROMK in the open state and which are modified by protons. We identify a highly conserved C-C-terminal arginine-glutamate (R-E) ion pair that forms an intermolecular salt bridge and responds to changes in proton concentration. Our results support the intermolecular model for pH gating of inward rectifier K channels.channel gating ͉ inward rectifier ͉ Kir1.1 ͉ pH sensing ͉ potassium channel

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Section: Resultssupporting

confidence: 89%

“…1) produced pH-insensitive channels (8,10,14); however, a more complete pH titration to 4 demonstrated that each of the three ROMK isoforms remained pH sensitive, with the N-terminal K3 M mutation ( Fig. 2 B and C).…”

Section: Resultsmentioning

confidence: 96%

Subunit–subunit interactions are critical for proton sensitivity of ROMK: Evidence in support of an intermolecular gating mechanism

MacGregor

Dong

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…These data suggest a possibility that pH i -sensitive K ϩ channels usually have the specific pH i -sensitive site in the channel protein. Although the mechanism of pH i -sensitivity in the BK channel of RPTECs is still unknown, our conclusion that H ϩ regulates the activity of this BK channel by binding to the site distinct from Ca 2ϩ sites is supported by findings in the above recent reports [35][36][37]. It is quite likely that H ϩ inhibited the channel activity mainly by the allosteric reduction of the Ca 2ϩ binding affinity.…”

Section: Discussionsupporting

confidence: 80%

“…On the other hand, a recent report using mSlo3, a member of the BK channel family sensitive to pH i rather than to Ca 2ϩ , provided evidence that the pH i -sensitivity of this channel is independent of Ca 2ϩ -sensitivity [35]. Furthermore, it has also been demonstrated in a cloned pH i -sensitive K ϩ channel, K ir 1.1 (ROMK), that an intracellular lysine close to the M1 segment, composing the pore region, has been identified as the structural element necessary for pH idependent gating [36,37]. These data suggest a possibility that pH i -sensitive K ϩ channels usually have the specific pH i -sensitive site in the channel protein.…”

Section: Discussionmentioning

confidence: 96%

Modulation of the Ca2+-Activated Large Conductance K+ Channel by Intracellular pH in Human Renal Proximal Tubule Cells.

Hirano

Nakamura²,

Itazawa³

et al. 2002

JJP

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sensitive large conductance (big) K ϩ channels (BK channels) have been reported in the cell membranes of a variety of tissues [1,2]. An elevation of cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] i ) and depolarization of the membrane commonly stimulates activity of the BK channels [1][2][3]. It has already been proved by the use of cloned BK channels that the channel protein possesses the Ca 2ϩ -binding sites [4,5] and the voltagesensing domain [6,7]. Besides Ca 2ϩ and voltage, other factors such as ATP [8-11], pH [8, 9, 12-21], and Mg 2ϩ [19,[22][23][24] were also reported to modulate BK channels. Among these factors, intracellular pH (pH i ) is one of the significant factors that affect channel activity [8,9,[12][13][14][15][16][17][18][19][20][21], i.e., channel activity is inhibited by intracellular acidification and stimulated by its alkalization. Although the mechanism for pH i effects on channel activity was not precisely understood, two different mechanisms have been proposed. One is that an increased cytoplasmic H ϩ can compete with Ca 2ϩ in binding to the same sites, thereby inhibiting channel activity [14][15][16], whereas the other is that the pH i -mediated change in channel activity is not competitive with Ca 2ϩ [17]. Moreover, it was demonstrated that the conductance of some BK channels was reduced by lowering pH i and enhanced by raising it [9,15,20].In the renal tubules, BK channels have been reported in cultured rabbit proximal tubule cells [25]

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“…We therefore examined the effect on glutamine uptake of lowering the internal pH of ASCT2-expressing oocytes by adding increasing concentrations of HCO 3 Ϫ to the incubation solution. Raising the concentration of HCO 3 Ϫ in the incubation solution from 0 to 90 mM for 15 min has been shown to lower the cytosolic pH of oocytes by ϳ1 unit (Fakler et al, 1996). Under these conditions we observed a modest decrease of glutamine uptake (Table 1).…”

Section: Glutamine Uptake In Asct2-expressing X Laevis Oocytesmentioning

confidence: 57%

The Astroglial ASCT2 Amino Acid Transporter as a Mediator of Glutamine Efflux

Bröer

Brookes²,

Ganapathy³

et al. 1999

Journal of Neurochemistry

207

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Glutamine release from astrocytes is an essential part of the glutamate-glutamine cycle in the brain. Uptake of glutamine into cultured rat astrocytes occurs by at least four different routes. In agreement with earlier studies, a significant contribution of amino acid transport systems ASC, A, L, and N was detected. It has not been determined whether these systems are also involved in glutamine efflux or whether specific efflux transporters exist. We show here that ASCT2, a variant of transport system ASC, is strongly expressed in rat astroglia-rich primary cultures but not in neuron-rich primary cultures. The amino acid sequence of rat astroglial ASCT2 is 83% identical to that of mouse ASCT2. In Xenopus laevis oocytes expressing rat ASCT2, we observed high-affinity uptake of [U-14 C]glutamine (K m ϭ 70 M) that was Na ϩ -dependent, concentrative, and unaffected by membrane depolarization. When oocytes were preloaded with [U-14 C]glutamine, no glutamine efflux was detected in the absence of extracellular amino acids. Neither lowering intracellular pH nor raising the temperature elicited efflux. However, addition of 0.1 mM unlabeled alanine, serine, cysteine, threonine, glutamine, or leucine to the extracellular solution resulted in a rapid release of glutamine from the ASCT2-expressing oocytes. Amino acids that are not recognized as substrates by ASCT2 were ineffective in this role. Extracellular glutamate stimulated glutamine release weakly at pH 7.5 but was more effective on lowering pH to 5.5, consistent with the pH dependence of ASCT2 affinity for glutamate. Our findings suggest a significant role of ASCT2 in glutamine efflux from astrocytes by obligatory exchange with extracellular amino acids. However, the relative contribution of this pathway to glutamine release from cells in vivo or in vitro remains to be determined.

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Identification of a titratable lysine residue that determines sensitivity of kidney potassium channels (ROMK) to intracellular pH.

Cited by 165 publications

References 39 publications

Subunit–subunit interactions are critical for proton sensitivity of ROMK: Evidence in support of an intermolecular gating mechanism

Subunit–subunit interactions are critical for proton sensitivity of ROMK: Evidence in support of an intermolecular gating mechanism

Modulation of the Ca2+-Activated Large Conductance K+ Channel by Intracellular pH in Human Renal Proximal Tubule Cells.

The Astroglial ASCT2 Amino Acid Transporter as a Mediator of Glutamine Efflux

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