Both the wild type and a functional isoform of CFTR are expressed in kidney

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

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Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

The Journal of General Physiology ◽

10.1085/jgp.200810056 ◽

2008 ◽

Vol 132 (6) ◽

pp. 681-692 ◽

Cited By ~ 54

Author(s):

Qinghuan Xiao ◽

Andrew Prussia ◽

Kuai Yu ◽

Yuan-yuan Cui ◽

H. Criss Hartzell

Keyword(s):

Amino Acids ◽

Density Functional ◽

Transmembrane Domain ◽

Channel Gating ◽

Regulatory Domain ◽

Acidic Amino Acids ◽

C Terminus ◽

Ef Hand ◽

Cl Channels

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl− ion channels that are activated by intracellular Ca2+. At present, the structures and mechanisms responsible for Ca2+ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca2+ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca2+ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca2+ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca2+. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca2+ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca2+-binding loop (312–323) reduced the apparent Ca2+ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca2+-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca2+ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca2+ and suggest a model in which Ca2+ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ∼100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca2+ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca2+.

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Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1

Biochemical Journal ◽

10.1042/bj20030884 ◽

2004 ◽

Vol 379 (1) ◽

pp. 31-38 ◽

Cited By ~ 63

Author(s):

Emily R. SLEPKOV ◽

Signy CHOW ◽

M. Joanne LEMIEUX ◽

Larry FLIEGEL

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Mammalian Cells ◽

Integral Membrane Protein ◽

Amide Hydrogen ◽

Wild Type ◽

Transmembrane Segment ◽

Mutant Proteins ◽

Transmembrane Segments ◽

Induced Changes

NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167→Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE.

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TMEM16A–TMEM16B chimaeras to investigate the structure–function relationship of calcium-activated chloride channels

Biochemical Journal ◽

10.1042/bj20130348 ◽

2013 ◽

Vol 452 (3) ◽

pp. 443-455 ◽

Cited By ~ 32

Author(s):

Paolo Scudieri ◽

Elvira Sondo ◽

Emanuela Caci ◽

Roberto Ravazzolo ◽

Luis J. V. Galietta

Keyword(s):

Chloride Channels ◽

Transmembrane Domain ◽

Amino Acid Residues ◽

Intracellular Loop ◽

Transmembrane Segments ◽

C Terminus ◽

Third Intracellular Loop ◽

Cl Channels ◽

Function Relationship ◽

Relationship Of

TMEM16A and TMEM16B proteins are CaCCs (Ca2+-activated Cl− channels) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a 10-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimaeric proteins in which different domains of the TMEM16A protein were replaced by the equivalent domains of TMEM16B. Replacement of the N-terminus, TMD (transmembrane domain) 1–2, the first intracellular loop and TMD3–4 did not change the channel's properties. Instead, replacement of intracellular loop 3 decreased the apparent Ca2+ affinity by nearly 8-fold with respect to wild-type TMEM16A. In contrast, the membrane currents derived from chimaeras containing TMD7–8 or the C-terminus of TMEM16B showed higher activation and deactivation rates without a change in Ca2+ sensitivity. Significantly accelerated kinetics were also found when the entire C-terminus of the TMEM16A protein (77 amino acid residues) was deleted. Our findings indicate that the third intracellular loop of TMEM16A and TMEM16B is the site involved in Ca2+-sensitivity, whereas the C-terminal part, including TMD7–8, affect the rate of transition between the open and the closed state.

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Determinants of cardiac Na+/Ca2+exchanger temperature dependence: NH2-terminal transmembrane segments

AJP Cell Physiology ◽

10.1152/ajpcell.00558.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. C512-C520 ◽

Cited By ~ 8

Author(s):

Christian Marshall ◽

Chadwick Elias ◽

Xiao-Hua Xue ◽

Hoa Dinh Le ◽

Alexander Omelchenko ◽

...

Keyword(s):

Temperature Dependence ◽

Temperature Sensitivity ◽

Xenopus Oocytes ◽

Energy Of Activation ◽

Wild Type ◽

Transmembrane Segment ◽

Inhibitory Peptide ◽

Transmembrane Segments ◽

Lower Temperature ◽

Patch Technique

The cardiac Na+/Ca2+ exchanger (NCX) in trout exhibits profoundly lower temperature sensitivity in comparison to the mammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout and canine NCX cDNA and expressed in Xenopus oocytes. NCX-mediated currents were measured at 7, 14, and 30°C using the giant excised-patch technique. By using this approach, the differential temperature dependence of NCX was found to reside within the NH2-terminal region of the molecule. Specifically, we found that ∼75% of the Na+/Ca2+ exchange differential energy of activation is attributable to sequence differences in the region that include the first four transmembrane segments, and the remainder is attributable to transmembrane segment five and the exchanger inhibitory peptide site.

