Nonselective and G
 βγ
 -Insensitive
 
 weaver
 
 K
 +
 Channels

Navarro, Betsy; Kennedy, Matthew; Velimirovic, Bratislav M.; Bhat, Deepti; Peterson, Andrew S.; Clapham, David E.

doi:10.1126/science.272.5270.1950

Cited by 153 publications

(128 citation statements)

References 18 publications

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“…3). Acidic substitution reduced the P Na ͞P K ratio to values comparable with WT Kir3.2 channels, whereas hydrophobic, hydrophilic, and basic substitution resulted in nonselective channels (Table 1) (21). These experiments reveal that only acidic residues at this pore-facing site in the channel cavity are capable of reinstating K ϩ selectivity to the nonselective S177W channel.…”

Section: Effect Of the M2 Helix Mutations On The Geometry Of The Selementioning

confidence: 81%

Electrostatic interactions in the channel cavity as an important determinant of potassium channel selectivity

Bichet

Grabe

Jan

2006

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

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Potassium channels are membrane proteins that allow the passage of potassium ions at near diffusion rates while severely limiting the flux of the slightly smaller sodium ions. Although studies thus far have focused on the narrowest part of the channel, known as the selectivity filter, channels are long pores with multiple ions that traverse the selectivity filter, the water-filled central cavity, and the rest of the pore formed by cytoplasmic domains. Here, we present experimental analyses on Kir3.2 (GIRK2), a G proteinactivated inwardly rectifying potassium (Kir) channel, showing that a negative charge introduced at a pore-facing position in the cavity (N184) below the selectivity filter restores both K ؉ selectivity and inward rectification properties to the nonselective S177W mutant channel. Molecular modeling demonstrates that the negative residue has no effect on the geometry of the selectivity filter, suggesting that it has a local effect on the cavity ion. Moreover, restoration of selectivity does not depend on the exact location of the charge in the central cavity as long as this residue faces the pore, where it is in close contact with permeant ions. Our results indicate that interactions between permeant ions and the channel cavity can influence ion selectivity and channel block by means of an electrostatic effect.inward rectification ͉ permeation ͉ permeability ͉ GIRK modeling ͉ Kir3 P otassium channels are found in all kingdoms and domains and serve a wide range of important physiological functions, such as volume regulation and potassium transport in plants (1, 2) and the control of insulin release, blood flow, heart rate, neuronal signaling, and potassium secretion in the kidney and inner ear of animals (3-5). These physiological functions rely critically on the channel's ability to permeate K ϩ and not Na ϩ , which is smaller and more abundant in nature. To understand how K ϩ channels fulfill their physiological functions, and to gain some appreciation as to how their remarkable selectivity for K ϩ permeation might have evolved, it is important to characterize all of the features of the K ϩ channel pore that contribute to its ability to discriminate between potassium and other ions.Our understanding of K ϩ channel selectivity has gained considerable insight from the KcsA potassium channel crystal structure, which revealed that four identical subunits come together to form a central pore (6). The pore is most constricted over a narrow span, termed the selectivity filter, which is formed by four loops bearing the amino acid sequence GYG near the extracellular side of the membrane. This sequence is highly conserved throughout the K ϩ channel superfamily and is thought to underlie the basis of K ϩ selectivity; therefore, it is called the K ϩ channel signature sequence (7). Although it is clear that this sequence plays a key role in selectivity, it makes up only a fraction of the channel's entire permeation pathway, and the relative contributions to K ϩ selectivity from the selectivity filter and the r...

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Section: Effect Of the M2 Helix Mutations On The Geometry Of The Selementioning

confidence: 81%

Electrostatic interactions in the channel cavity as an important determinant of potassium channel selectivity

Bichet

Grabe

Jan

2006

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

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“…[5][6][7][8][9][10][11][12] It is generally assumed that target cells in weaver mice die as a result of chronic depolarization; however, the pathogenetic mechanisms that lead to selective activation of apoptosis in cerebellar granule cell precursors but not in dopaminergic neurons of midbrain are not completely understood. Here we show that abnormalities in the control of cell cycle in wv/wv cerebellum represent a major and early consequence of the altered GIRK2 channel function that may strongly influence the susceptibility of EGL cells to undergo apoptosis.…”

Section: Discussionmentioning

confidence: 99%

“…4 In vitro, the mutation leads to a complex series of changes in channel activity, ranging from a loss of G-proteindependent inward rectifier current regulation 5,6 to reduced selectivity for potassium over sodium and calcium ions. [7][8][9][10][11][12] This alteration results in chronic depolarization and cell death. In vivo studies show that the granule cell precursors of the external germinal layer (EGL) of the cerebellum die massively via apoptosis within the first 3 postnatal weeks.…”

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confidence: 99%

A Cell Cycle Alteration Precedes Apoptosis of Granule Cell Precursors in the Weaver Mouse Cerebellum

