Thomas Kjær Klausen:
Volume Regulated Channels

Date: 02-10-2009    Supervisor: Else Kay Hoffmannand Stine Falsig Pedersen

Despite the relative stability of the extracellular space of healthy higher vertebrates, mammalian cell volume homeostasis is constantly challenged by intracellular dynamics, pathological events or the migration of cells between environments of varying osmotic strength. To counter the effects of volume perturbations evolution have developed system of channels and transporters to tightly control volume homeostasis.

In the past decades evidence has been mounting, that the importance of these volume regulated channels and transporters are not restricted to the defense of cellular volume but are also essential for a number of physiological 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 the transient receptor potential Vanilloid (TRPV4) channel.

I: Cl- serves a multitude of functions in the mammalian cell, regulating the membrane potential (Em), cell volume, protein activity and the driving force for facilitated transporters giving Cl- and Cl- channels a major potential of regulating cellular function. These functions include control of the cell cycle, controlled cell death and cellular migration. Volume regulatory mechanisms has long been in focus for regulating cellular proliferation and my thesis work have been focusing on the role of Cl- channels in proliferation with specific emphasis on ICl, swell. Pharmacological blockage of the ubiquitously expressed ICl, swell will decrease proliferation in several cell types, including Ehrlich cells. A differentiated expression of ICl, swell in the cell cycle 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 the role 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 nonselective cation channel, which is activated by cell swelling. Besides cell swelling, however, TRPV4 is also activated by heat and a number of synthetic compounds. Despite of intense investigation of TRPV4, the structure function relationship is still rudimentary understood. Potential agonist binding sites have been proposed 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 is further elaborate the structure/function relationship between TRPV4 and its agonists. I identifies new essential residues for agonist activation, which has the potential of explaining agonist gating in TRPV4. The thesis will further complex the understanding of the TRPV4 pharmacore as new synthetic compounds are identified to interact with TRPV4. Understanding the structure/function relationship of TRPV4 is essential for 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 TRPV4 mutations.