Inhibition of ATP-induced increase in muscarinic K<sup>+</sup>current by trypsin, alkaline pH, and anions

Pleumsamran, Apisate; Wolak, Michael L.; Kim, Dong‐Hee

doi:10.1152/ajpheart.1998.275.3.h751

Cited by 8 publications

(8 citation statements)

References 28 publications

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“…Therefore, the inhibition of whole-cell GIRK currents seems to attribute mainly to the inhibition of P o . Intracellular acidification has been previously shown to inhibit cell-endogenous K ACh channels in atrial myocardium (48). Consistent with our current observations, intracellular protons act on the P o in the cardiac K ϩ channels with modest inhibitory effect on single-channel conductance.…”

Section: Discussionsupporting

confidence: 92%

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Inhibition of G-protein-coupled Inward Rectifying K+Channels by Intracellular Acidosis

Mao

Chen

et al. 2003

Journal of Biological Chemistry

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G-protein-coupled inward rectification K؉ (GIRK) channels play an important role in modulation of synaptic transmission and cellular excitability. The GIRK channels are regulated by diverse intra-and extracellular signaling molecules. Previously, we have shown that GIRK1/GIRK4 channels are activated by extracellular protons. The channel activation depends on a histidine residue in the M1-H5 linker and may play a role in neurotransmission. Here, we show evidence that the heteromeric GIRK1/GIRK4 channels are inhibited by intracellular acidification. This inhibition was produced by selective decrease in the channel open probability with a modest drop in the single-channel conductance. The inhibition does not seem to require G-proteins as it was seen in two G-protein coupling-defective GIRK mutants and in excised patches in the absence of exogenous Gproteins. Three histidine residues in intracellular domains were critical for the inhibition. Individual mutation of His-64, His-228, or His-352 in GIRK4 abolished or greatly diminished the inhibition in homomeric GIRK4. Mutations of any of these histidine residues in GIRK4 or their counterparts in GIRK1 were sufficient to eliminate the pH i sensitivity of the heteromeric GIRK1/GIRK4 channels. Thus, the molecular and biophysical bases for the inhibition of GIRK channels by intracellular protons are illustrated. Because of the inequality of the pH i and pH o in most cells and their relatively independent controls by cellular versus systemic mechanisms, such pH i sensitivity may allow these channels to regulate cellular excitability in certain physiological and pathophysiological conditions when intracellular acidosis occurs.G-protein-coupled inward rectifier K ϩ (GIRK) 1 channels play an important role in regulating cellular excitability and synaptic transmission (1, 2). Activation of GIRK channels leads to inhibition of neurons, cardiac myocytes, and endocrine cells (3). It is known that the activation of GIRK channels depends on G-protein ␤␥ subunits (G␤␥) that are released from the G␣␤␥ heterotrimer when G-protein-coupled receptors are activated by extracellular hormones or neurotransmitters. The G␤␥-dependent activation can be eliminated by disrupting certain protein domains and amino acid residues that may be involved in channel gating or the interaction with the G␤␥ (4 -8). In addition to the G␤␥ dependence, GIRK channels are modulated by a number of cellular signaling molecules, such as phospholipids (9, 10), arachidonic acid (11, 12), Na ϩ (13, 14), Mg 2ϩ (15), redox agents (16, 17), and protein kinases (18,19). Such complex regulations suggest that GIRK channels are capable of integrating extra-and intracellular signals and coupling the information to cellular excitability.One important signaling molecule is the hydrogen ion. Our recent studies have shown that the heteromeric GIRK1/GIRK4 channels are stimulated by a drop in extracellular pH (pH o ) (20). The pH o sensitivity depends on a histidine residue located in the M1-H5 linker and may play a role in enhanci...

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Section: Discussionsupporting

confidence: 92%

“…Previous studies have indicated that pH differentially modulates the effects of GTP and ATP on GIRK channels (48). Our recent studies have shown that protonation of a histidine residue in the intracellular C terminus affects the ATP sensitivity of the ATP-sensitive K ϩ channels through the allosteric mechanism (36,53).…”

Section: Discussionmentioning

confidence: 92%

Inhibition of G-protein-coupled Inward Rectifying K+Channels by Intracellular Acidosis

Mao

Chen

et al. 2003

Journal of Biological Chemistry

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“…In our recent study, we found that intracellular application of trypsin prevented the increase in channel activity and open time duration normally produced by ATP (Pleumsamran et al 1998). Although we do not know at what step(s) in the pathway trypsin blocked the ATP effect, it was of interest to examine whether the similar modulatory effects of phospholipids on the K ACh channel activity and open time duration could also be blocked by trypsin.…”

