Volume Regulated Channels:  - ICl , swel l Regulation of Cell Physiology- Structure/Function Relation of TRPV4 and Chemical Agonists

Thomas Kjær Klausen

Volume Regulated Channels: - ICl , swel l Regulation of Cell Physiology- Structure/Function Relation of TRPV4 and Chemical Agonists

Thomas Kjær Klausen

Cellebiologi og Fysiologi

Abstract

På trods a den relative stabilitet af det ekstracellulære milieu i dunde pattedyr, vil volumen homeostasen af

celler konstant blive udfordret af den intracellulære dynamik, patologiske begivenheder eller cellens

vandring mellem milieuer af varierende osmotisk styrke. For at modvirke disse udfordringer for volumen

homeostasen har evolutionen udviklet et system a kanaler og transportere der stringent kontrollerer

cellens volumen.

I de senste årtier er der opbygget evidens for, at vigtigheden af disse volumenregulerede kanaler og

transportere ikke er begrænset til forsvaret af den cellulære volumen, men tillige er essential for en række

fysiologiske processer, som proliferation, kontrolleret celledød, migration og nyrernes funktion.

Denne tese fokuserer hovedsageligt på to kanaler: Den svulmnings aktiverede Cl- kanal (ICl, swell) og transient

receptor potential vanilloid kanal (TRPV4).

I: Cl- er vigtig for en række cellulære funktioner, hvor Cl- regulerer membran potentialet, cellens volumen,

protein aktivitet og drivenergien for en række sekundære transporter, hvilket giver Cl- og Cl- kanaler en stor

mulighed for at regulere cellulære funktioner. Disse funktioner inkluderer kontrol af celle cyclus,

controlleret celledød og cellulær migration. Volumen regulatoriske mekanismer har længe været i fokus i

reguleringen af den cellulære proliferation og min tese har fokuseret på Cl- kanalers rolle i proliferation

med speciel fokus på ICl, swell. Farmakologisk modulering a den allestedsnærværende ICl, swell vil medføre

nedsat proliferationshastighed i adskillige celle typer. Ligeledes har man i adskillige celletyper beskrevet en

varieret udtryksgrad af ICl, swell igennem celle cyklus, hvilket kunne antyde en regulerende rolle. Mine data i

Ehrlich celler antyder dog at ICl, swell spiller en beskyttene snarere end en regulerende rolle, mens Cl-

regulerer proliferationshastigheden via membran potentialet gennem andre kanaler.

II: I hovedparten af epitelceller vil det volumen regulerede respons til celle swulmning være afhængig af

Ca2+. TRPV4, et medlem af transient receptor potential familien af kkation kanaler, er permeable for Ca2+og

aktiveres af cellesvulmning. Ud over aktivering ved cellesvulmning vil TRPV4 ligeledes aktiveres af varme og

en række syntetiske stoffer. På trods af en intens udforskning af TRPV4, så kender man stadig ikke de

struktur/funktion relaterede mekanismer, der danner basis for aktivering af kanalen.Enkelte associations

for syntetiske stoffer positioner i TRPV4 strukturen er blevet identificeret, men disse bidrager ikke

tilstrækkeligt til forståelsen af TRPV4 aktiverin. I denne tese identificerer nye bindingssties for syntetiske

stoffer og udvider komplexiteten af aktiverende stoffer. Forståelsen af denne struktur/funktion relation er

essentiel for udviklingen af nye specifikke medikamenter mod TRPV4 relaterede sygdomme som f.eks

nedarvet ben dysplasia.

Originalsprog	Engelsk

Udgivelsessted	Biologisk Institut
Forlag	Museum Tusculanum
Status	Udgivet - 2009

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http://www.bi.ku.dk/bibliotek/phd/Thomas%20Kj%E6r%20Klausen.pdf

