STRUCTURAL BASIS FOR THE FUNCTION AND REGULATION
STRUCTURAL BASIS FOR THE FUNCTION AND REGULATION OF THEEPITHELIAL SODIUM CHANNELPradeep KotaA dissertation submitted to the faculty of the University of North Carolina at Chapel Hill inpartial fulfillment of the requirements for the degree of Doctor of Philosophy in theDepartment of Biochemistry and BiophysicsChapel Hill2012Approved by:Nikolay V. Dokholyan, PhDJohn R. Riordan, PhDM. Jackson Stutts, PhDBrian A. Kuhlman, PhDSharon L. Campbell, PhD
2012Pradeep KotaALL RIGHTS RESERVEDii
ABSTRACTPRADEEP KOTA: Structural Basis for The Function and Regulation of theEpithelial Sodium Channel(Under the direction of Dr. Nikolay V. Dokholyan)Epithelial sodium channels (ENaC) mediate sodium transport acrossepithelia. Functional channels are assembled from three homologous α, β and γsubunits with 30% similarity in amino acid sequence. Mutations in differentsubunits of this channel are responsible for diseases including Liddle’s syndromeand type I pseudohypoaldosteronism. ENaC is synthesized on the ERmembrane, aquires complex N-linked glycosylation in the Golgi and is traffickedto the plasma membrane where it is activated upon cleavage by numerousmembrane-anchored and/or soluble serine proteases secreted into theextracellular milieu. Although it has been established that exogenous expressionof all three subunits in oocytes is required for robust channel activity, the numberand stoichiometry of subunits comprising one functional channel remainsunclear. Different biophysical and electrophysiological studies have concludedthat ENaC assembles as a trimer or a tetramer with possible larger molecularweight oligomers arising from higher order assembly of trimers or tetramers. Dueto the lack of structural information on ENaC, the molecular aspects of channelactivation and regulation of function remain less well understood. In the currentstudy, using a battery of computational and experimental techniques, we addressiii
specific questions concerning the structural aspects of regulation of channelactivation and function by constructing a structural model of the channel.Significant advances through this study include determination of oligomerizationstate of ENaC using native gel electrophoresis and identification of allostericcommunication within the channel and modulating channel activity by rationalmutagenesis of the identified allosteric sites. In this study, we conclude thatENaC assembles as both trimers and tetramers in the same cell. The amount oftetramers correlates well with increase in function and more importantly, thegamma subunit plays a crucial role in the formation of tetramers in oocytes. Webelieve that the results presented here would be immensely helpful in the futurefor understanding the cellular aspects of channel regulation and function at themolecular level.iv
To my parentsv
ACKNOWLEDGEMENTSMore people than I can count, deserve credit for my successful completionof this dissertation. Due to limited space, I would like to acknowledge thecontributions of the most important people in my life as well as my professionalcareer.ProfessionalI would like to thank Prof. Barry Lentz for giving me a position in theMolecular and Cellular Biophysics program and providing me with an opportunityto identify my interests in protein biochemistry and computational biophysics. Iwould like to thank my advisor Prof. Nikolay V. Dokholyan for inculcating passionand perseverance in me and motivating me at all times to succeed in completionof my dissertation. Dr. Brian Kuhlman and Dr. Sharon Campbell have providedvaluable suggestions throughout my career as a graduate student, by servingcrucial roles on my thesis committee. I would like to thank Dr. John Riordan forextending the honor of being the chair of my committee and for inspiring me to bea scientist. Dr. Jack Stutts has taken special interest in my professionaldevelopment as a scientist. Working closely with him has given me a rich insightinto the physiological aspects of epithelial sodium channel function. I would liketo extend my heartfelt thanks to Dr. Martina Gentzsch for providing her supportand motivation and aiding my development as an experimental biologist.vi
