Studies of ligand- and temperature-gated ion channels using microfluidic methods

Doktorsavhandling, 2009

This thesis describes the studies of the GABAA receptor, a ligand-gated ion channel, and the ligand- and temperature-gated ion channel TRPV1. The desensitization behavior of both of these proteins has been studied for different conditions, using a microfluidic device for superfusion. Also, temperature control has been added to this system in order to enable studies of thermodynamic and kinetic properties of ion channels, as well as for studies of temperature-gated channels. We examine the effect of desensitization on dose-response curves for the GABAA receptor, and also compares two different GABAA receptor agonists, GABA and β-alanine, which display different affinities but similar efficacies and desensitization behavior. The results show that the dose-response curves depend greatly on the order of application, and computer simulations using a proposed kinetic model show that this is due to different distributions of the slow and fast desensitized states. This leads to a memory effect for the receptor population that can last up to minutes. We also describe the differential blocking effect of the competitive antagonist bicuculline on the different GABAA receptor state distributions and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. A concept for the post-production modification of commercially available microfluidic devices to incorporate local temperature control is presented and the resulting system characterized. This device was used to demonstrate that temperature affects the dose-response curves for the GABAA receptor in combination with GABA, but not with β-alanine, and this could be due to their different activation energies for binding and dissociation. The device was also used to characterize desensitization of the temperature-gated ion channel TRPV1 in response to low pH. Our results indicate that temperature affects the rate of both TRPV1 activation and desensitization, and that tachyphylaxis is both pH and temperature dependent. Also, if TRPV1 is exposed several times to low pH, the desensitization becomes slower, whereas the activation rate is constant.