Migheli

Piva

Casolino

et al. 1999

Journal of Neuropathology and Experimental Neurology

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A missense mutation in the gene coding for the Gprotein-activated inwardly rectifying potassium (GIRK) channel , GIRK2 , is responsible for apoptosis in the external germinal layer (EGL) of the cerebellum and a nonapoptotic death of midbrain dopaminergic neurons in the weaver (wv) mouse. Failure of axonogenesis and migration are considered to be the primary consequences of GIRK2 channel malfunction in the cerebellum. We investigated whether a disruption of the cell cycle precedes the failure of migration and axonogenesis and leads to massive apoptosis. To this end , immunohistochemistry and immunoblotting for PCNA , Cdk4 , cyclin D , cyclin A , and the Cdk inhibitor p27/kip1 , as well as in situ end-labeling for apoptotic DNA fragmentation , were applied to cerebella of P7-P21؉/؉, wv/؉, and wv/wv mice. In ؉/؉ and wv/؉ mice , the expression of cell cycle proteins was limited to the outer , premigratory zone of the EGL. Antibodies to p27 , a marker of cell differentiation , gave a reverse staining pattern. Due to migration delay, patches of p27-positive cells persisted in the outer EGL in P21 wv/؉ mice. On the contrary , marked cell cycle up-regulation and absence of p27 occurred throughout the EGL at all ages in wv/wv mice , indicating an inability to switch off the cell cycle. Mitotic index evaluation showed that cell cycle activation was unrelated to proliferative events. Cell cycle proteins were not expressed in the substantia nigra , suggesting that nonapoptotic death of mature dopaminergic neurons is not preceded by abortive cell cycle reentry. Our data show that abnormalities of the cell cycle in wv/wv cerebellum represent a major and early consequence of GIRK2 channel malfunction and may strongly influence the susceptibility of EGL cells to apoptosis. These observations may help in understanding the pathogenesis of human neurological channelopathies. (Am J Pathol 1999, 155:365-373)The weaver (wv) mutation in the homozygous (wv/wv) mouse is associated with a loss of granule cells in the cerebellum, dopaminergic neurons in the midbrain, and germinal epithelium in the testis.

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“…Indeed, the presence of other residues at this site might very well have adverse consequences in vivo because mutant channels containing a Lys or Gln at this position are sufficiently active at 37°C to promote the death of transfected mammalian cells in culture. Such hyperactivating mutations in the VR1 gene might result in loss of nociceptors, in much the same way that constitutively activating mutations of other ion channels lead to neurodegeneration (29)(30)(31)(32)(33)(34), or systemic treatment of neonatal rats with capsaicin induces death of VR1-expressing primary afferent neurons (35). Among rare individuals who suffer from congenital insensitivity to pain, some lack a subset of unmyelinated small-diameter nociceptors as a consequence of defects in the high-affinity nerve growth factor receptor (trkA) gene (36)(37)(38).…”

Section: Discussionmentioning

confidence: 99%

Acid potentiation of the capsaicin receptor determined by a key extracellular site

Jordt

Tominaga

Julius

2000

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

575

415

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The capsaicin (vanilloid) receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. The response of VR1 to capsaicin or noxious heat is dynamically potentiated by extracellular protons within a pH range encountered during tissue acidosis, such as that associated with arthritis, infarction, tumor growth, and other forms of injury. A molecular determinant for this important physiological activity was localized to an extracellular Glu residue (E600) in the region linking the fifth transmembrane domain with the putative pore-forming region of the channel. We suggest that this residue serves as a key regulatory site of the receptor by setting sensitivity to other noxious stimuli in response to changes in extracellular proton concentration. We also demonstrate that protons, vanilloids, and heat promote channel opening through distinct pathways, because mutations at a second site (E648) selectively abrogate proton-evoked channel activation without diminishing responses to other noxious stimuli. Our findings provide molecular evidence for stimulus-specific steps in VR1 activation and offer strategies for the development of novel analgesic agents. P ain is initiated when noxious thermal, mechanical, or chemical stimuli excite the peripheral terminals of specialized primary afferent neurons called nociceptors (1). Tissue damage, such as that associated with infection, inflammation, or ischemia, produces an array of chemical mediators that activate or sensitize nociceptor terminals to elicit pain and promote tenderness at the site of injury. An important component of this proalgesic response is local acidosis, namely, a reduction in extracellular pH to levels below the physiological norm of Ϸ7.6 (2). Protons are capable of modulating the activity of a number of receptors and ion channels expressed by primary afferent nociceptors, including acid-sensitive channels of the degenerin family (3), ATPgated channels (4, 5), and vanilloid receptors (6, 7). Analyses of native and cloned vanilloid receptors suggest that they play a role in mediating sustained proton responses in vivo (8)(9)(10)(11)(12). This is further supported by the profoundly reduced responses to acid in cultured dorsal root ganglion neurons and isolated skin nerve preparations of mice with a targeted disruption of the gene encoding for the cloned capsaicin (vanilloid) receptor VR1 (13).Vanilloid receptors are nociceptor-specific cation channels that serve as the molecular target for capsaicin, the main pungent ingredient in ''hot'' chili peppers. VR1 is homologous to members of the transient receptor potential family of ion channels first identified in the Drosophila phototransduction pathway (6,14). We have shown that, when expressed in heterologous systems, VR1 is also activated by noxious heat (6, 7) with a threshold (Ͼ43°C) resembling that of native heat-activated currents in cultured sensory neurons (15). Acidification of the extracellular milieu has two primary effects on V...

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Nonselective and G _βγ -Insensitive weaver K ⁺ Channels

Cited by 153 publications

References 18 publications

Electrostatic interactions in the channel cavity as an important determinant of potassium channel selectivity

Electrostatic interactions in the channel cavity as an important determinant of potassium channel selectivity

A Cell Cycle Alteration Precedes Apoptosis of Granule Cell Precursors in the Weaver Mouse Cerebellum

Acid potentiation of the capsaicin receptor determined by a key extracellular site

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Nonselective and G βγ -Insensitive weaver K + Channels

Cited by 153 publications

References 18 publications

Electrostatic interactions in the channel cavity as an important determinant of potassium channel selectivity

Electrostatic interactions in the channel cavity as an important determinant of potassium channel selectivity

A Cell Cycle Alteration Precedes Apoptosis of Granule Cell Precursors in the Weaver Mouse Cerebellum

Acid potentiation of the capsaicin receptor determined by a key extracellular site

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Product

Resources

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Nonselective and G _βγ -Insensitive weaver K ⁺ Channels