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Both CFTR and outwardly rectifying chloride channels contribute to cAMP-stimulated whole cell chloride currents

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.5.c1464 ◽

1994 ◽

Vol 266 (5) ◽

pp. C1464-C1477 ◽

Cited By ~ 91

Author(s):

E. M. Schwiebert ◽

T. Flotte ◽

G. R. Cutting ◽

W. B. Guggino

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Cell Lines ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Wild Type ◽

Whole Cell ◽

Cyclic Monophosphate ◽

Cl Channel ◽

Cl Channels

From whole cell patch-clamp recordings at 35 degrees C utilizing either nystatin perforation or conventional methods with 5 mM MgATP in the pipette solution, it was demonstrated that both cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels and outwardly rectifying Cl- channels (ORCC) contribute to adenosine 3',5'-cyclic monophosphate (cAMP)-activated whole cell Cl- currents in cultured human airway epithelial cells. These results were similar whether recordings were performed on two normal human cell lines or on two cystic fibrosis (CF) cell lines stably complemented with wild-type CF gene. These results were obtained by exploiting dissimilar biophysical properties of CFTR and ORCC currents such as the degree of rectification of the current-voltage relationship, the difference in sensitivity to Cl- channel-blocking drugs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), calixarenes, and diphenylamine carboxylic acid (DPC), and the opposing Cl- relative to I- permeabilities of the two channels. In normal cells or complemented CF cells, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate stimulated outwardly rectifying whole cell Cl- currents. Addition of DIDS in the presence of cAMP inhibited the outwardly rectifying portion of the cAMP-activated Cl- current. The remaining cAMP-activated, DIDS-insensitive, linear CFTR Cl- current was inhibited completely by DPC. Additional results showed that not only do ORCC and CFTR Cl- channels contribute to cAMP-activated Cl- currents in airway epithelial cells where wild-type CFTR is expressed but that both channels fail to respond to cAMP in delta F508-CFTR-containing CF airway cells. We conclude that CFTR not only functions as a cAMP-regulated Cl- channel in airway epithelial cells but also controls the regulation of ORCC.

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Regulation of Greatwall kinase during Xenopus oocyte maturation

Molecular Biology of the Cell ◽

10.1091/mbc.e11-01-0008 ◽

2011 ◽

Vol 22 (13) ◽

pp. 2157-2164 ◽

Cited By ~ 19

Author(s):

Tomomi M. Yamamoto ◽

Kristina Blake-Hodek ◽

Byron C. Williams ◽

Andrea L. Lewellyn ◽

Michael L. Goldberg ◽

...

Keyword(s):

Oocyte Maturation ◽

Mammalian Cells ◽

Xenopus Oocytes ◽

Kinase Activity ◽

Wild Type ◽

M Phase ◽

Phosphatase 2A ◽

Greatwall Kinase ◽

Related Proteins

Greatwall kinase has been identified as a key element in M phase initiation and maintenance in Drosophila, Xenopus oocytes/eggs, and mammalian cells. In M phase, Greatwall phosphorylates endosulfine and related proteins that bind to and inhibit protein phosphatase 2A/B55, the principal phosphatase for Cdk-phosphorylated substrates. We show that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates from it when progesterone-treated oocytes reach M phase. This dissociation does not require Greatwall kinase activity or phosphorylation at T748 in the presumptive T loop of the kinase. A mutant K71M Greatwall, also known as Scant in Drosophila, induces M phase in the absence of progesterone when expressed in oocytes, despite its reduced stability and elevated degradation by the proteasome. M phase induction by Scant Greatwall requires protein synthesis but is not associated with altered binding or release of PP2A/B55 as compared to wild-type Greatwall. However, in vitro studies with Greatwall proteins purified from interphase cells indicate that Scant, but not wild-type Greatwall, has low but detectable activity against endosulfine. These results demonstrate progesterone-dependent regulation of the PP2A/B55–Greatwall interaction during oocyte maturation and suggest that the cognate Scant Greatwall mutation has sufficient constitutive kinase activity to promote M phase in Xenopus oocytes.