Citationsformater

@phdthesis{fcdb41406d6b11df928f000ea68e967b,

title = "Volume Regulated Channels: - ICl , swel l Regulation of Cell Physiology- Structure/Function Relation of TRPV4 and Chemical Agonists",

abstract = "Despite the relative stability of the extracellular space of healthy higher vertebrates, mammalian cellvolume homeostasis is constantly challenged by intracellular dynamics, pathological events or themigration of cells between environments of varying osmotic strength. To counter the effects of volumeperturbations evolution have developed system of channels and transporters to tightly control volumehomeostasis.In the past decades evidence has been mounting, that the importance of these volume regulated channelsand transporters are not restricted to the defense of cellular volume but are also essential for a number ofphysiological processes such as proliferation, controlled cell death, migration and endocrinology.The thesis have been focusing on two Channels, namely the swelling activated Cl- channel (ICl, swell) and thetransient receptor potential Vanilloid (TRPV4) channel.I: Cl- serves a multitude of functions in the mammalian cell, regulating the membrane potential (Em), cellvolume, protein activity and the driving force for facilitated transporters giving Cl- and Cl- channels a majorpotential of regulating cellular function. These functions include control of the cell cycle, controlled celldeath and cellular migration. Volume regulatory mechanisms has long been in focus for regulating cellularproliferation and my thesis work have been focusing on the role of Cl- channels in proliferation with specificemphasis on ICl, swell. Pharmacological blockage of the ubiquitously expressed ICl, swell will decreaseproliferation in several cell types, including Ehrlich cells. A differentiated expression of ICl, swell in the cellcycle has been described in different cell types indicating a regulating role. In Ehrlich Lettr{\'e} ascites (ELA)cells we suggest the differentiated expression of ICl, swell to be protective rather than regulating, while therole of Cl- in proliferation is due to other Cl- channels regulating Em.II: The volume regulated response to hypotonic stimuli is Ca2+ dependent in the majority of endothelial cells.TRPV4, a member of the Transient Receptor Potential (TRP) channel family is a Ca2+ permeable nonselectivecation channel, which is activated by cell swelling. Besides cell swelling, however, TRPV4 is alsoactivated by heat and a number of synthetic compounds. Despite of intense investigation of TRPV4, thestructure function relationship is still rudimentary understood. Potential agonist binding sites have beenproposed in transmembrane domains 3 and 4, in congruence with agonist binding sites of TRPV1. However,the functional relationship between TRPV4 and agonist binding is not yet understood. In this thesis isfurther elaborate the structure/function relationship between TRPV4 and its agonists. I identifies newessential residues for agonist activation, which has the potential of explaining agonist gating in TRPV4. Thethesis will further complex the understanding of the TRPV4 pharmacore as new synthetic compounds areidentified to interact with TRPV4. Understanding the structure/function relationship of TRPV4 is essentialfor future development of specific TRPV4 agonist for treatment of diseases causes by dysfunctional TRPV4.E.g. two inherited bone dysplasias have recently been demonstrated in humans to originate from TRPV4mutations.",

author = "Klausen, {Thomas Kj{\ae}r}",

note = "Academic advisors: Professor Else Kay Hoffmann Lektor Stine Falsig Pedersen",

year = "2009",

language = "English",

publisher = "Museum Tusculanum",

}

TY - BOOK

T1 - Volume Regulated Channels

T2 - - ICl , swel l Regulation of Cell Physiology- Structure/Function Relation of TRPV4 and Chemical Agonists

AU - Klausen, Thomas Kjær

N1 - Academic advisors: Professor Else Kay Hoffmann Lektor Stine Falsig Pedersen

PY - 2009

Y1 - 2009

N2 - Despite the relative stability of the extracellular space of healthy higher vertebrates, mammalian cellvolume homeostasis is constantly challenged by intracellular dynamics, pathological events or themigration of cells between environments of varying osmotic strength. To counter the effects of volumeperturbations evolution have developed system of channels and transporters to tightly control volumehomeostasis.In the past decades evidence has been mounting, that the importance of these volume regulated channelsand transporters are not restricted to the defense of cellular volume but are also essential for a number ofphysiological processes such as proliferation, controlled cell death, migration and endocrinology.The thesis have been focusing on two Channels, namely the swelling activated Cl- channel (ICl, swell) and thetransient receptor potential Vanilloid (TRPV4) channel.I: Cl- serves a multitude of functions in the mammalian cell, regulating the membrane potential (Em), cellvolume, protein activity and the driving force for facilitated transporters giving Cl- and Cl- channels a majorpotential of regulating cellular function. These functions include control of the cell cycle, controlled celldeath and cellular migration. Volume regulatory mechanisms has long been in focus for regulating cellularproliferation and my thesis work have been focusing on the role of Cl- channels in proliferation with specificemphasis on ICl, swell. Pharmacological blockage of the ubiquitously expressed ICl, swell will decreaseproliferation in several cell types, including Ehrlich cells. A differentiated expression of ICl, swell in the cellcycle has been described in different cell types indicating a regulating role. In Ehrlich Lettré ascites (ELA)cells we suggest the differentiated expression of ICl, swell to be protective rather than regulating, while therole of Cl- in proliferation is due to other Cl- channels regulating Em.II: The volume regulated response to hypotonic stimuli is Ca2+ dependent in the majority of endothelial cells.TRPV4, a member of the Transient Receptor Potential (TRP) channel family is a Ca2+ permeable nonselectivecation channel, which is activated by cell swelling. Besides cell swelling, however, TRPV4 is alsoactivated by heat and a number of synthetic compounds. Despite of intense investigation of TRPV4, thestructure function relationship is still rudimentary understood. Potential agonist binding sites have beenproposed in transmembrane domains 3 and 4, in congruence with agonist binding sites of TRPV1. However,the functional relationship between TRPV4 and agonist binding is not yet understood. In this thesis isfurther elaborate the structure/function relationship between TRPV4 and its agonists. I identifies newessential residues for agonist activation, which has the potential of explaining agonist gating in TRPV4. Thethesis will further complex the understanding of the TRPV4 pharmacore as new synthetic compounds areidentified to interact with TRPV4. Understanding the structure/function relationship of TRPV4 is essentialfor future development of specific TRPV4 agonist for treatment of diseases causes by dysfunctional TRPV4.E.g. two inherited bone dysplasias have recently been demonstrated in humans to originate from TRPV4mutations.