I would like to thank Dr. Douglas Cyr, Dr. Klaus Hahn and Dr. AravindAsokan for very productive collaborations. I enjoyed working with Dr. AndreiAleksandrov (Riordan lab), Dr. Lihua He (Riordan lab), Tim Jensen (Riordan lab),Dr. Andrei Karginov (Hahn lab), Dr. Dan Summers (Cyr lab) and Dr. PulicherlaNagesh (Asokan lab).I would like to extend special thanks to every member of the Dokholyanlab for making such a close-knit team and creating a friendly and motivatingenvironment to be joyful and productive at all times.PersonalI am indebted to my parents for providing me with the right opportunity andeducation at the right time. I am very grateful to them for being supportive ofevery step I took throughout my career so far. I would forever remember themomentous emotional support my wife, Dhivya Ramalingam, has provided mewith, to stay sane and be productive in my graduate school. Finally, I would liketo thank my paternal grand father for inculcating interest in science at a veryearly age.vii
PREFACEPart of the work described in this dissertation was published as articles inBioinformatics and Proteins: Structure, Function and Bioinformatics:Pradeep Kota, Feng Ding, Srinivas Ramachandran and Nikolay V. Dokholyan,Gaia: Automated quality assessment of protein structure models, Bioinformatics(2011) 27:2209-2215Srinivas Ramachandran, Pradeep Kota, Feng Ding and Nikolay V. Dokholyan,Automated minimization of steric clashes in protein structures, Proteins: Structure,Function and Bioinformatics (2011) 79:261-270Required permission to reuse figures and text extracts from the article has beenobtained from all the authors and Wiley-Blackwell and Oxford University Press (JournalPublishers).viii
Table of ContentsCHAPTER 1. Introduction . 11.1 Molecular architecture of ENaC .21.2 Subunit stoichiometry of ENaC.31.4 Activation of ENaC by proteases .61.5 Structural aspects of functional regulation of ENaC .81.6 Motivation.12CHAPTER 2. Materials and Methods . 162.1 Homology Model Building .162.2 Structure Refinement .162.3 Computational Methods.282.4 Biochemical Characterization .31CHAPTER 3. Structural modeling and biochemical characterization . 363.1 Homology model building of ENaC .363.2 Structual models of the N- and C-terminal segments.393.3 Refinement of the structural model of ENaC .413.4 ENaC appears as both trimers and tetramers on the mammaliancell surface .433.5 Tetramers are more functional than the trimers.473.6 Expression of γ-ENaC is critical for formation of tetramers .49ix
CHAPTER 4. Energetic and structural basis for activation of ENaC . 544.1 CAP3 has less stringent sequence requirement for cleavage than furin .554.2 Catalytic activity of matriptase is required for activation of ENaC.614.3 CAP3 cleaves γENaC at an alternate site N-terminal to the furin site .644.4 CAP3 cleaves ENaC at multiple sites C-terminal to the furin site .70CHAPTER 5. Allosteric signal propagation within ENaC . 735.1 Role of the N-terminus in activation of ENaC .765.2 Long-range interaction networks within γENaC.785.3 Interaction of the N-terminus with PIP2 .81CHAPTER 6. Conclusions and future directions. 84Bibliography . 87x
List of FiguresFigure 1.1 Activation and regulation of epithelial sodium channels. 12Figure 3.1: Sequence alignment of rat alpha, beta and gamma ENaCwith chicken ASIC. . 37Figure. 3.2: Structural model of a-ENaC . 38Figure 3.3. Structural models of N-terminal segments of ENaC . 40Figure 3.4 Detergent solubilization of ENaC . 43Figure 3.5 Oligomerization state of ENaC in 3T3 cells. 44Figure 3.6 Oligomerization state of ENaC in Xenopus oocytes . 46Figure 3.7 All three subunits are required for functional recapitulation . 48Figure 3.8 Correlation between tetramer formation and expression ofsubunits. 48Figure 3.9 Modulation of γ-ENaC levels modulates whole cell currents . 50Figure 3.10 Gamma subunit expression levels regulate maturation . 52Figure 3.11 Increase in γENaC expression level promotestetramer assembly . 53Figure 4.1 Disorder prediction for the hypervariable region in ratENaC subunits . 56Figure 4.2. Simulation system and protocol . 57Figure 4.3. Structural models of peptides from γ-ENaC bound tofurin and CAP3. 58Figure 4.4. Energetic basis for peptide binding to furin and CAP3. 60Figure 4.5. Catalytic activity of matriptase/CAP3 is required forxi
