Second messenger modulation of the human ether a go-go related gene (HERG) potassium channel
thesisposted on 15.12.2014, 10:34 by Sarah Louise Cockerill
HERG (human ether-a-go-go related gene) encodes the major pore-funding subunit of IKr, a current which is vital for normal repolarisation of the cardiac action potential. Attenuation of IKr can lead to long QT syndrome, which can predispose individuals to arrhythmias and sudden cardiac death. Given the physiological importance of HERG potassium currents, it is important to understand how they are regulated by intracellular signalling pathways. Whole cell voltage clamp and calcium imaging techniques were used to investigate modulation of HERG channels expressed in HEK 293 cells by second messenger pathways. Stimulating protein kinase C (PKC) by Gaq/11-coupled muscarinic receptor stimulation to elevate diacylglycerol (DAG) and calcium, using OAG (an analogue of DAG), or elevating calcium using ionomycin resulted in a sustained decrease of HERG current. This HERG current response is likely to be mediated by a or b (calcium-sensitive) isoforms of PKC. 32P labelled phosphate incorporation into HERG in OAG treated and non-treated cells was used to determine if PKC directly phosphorylates the channel. Two protein bands at 155 and 135 kDa, corresponding to mature and core glycosylated forms of HERG respectively, were observed in untreated cells, indicating phosphorylation under basal conditions. Stimulation of PKC significantly increased phosphorylation of both bands. HERG currents were also attenuated by activation of cAMP dependent protein kinase (PKA). However, in contrast to PKC, PKA stimulation resulted in a net dephosphorylation of HERG. Overall, the phosphorylation assays suggest HERG channel phosphorylation is dynamically regulated by PKC, PKA and protein phosphatases. In the course of this study it was also found that HERG is directly blocked by caffeine. Caffeine block is open/inactivation state dependent. Caffeine binds within the inner cavity, to sites that include Phe656 and Tyr652.