AB - Despite the relative stability of the extracellular space of healthy higher vertebrates, mammalian cellvolume homeostasis is constantly challenged by intracellular dynamics, pathological events or themigration of cells between environments of varying osmotic strength. To counter the effects of volumeperturbations evolution have developed system of channels and transporters to tightly control volumehomeostasis.In the past decades evidence has been mounting, that the importance of these volume regulated channelsand transporters are not restricted to the defense of cellular volume but are also essential for a number ofphysiological processes such as proliferation, controlled cell death, migration and endocrinology.The thesis have been focusing on two Channels, namely the swelling activated Cl- channel (ICl, swell) and thetransient receptor potential Vanilloid (TRPV4) channel.I: Cl- serves a multitude of functions in the mammalian cell, regulating the membrane potential (Em), cellvolume, protein activity and the driving force for facilitated transporters giving Cl- and Cl- channels a majorpotential of regulating cellular function. These functions include control of the cell cycle, controlled celldeath and cellular migration. Volume regulatory mechanisms has long been in focus for regulating cellularproliferation and my thesis work have been focusing on the role of Cl- channels in proliferation with specificemphasis on ICl, swell. Pharmacological blockage of the ubiquitously expressed ICl, swell will decreaseproliferation in several cell types, including Ehrlich cells. A differentiated expression of ICl, swell in the cellcycle has been described in different cell types indicating a regulating role. In Ehrlich Lettré ascites (ELA)cells we suggest the differentiated expression of ICl, swell to be protective rather than regulating, while therole of Cl- in proliferation is due to other Cl- channels regulating Em.II: The volume regulated response to hypotonic stimuli is Ca2+ dependent in the majority of endothelial cells.TRPV4, a member of the Transient Receptor Potential (TRP) channel family is a Ca2+ permeable nonselectivecation channel, which is activated by cell swelling. Besides cell swelling, however, TRPV4 is alsoactivated by heat and a number of synthetic compounds. Despite of intense investigation of TRPV4, thestructure function relationship is still rudimentary understood. Potential agonist binding sites have beenproposed in transmembrane domains 3 and 4, in congruence with agonist binding sites of TRPV1. However,the functional relationship between TRPV4 and agonist binding is not yet understood. In this thesis isfurther elaborate the structure/function relationship between TRPV4 and its agonists. I identifies newessential residues for agonist activation, which has the potential of explaining agonist gating in TRPV4. Thethesis will further complex the understanding of the TRPV4 pharmacore as new synthetic compounds areidentified to interact with TRPV4. Understanding the structure/function relationship of TRPV4 is essentialfor future development of specific TRPV4 agonist for treatment of diseases causes by dysfunctional TRPV4.E.g. two inherited bone dysplasias have recently been demonstrated in humans to originate from TRPV4mutations.

M3 - Ph.D. thesis

BT - Volume Regulated Channels

PB - Museum Tusculanum

CY - Biologisk Institut

ER -

Volume Regulated Channels: - ICl , swel l Regulation of Cell Physiology- Structure/Function Relation of TRPV4 and Chemical Agonists

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