stimulation of ENaC. 62Figure 4.6. CAP3 coexpression stimulates ENaC containing mutantfurin sites . 64Figure 4.7. CAP3 mediates neither activation nor cleavage ofγ-135QQQQ ENaC. 66Figure 4.8. Residue 135R in γ-ENaC can form a CAP3-sensitivecleavage site with 132K. 69Figure 4.9. The basic tract 178-RKRK in γ-ENaC is not essential forCAP3 stimulation of INa . 71Figure 5.1 N-terminal basic stretch is critical for channel function . 77Figure 5.2 Allosteric networks in gamma ENaC . 79Figure 5.3 Y370 is a critical residue mediating allosteric propagationwithin ENaC . 80Figure 5.4 N-terminus of γENaC forms an α/β fold in presence of PIP2 . 81Figure 5.5. N-terminus changes secondary structural content uponbinding PIP2. 83xii
List of AbbreviationsENaCEpithelial sodium channelDEGDegenerinBNaCBrain sodium channelFaNaChFMRFamide gated sodium channelPHA-1Type 1 pseudohypoaldosteronismCAPChannel activating ammonium- Oethyl]methanethiosulfonatebromidePOOpen probabilityCAPChannel-activating proteasehNEHuman neutrophil elastaseMAPMembrane associated proteasePIP2Phosphatidylinositol 4,5-bisphosphatePIP3Phosphatidylinositol 3,4,5-trisphosphatePSPhosphatidylserineCDCircular dichroism spectroscopyBN-PAGEBlue native polyacrylamide gel electrophoresisCN-PAGEClear native polyacrylamide gel electrophoresisSASASolvent accessible surface areaMSAMolecular surface areaxiii
MDMolecular dynamicsDMDDiscrete molecular dynamicsPDBProtein data bankEEF1Effective energy function 1CGConjugate gradientBLASTBasic local alignment search toolPMFPotential of mean forcexiv
CHAPTER 1IntroductionThe epithelial sodium channel (ENaC) is a prototypic member of theDEG/ENaC superfamily of ion channels (Canessa et al., 1994b). The DEG/ENaCsuperfamily can be classified as, (i) amiloride-sensitive ENaCs involved in Na reabsorption in epithelia including the distal colon, distal nephron and sweatglands (Duc et al., 1994; Renard et al., 1995), (ii) voltage-independent brain Na channels (BNaC1 and BNaC2) (Garcia-Anoveros et al., 1997), (iii) degenerins(MEC-4, MEC-10 and DEG-1) that form part of a mechanotranduction complexfor touch sensitivity in Caenorhabditis elegans (Driscoll and Tavernarakis, 1997;Garcia-Anoveros and Corey, 1997), and (iv) peptide neurotransmitter Phe-MetArg-Phe-NH2 (FMRF) amide-gated sodium channels (FaNaCh) expressed in theganglion of the snail Helix aspersa (Lingueglia et al., 1995). Other members ofthis superfamily include the acid-sensing ion channel (ASIC), dorsal root gangliaacid-sensing ion channel (DRASIC), and other mechanosensitive cationchannels expressed in cochlear hair cells and oocytes (Benos et al., 1995; Coreyand Garcia-Anoveros, 1996; Garty, 1994; Rossier et al., 1994). Due to itsparticularly crucial role in Na transport across the aldosterone-sensitive distalnephron, regulation of expression and function of ENaC is critical in control of
blood pressure. Hormones such as aldosterone, vasopressin and insulin as wellas PKA/cAMP, PKC, Ca2 and G-proteins tightly regulate ENaC expression andfunction in kidneys (Benos et al., 1995; Garty and Palmer, 1997). Thepathophysiological importance of ENaC has been evidenced by the identificationof mutations in the channel responsible for diseases like Liddle’s syndrome, anautosomal dominant variant of hypertension (Shimkets et al., 1994), and for type1 pseudohypoaldosteronism (PHA-1), a salt-losing syndrome (Chang et al.,1996).1.1 Molecular architecture of ENaCENaC is a heteromultimeric ion channel made of homologous ( 30-40%sequence identity) α, β and γ glycoprotein subunits (75-90 kDa each)surrounding the channel pore (Canessa et al., 1993; Canessa et al., 1994b). Theδ-subunit has functional similarities with the α-subunit, but its physiological role isless well-understood (Waldmann et al., 1995). All members of the DEG/ENaCsuperfamily share a common structural topology. Hydropathy analysis indicatesthat all ENaC subunits have two hydrophobic membrane-spanning regionsseparated by a large ( 500 residues) hydrophilic loop (Canessa et al., 1994a). Nand C-termini are intracellular while the large hydrophilic loop is extracellular withhighly conserved cysteine residues and multiple N-glycosylation sites (Canessaet al., 1994a; Snyder et al., 1994). α-subunits alone can assemble as channels atthe plasma membrane, albeit of low conductance, while β and Υ subunits cannotform functional channels when expressed alone (Harris et al., 2008). Moreover,2