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Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00465.2010 ◽

2011 ◽

Vol 301 (4) ◽

pp. L587-L597 ◽

Cited By ~ 13

Author(s):

Louise C. Pyle ◽

Annette Ehrhardt ◽

Lisa High Mitchell ◽

LiJuan Fan ◽

Aixia Ren ◽

...

Keyword(s):

Cystic Fibrosis ◽

High Throughput Screening ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Regulatory Domain ◽

Thyroid Cells ◽

Binding Domains ◽

Mechanistic Basis ◽

Rat Thyroid

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UCCF-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UCCF-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o− and Fischer rat thyroid cells, expressing various CFTR forms. UCCF-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UCCF-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR.

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Identification of Cys140 in helix 4 as an exofacial cysteine residue within the substrate-translocation channel of rat equilibrative nitrobenzylthioinosine (NBMPR)-insensitive nucleoside transporter rENT2

Biochemical Journal ◽

10.1042/bj3530387 ◽

2001 ◽

Vol 353 (2) ◽

pp. 387-393 ◽

Cited By ~ 21

Author(s):

Sylvia Y. M. YAO ◽

Manickavasagam SUNDARAM ◽

Eugene G. CHOMEY ◽

Carol E. CASS ◽

Stephen A. BALDWIN ◽

...

Keyword(s):

Xenopus Oocytes ◽

Cysteine Residue ◽

Integral Membrane Proteins ◽

Structural Domain ◽

Nucleoside Transporter ◽

Vasoactive Drugs ◽

Wild Type ◽

Functional Protein ◽

Equilibrative Nucleoside Transporter ◽

Transmembrane Segments

The human and rat equilibrative nucleoside transporter proteins hENT1, rENT1, hENT2 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs), and are distinguished functionally by differences in transport of nucleobases and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs. In the present study, we have produced recombinant hENT1, rENT1, hENT2 and rENT2 in Xenopus oocytes and investigated uridine transport following exposure to the impermeant thiol-reactive reagent p-chloromercuriphenyl sulphonate (PCMBS). PCMBS caused reversible inhibition of uridine influx by rENT2, but had no effect on hENT1, hENT2 or rENT1. This difference correlated with the presence in rENT2 of a unique Cys residue (Cys140) in the outer half of TM4 that was absent from the other ENTs. Mutation of Cys140 to Ser produced a functional protein (rENT2/C140S) that was insensitive to inhibition by PCMBS, identifying Cys140 as the exofacial Cys residue in rENT2 responsible for PCMBS inhibition. Uridine protected wild-type rENT2 against PCMBS inhibition, suggesting that Cys140 in TM4 lies within or is closely adjacent to the substrate-translocation channel of the transporter. TM4has been shown previously to be within a structural domain (TMs 3Ő6) responsible for interactions with NBMPR, vasoactive drugs and nucleobases.

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Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vpu Transmembrane Domain That Promotes the Enhanced Release of Virus-Like Particles from the Plasma Membrane of Mammalian Cells

Journal of Virology ◽

10.1128/jvi.72.2.1270-1279.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 1270-1279 ◽

Cited By ~ 43

Author(s):

Mousumi Paul ◽

Suparna Mazumder ◽

Nicholas Raja ◽

M. Abdul Jabbar

Keyword(s):

Amino Acids ◽

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Hela Cells ◽

Mammalian Cells ◽

Transmembrane Domain ◽

Wild Type ◽

Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.

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Rescue of Volume-regulated Anion Current by Bestrophin Mutants with Altered Charge Selectivity

The Journal of General Physiology ◽

10.1085/jgp.200810065 ◽

2008 ◽

Vol 132 (5) ◽

pp. 537-546 ◽

Cited By ~ 24

Author(s):

Li-Ting Chien ◽

H. Criss Hartzell

Keyword(s):

Mammalian Cells ◽

Anion Channel ◽

Peritoneal Macrophages ◽

Hek293 Cells ◽

S2 Cells ◽

Wild Type ◽

Biophysical Properties ◽

Ionic Selectivity ◽

Charge Selectivity ◽

Cl Channels

Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca2+-activated Cl− channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca2+-activated Cl− currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES−) (PCs/PCl = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs+ than Cl−. The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES− treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1−/−/best2−/−). VRACs were identical in wild-type and best1−/−/best2−/− mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.

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