AbstractIn glia inward rectifying potassium channels (Kir) are predominantly responsible for the high selective membrane permeability to K+, for the maintenance of the RMP close to the EK and for the clearance of excess K+ released during action potentials by the process of K+ buffering.
In this study I have investigated the expression, subcellular localisation and heteromer forming ability of Kir4.1, Kir5.1 and Kir2.1 subunits in optic nerve glia. Immunocytochemistry results demonstrated the expression ofKir4.1, Kir5.1 and Kir2.1 subunits in the optic nerve astrocytes and oligodendrocytes. I used immunocytochemical approach and Western blot analysis of the optic nerve plasma membrane fraction to prove the invitro and in vivo functional expression of Kir4.1, Kir5.1 and Kir2.1 subunits inoptic nerve glia. As Kir4.1 is known to form heteromeric channels with theKir5.1 and Kir2.1 subunit I investigated the in vivo and in vitro heteromerforming ability of these Kir subunits using co-immunoprecipitation and immunocytochemistry techniques and found evidence for heteromericKir4.1/Kir5.1, Kir4.1/Kir2.1 and Kir5.1/Kir2.1 and homomeric Kir4.1channels in optic nerve astrocytes and oligodendrocyte.
By using shRNA and genetic ablation of the Kir4.1 subunit I found that Kir4.1 regulates the expression and subcellular localisation of the Kir5.1 and Kir2.1 subunits.
With Western blot analysis I demonstrated that in the absence of theKir4.1 subunit Kir2.1, Kir6.1 and Na/K ATPase α1, the subunits involved in the K+ buffering, compensate for the lack of functional Kir4.1. However, as a result of the metabolic stress due to the increased ATP consumption by the Na/K ATPase the mitochondrial fraction and the activity of KATP channels were found to be increased in the brain of the Kir4.1 knock outmice. The increased expression of Na/K ATPase α1 subunit, known to be involved in K+ buffering, in the absence of the Kir4.1 subunit affirms the role of Kir4.1 in K+ buffering. In astrocytes homomeric Kir4.1, Kir2.1and heteromeric Kir4.1/Kir2.1, Kir4.1/Kir5.1 channels are believed to be involved in K+ buffering and their disfunction has been associated with epilepsy. Heteromeric Kir4.1/Kir5.1 channels are suggested to be involved in CO2/H+ chemosensation in the optic nerve. Due to the unique pH sensitivity of the Kir4.1/Kir5.1 channel, it has been identified as an astrocytic CO2/H+ chemoreceptor in the retrotrapezoid nucleus, associated with the control of respiration. Silent Kir5.1/Kir2.1 channels may regulate these processes. As optic nerve oligodendrocytes also express Kir4.1,Kir5.1 and Kir2.1 subunits, they may also be involved in K+ buffering and/or CO2/H+ chemosensation via these Kir channels, and therefore important for maintaining ionic homeostasis and normal cellular physiology.
|Date of Award||Jan 2013|
|Supervisor||Arthur Butt (Supervisor) & Geoff Pilkington (Supervisor)|