Section: Resultsmentioning

confidence: 99%

Modulation of rat atrial G protein‐coupled K⁺ channel function by phospholipids

Kim

Bang

1999

The Journal of Physiology

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G protein‐gated K+ channels (KACh channels) in the heart and brain are activated by the βγ subunit of inhibitory G protein. Phosphatidylinositol‐4,5‐bisphosphate (PIP2) has recently been reported to directly activate KACh channels (GIRK) expressed in oocytes, as well as to support activation by the βγ subunit in the presence of Na+. We examined the effect of Na+, PIP2 and other phospholipids on the KACh channel to understand better their role in KACh channel activation and modulation. In atrial membrane patches, none of the phospholipids tested including PIP2 caused activation of the KACh channel in either the presence or the absence of 30 mM Na+. PIP2 (3 μM) and other phospholipids (30 μM) blocked acetylcholine‐induced activation of the KACh channel. When KACh channels were first activated with GTPγS, however, all phospholipids (100 μM) tested augmented the KACh channel activity 1·5‐ to 2‐fold. Phosphatidylinositol‐4‐phosphate (PIP) and PIP2 were an order of magnitude more potent than other phospholipids. The increase in KACh channel activity was the result of a shift in the gating mode of the channel from a short‐lived to a longer‐lived open state. Such a modulatory effect was qualitatively similar to that produced by intracellular ATP. Trypsin blocked the ATP effect but not the phospholipid effect on the KACh channel kinetics. The phosphate group linked to the glycerol backbone was important for KACh channel modulation by phospholipids. The higher potency of PIP and PIP2 was due to the presence of inositol phosphates. Intracellular Na+ (30 mM) increased the frequency of KACh channel opening ≈2‐fold if the channels were already active, but did not affect modulation by phospholipids. The effects of Na+ and phospholipids on KACh channel activity were additive. A low concentration of ATP (20 μM), which had no effect on the KACh channel by itself, potentiated the stimulatory action of phospholipids, indicating that ATP and phospholipids interacted to modulate KACh channel function. We conclude that exogenously applied PIP2 and other phospholipids block agonist‐mediated KACh channel activation. However, if the KACh channel is already activated with GTPγS, phospholipids augment the existing activity by increasing the number of longer‐lived channel openings. The evidence for and against the role of PIP and PIP2 in the stimulatory effect of ATP on the KACh channel is presented and discussed.

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“…The influence of citrate remains elusive. Apparently citrate may affect K + and Ca 2+ channels [17, 18], and, surprisingly in this context, even block K + channels [17]. The separation with NP was unchanged after removal of extracellular citrate by repeated washings, which could indicate an indirect effect of citrate or its breakdown products.…”

Section: Resultsmentioning

confidence: 99%

Separation of Human Lymphocytes from Citrated Blood by Density Gradient (NycoPrep) Centrifugation: Monocyte Depletion Depending upon Activation of Membrane Potassium Channels

Bøyum¹,

Fjerdingstad²,

Martinsen³

et al. 2002

Scandinavian Journal of Immunology

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Routine one-step centrifugation procedures (Lymphoprep LP, Percoll) commonly used for separation of blood cells split the cells into two major fractions. After centrifugation the mononuclear cells (MNC monocytes and lymphocytes) are located on the top of the separation fluid, whereas erythrocytes and granulocytes have sedimented to the bottom. We now show that a relatively pure lymphocyte suspension can be obtained by one-step centrifugation of citrated blood by using NycoPrep (NP iohexol), a nonionic X-ray contrast agent. With this gradient medium also the monocytes pass to the bottom, leaving lymphocytes on the top. In parallel separations with LP, which contains Ficoll and a fully dissociated sodium salt of a contrast medium, the results were as usual, i.e. $70±85% lymphocytes and 30±15% monocytes in the top fraction. The monocyte depletion with NP depended upon the use of citrated (ACD) blood and a proper balance of density and osmolality of the gradient medium, and was enhanced by 20 min preincubation with CaCl 2 at room temperature. Monocyte depletion could not be obtained with LP. Under optimal conditions (density 1.075 g/ml, osmolality 280±300 mOsm/kg), the monocyte admixture amounted to $1 (0±2)%, in separations with buffy coat samples. For freshly drawn blood, it was necessary to slightly modify the NP solution. The monocyte depletion was counteracted by blockers of K channels or by KCl in the cell suspension. Following incubation in NP of Percoll-separated cells, an enhanced release of K was observed. The results are interpreted as follows: NP mediates the opening of K channels of MNC, which leads to efflux of K , accompanied with associated anions (Cl ± ). This reduces the osmolality inside the cells which therefore expel water to maintain osmotic equilibrium. In this regard it appears that monocytes are more sensitive than lymphocytes, their density therefore increasing more, so that they are able to pass the density barrier otherwise exerted by the gradient medium.

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Inhibition of ATP-induced increase in muscarinic K⁺current by trypsin, alkaline pH, and anions

Cited by 8 publications

References 28 publications

Inhibition of G-protein-coupled Inward Rectifying K+Channels by Intracellular Acidosis

Inhibition of G-protein-coupled Inward Rectifying K+Channels by Intracellular Acidosis

Modulation of rat atrial G protein‐coupled K⁺ channel function by phospholipids

Separation of Human Lymphocytes from Citrated Blood by Density Gradient (NycoPrep) Centrifugation: Monocyte Depletion Depending upon Activation of Membrane Potassium Channels

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Inhibition of ATP-induced increase in muscarinic K+current by trypsin, alkaline pH, and anions

Cited by 8 publications

References 28 publications

Inhibition of G-protein-coupled Inward Rectifying K+Channels by Intracellular Acidosis

Inhibition of G-protein-coupled Inward Rectifying K+Channels by Intracellular Acidosis

Modulation of rat atrial G protein‐coupled K+ channel function by phospholipids

Separation of Human Lymphocytes from Citrated Blood by Density Gradient (NycoPrep) Centrifugation: Monocyte Depletion Depending upon Activation of Membrane Potassium Channels

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Inhibition of ATP-induced increase in muscarinic K⁺current by trypsin, alkaline pH, and anions

Modulation of rat atrial G protein‐coupled K⁺ channel function by phospholipids