Function recovery after chemobleaching (FRAC): Evidence for activity silent membrane receptors on cell surface

Sun, Haiyan; Shikano, Sojin; Xiong, Qiaojie; Li, Min

doi:10.1073/pnas.0404178101

Cited by 24 publications

(31 citation statements)

References 29 publications

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“…Changing the hNav1.5 amino acid 373 from cysteine to tyrosine is expected to decrease channel sensitivity to extracellular application of [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET), which blocks channels by binding to cysteine at residue 373. Using MTSET reagents to bind to pore cysteines is an established technique for preferentially blocking sodium channels [15][16][17] and other channels such as Kir2.1 potassium channels 18 to assess for their presence and functionality of the channel in the cell membrane. C373 is located in domain 1 of hNav1.5 in the pore-forming loop (SS2) before S6 ( Figure 1B).…”

Section: R282h-scn5a Protein Traffickingmentioning

confidence: 99%

SCN5A Polymorphism Restores Trafficking of a Brugada Syndrome Mutation on a Separate Gene

et al. 2006

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Background-Brugada syndrome is associated with a high risk of sudden cardiac death and is caused by mutations in the cardiac voltage-gated sodium channel gene. Previously, the R282H-SCN5A mutation in the sodium channel gene was identified in patients with Brugada syndrome. In a family carrying the R282H-SCN5A mutation, an asymptomatic individual had a common H558R-SCN5A polymorphism and the mutation on separate chromosomes. Therefore, we hypothesized that the polymorphism could rescue the mutation. Methods and Results-In heterologous cells, expression of the mutation alone did not produce sodium current. However, coexpressing the mutation with the polymorphism produced significantly greater current than coexpressing the mutant with the wild-type gene, demonstrating that the polymorphism rescues the mutation. Using immunocytochemistry, we demonstrated that the R282H-SCN5A construct can traffic to the cell membrane only in the presence of the H558R-SCN5A polymorphism. Using fluorescence resonance energy transfer and protein fragments centered on H558R-SCN5A, we demonstrated that cardiac sodium channels preferentially interact when the polymorphism is expressed on one protein but not the other. Conclusions-This study suggests a mechanism whereby the Brugada syndrome has incomplete penetrance. More importantly, this study suggests that genetic polymorphisms may be a potential target for future therapies aimed at rescuing specific dysfunctional protein channels. (Circulation. 2006;114:368-376.)

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Section: R282h-scn5a Protein Traffickingmentioning

confidence: 99%

SCN5A Polymorphism Restores Trafficking of a Brugada Syndrome Mutation on a Separate Gene

et al. 2006

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“…Functional recovery after chemobleaching of TRPV5 activity is regulated by extracellular pH. MDCK cells stably expressing TRPV5 were used to assess the pH dependence of TRPV5 trafficking by FRAC (20). MDCK cells are convenient for imaging EGFP-TRPV5-containing vesicles in contrast to transfected HEK293T cells that display a high density of EGFP-TRPV5-containing vesicles (Fig.…”

Section: Extracellular Ph Determines Trpv5-mediated Camentioning

confidence: 99%

Extracellular pH Dynamically Controls Cell Surface Delivery of Functional TRPV5 Channels

