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 -