α-subunits are hypothesized to chaperone the assembly and trafficking of theheteromultimer to the plasma membrane (Harris et al., 2008).1.2 Subunit stoichiometry of ENaCGenes encoding different subunits of ENaC have been cloned 20 yearsago and yet the subunit composition of function epithelial sodium channelsremains unsettled. Many groups have addressed this important issue regardingthe subunit stoichiometry of ENaC and related channels. Although the recentlysolved crystal structures of related ion channels and receptors (Gonzales et al.,2009; Jasti et al., 2007; Kawate et al., 2009) suggest a trimeric organization ofthe channel, some groups propose a tetrameric structure (Anantharam andPalmer, 2007; Firsov et al., 1998; Kosari et al., 1998) while others suggest thatfunctional ENaC channels are composed of three, six or nine subunits (Cheng etal., 1998; Snyder et al., 1998; Staruschenko et al., 2005; Staruschenko et al.,2004; Stewart et al., 2011). Quantitative analysis of cell surface expression ofENaC showed that assembly follows fixed stoichiometry with the α-ENaC as themost abundant subunit.Firsov et al., provided several lines of evidence to argue that ENaC is atetramer (Firsov et al., 1998). They developed a quantitative assay based on thebinding of125I-labeled M2 anti-FLAG monoclonal antibody directed against aFLAG reporter epitope introduced in the extracellular loop of different ENaCsubunits (Firsov et al., 1996). Using this assay, they determined that channelshave equal number of β and γ subunits and twice the number of α subunits,suggesting α2β1γ1 to be the most likely stoichiometry (Firsov et al., 1998; Firsov3
et al., 1996). Kosari et al., independently concluded that ENaC is a tetramer byperforming functional studies on Xenopus oocytes expressing mutant subunits(αS583C, βG525C, γG542C – based on the sequence of mouse ENaC subunits)with lower affinity to amiloride than the wildtype subunits (Kosari et al., 1998).Coscoy et al., performed sedimentation of the peptide-activated FaNaChchannels in sucrose gradients to determine subunit composition. They reportedthat FaNaCh was observed in the fraction corresponding to a molecular mass of 350 kDa (Coscoy et al., 1998). Based on the molecular weights of individualsubunits, which ranges between 75 and 90 kDa, they concluded that FaNaCh isa tetramer (Coscoy et al., 1998). Given that FaNaCh is a close member of ENaCin the DEG/ENaC superfamily, they hypothesized that ENaC could be a tetramer.The heterotetrameric assembly is particularly attractive because of its four-foldsymmetry around a central conducting pore; a hallmark feature of severalpotassium (K ) channels (Doyle et al., 1998).In contrast to the tetrameric architecture proposed by Firsov et al., andKosari et al., Snyder et al., proposed that ENaC is formed by nine subunits, witha stoichiometry of α3β3γ3 (Snyder et al., 1998). Methanethiosulfonate (MTS)derivatives have been traditionally used to study the gating properties,accessibility and structure of ion channel pore regions (Akabas et al., 1992). Thechemical modification of an introduced cysteine by a charged MTS reagent mayproduce a measurable change in the function of the ion channel/transportprotein, which can be measured by electrical recording or isotope flux. Such datagive information concerning the time-course, state dependance and membrane4
sidedness of the accessibility of the cysteine (Akabas et al., 1992; Stauffer andKarlin, 1994).Snyder et al., used the mutant γG537C (according to thesequence of rat ENaC), in which C537 can be modified by the positively yl]methanethiosulfonatebromide). Modification by MTSET produces inhibition of the current of mutantchannels but not of wild-type channels. They coexpressed several combinationsof wildtype γ and the mutant γG537C subunits and measured the fraction ofcurrent blocked by MTSET. The reagent MTSET decreased ENaC currents in amuch l
PRADEEP KOTA: Structural Basis for The Function and Regulation of the Epithelial Sodium Channel (Under the direction of Dr. Nikolay V. Dokholyan) Epithelial sodium channels (ENaC) mediate sodium transport across epithelia. Functional
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