Lambers

Oancea

Groot

et al. 2007

Molecular and Cellular Biology

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Extracellular pH has long been known to affect the rate and magnitude of ion transport processes among others via regulation of ion channel activity. The Ca 2؉ -selective transient receptor potential vanilloid 5 (TRPV5) channel constitutes the apical entry gate in Ca 2؉ -transporting cells, contributing significantly to the overall Ca 2؉ balance. Here, we demonstrate that extracellular pH determines the cell surface expression of TRPV5 via a unique mechanism. By a comprehensive approach using total internal reflection fluorescence microscopy, cell surface protein labeling, electrophysiology, 45 Ca 2؉ uptake assays, and functional channel recovery after chemobleaching, this study shows that upon extracellular alkalinization, a pool of TRPV5-containing vesicles is rapidly recruited to the cell surface without collapsing into the plasma membrane. These vesicles contain functional TRPV5 channels since extracellular alkalinization is accompanied by increased TRPV5 activity. Conversely, upon subsequent extracellular acidification, vesicles are retrieved from the plasma membrane, simultaneously resulting in decreased TRPV5 activity. Thus, TRPV5 accesses the extracellular compartment via transient openings of vesicles, suggesting that rapid responses of constitutive active TRP channels to physiological stimuli rely on vesicular "kiss and linger" interactions with the plasma membrane.The superfamily of transient receptor potential (TRP) channels is involved in diverse physiological processes, ranging from sensory activity to fertility and epithelial ion transport (15). The highly Ca 2ϩ -selective TRP vanilloid 5 (TRPV5) channel constitutes the apical entry gate in Ca 2ϩ -transporting cells and facilitates renal Ca 2ϩ influx from the prourine (10). Several lines of evidence indicate that TRPV5 activity is sensitive to pH. First, acid-based homeostasis is known to affect renal Ca 2ϩ handling as reflected by altered Ca 2ϩ excretion in kidneys during chronic acidosis or alkalosis, which is mediated at least in part by changes in TRPV5 gene expression (16). Second, in vitro studies indicated that intra-and extracellular pH directly regulate the activity of TRPV5. Acidification inhibited, whereas alkalinization stimulated, TRPV5 activity, likely mediated by conformational changes of the channel pore helix (24-26). An intrinsic physiological effect of extracellular pH is the regulation of trafficking processes like endo-and exocytosis and lysosomal trafficking (8,12,14). Since several TRP channels display constitutive activity, controlled recruitment of these channels towards the plasma membrane is important for the translation of physiological stimuli into increased ion permeability of the plasma membrane. For instance, an essential process during insulin-like growth factor-I stimulation of cell growth is TRPV2 recruitment facilitating Ca 2ϩ entry during progression through the cell cycle (11). In Drosophila photoreceptors, the TRP-like subunit is shuttled between the plasma membrane and an intracellular compartment by a light...

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“…؉ Flux Assay A screen of the ChemBridge Diverset TM library of 20,000 compounds for KCNQ2 channel modulators was performed using a Rb ϩ flux assay reported earlier (29,30). To validate the hits from the initial screen, compounds were cherry-picked from the original compound library and retested using the Rb ϩ efflux assay using wild type KCNQ2.…”

Section: High Throughput Library Screening and Hits Validation Using mentioning

confidence: 99%

Isoform-specific Prolongation of Kv7 (KCNQ) Potassium Channel Opening Mediated by New Molecular Determinants for Drug-Channel Interactions

Gao

Zhang

et al. 2010

Journal of Biological Chemistry

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Kv7 channels, especially Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3), are key determinants for membrane excitability in the brain. Some chemical modulators of KCNQ channels are in development for use as anti-epileptic drugs, such as retigabine (D-23129, N-(2-amino-4-(4-fluorobenzylamino)-phenyl)), which was recently approved for clinical use. In addition, several other compounds were also reported to potentiate activity of the Kv7 channels. It is therefore of interest to investigate compound-channel interactions, so that more insights may be gained to aid future development of therapeutics. We have conducted a screen of 20,000 compounds for KCNQ2 potentiators using rubidium flux combined with atomic absorption spectrometry. Here, we report the characterization of a series of new structures that display isoform specificity and induce a marked reduction of deactivation distinct from that of retigabine. Furthermore, KCNQ2(W236L), a previously reported mutation that abolishes sensitivity to retigabine, remains fully sensitive to these compounds. This result, together with mutagenesis and other studies, suggests that the reported compounds confer a unique mode of action and involve new molecular determinants on the channel protein, consistent with the idea of recognizing a new site on channel protein.

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Function recovery after chemobleaching (FRAC): Evidence for activity silent membrane receptors on cell surface

Cited by 24 publications

References 29 publications

SCN5A Polymorphism Restores Trafficking of a Brugada Syndrome Mutation on a Separate Gene

SCN5A Polymorphism Restores Trafficking of a Brugada Syndrome Mutation on a Separate Gene

Extracellular pH Dynamically Controls Cell Surface Delivery of Functional TRPV5 Channels

Isoform-specific Prolongation of Kv7 (KCNQ) Potassium Channel Opening Mediated by New Molecular Determinants for Drug-Channel Interactions

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