Ion channels in the smooth muscle of the cardiovascular system control blood pressure and cardiac contractility through changes in their membrane potential. Potassium channels, Ca2+-activated K+ (Slo 1 or BK), of arterial smooth muscle and ATP-sensitive K+ channels (Kir6.2 and SUR2A ) of cardiac muscle play vital roles in regulating muscle membrane potential are reregulated by many signalling pathways. The focus of this research is to explore the role of a novel signalling molecule heam which is required for coordinating signalling gases, such as nitic oxide.
Heme, a ubiquitous protoporphyrin with iron at its centre, is an essential co-factor in many proteins such as hemeoglobin and the iron can bind to gases such as O2, CO and NO. We are just beginning to widen our understanding to include heam as a dynamic signalling molecule. We have recently published convincing evidence, some of the first of its kind, to demonstrate that heme regulates potassium channel activity in vascular smooth muscle and cardiac myocytes and we have identified a common heme structural motif to account for this behaviour. This is opening up new directions in both the ion channel and heme fields. We are using this information to make the first in-roads into understanding the precise mechanisms of heme and heme – gas dependent regulation. This includes the specific binding of signalling gases to heme at ion channels such as signalling through cellular carbon monoxide which is produced naturally as a metabolite in cells. This is important because the role of heme in ion channel control has not been widely appreciated and we are now in a position to make real and insightful development on this key question.
ATP-sensitive potassium channels
ATP-sensitive potassium (KATP) channels are voltage-independent K+ channels formed by eight proteins, four of which are pore-forming, inwardly rectifying subunits and four sulphonylurea receptor subunits. This K+ channel is exquisitely regulated by intracellular ATP levels. The ATP sensitivity confers the crucial role of KATP channels in the heart and vascular smooth muscle as metabolic sensors that open when cellular energy levels fall. When ATP levels fall during periods of metabolic stress which occurs during cardiac ischemeia, KATP channel opening hyperpolarizes the membrane potential, shortening the action potential and so reduces Ca2+ loading in the myocyte and in this way are considered protective against cell death.
We have established that heme regulates KATP channels and the site at which heme binds to the SUR2A subunit. This represents a very significant step forward. We have shown that heme activates KATP channels at physiological concentrations (~100nM) in both whole cell and single channel experiments. We have preliminary data showing carbon monoxide is also a signaling molecule at KATP channels. We plan to test the hypothesis that heme binding at KATP channel is necessary for carbon monoxide binding at the channel and heme is essential for functional effects of signaling gases.
Ca2+-activated K+ (Slo 1 or BK) channels
Regulation of vascular smooth muscle contraction (tone) is fundamental to ensure adequate blood flow and to maintain blood pressure. Contraction, is tightly linked to membrane potential and regulated by BK channels.
Heme signalling to ion channels is a novel, important, but poorly understood area, and modulation of K+ channels by heme will have profound effects on cardiac and vascular tone. Deciphering heme regulation of K+ channels is essential to understand vascular function; for this reason this project will define whether heme binding sites on BK channels of arterial smooth muscle and test the second hypothesis that heme binding is necessary for the function effects of signalling gases such as nitric oxide and carbon monoxide.
Our approach uses electrophysiology, which allows us to examine heme-binding and signaling gas interaction events on single ion channels in a smooth muscle cell. Functional assays on vascular tone or ventricular cell contractility will also be performed to assess the physiological consequences of heme and gas signaling.
Techniques that will be undertaken during the project
-Cardiac muscle contraction assays
-Cell viability assays
-Fluorescent cell imaging for cell calcium and mitochondrial function
-Electrophysiological methods for assaying ion channel modulation in response to heme and signalling gases such as nitric oxide.
Available to UK/EU applicants only
Application information https://www2.le.ac.uk/research-degrees/doctoral-training-partnerships/bbsrc