Gating Mechanism of a Potassium Channel, Experimental and Theoretical Studies

Abstract

Inwardly-rectifying potassium (Kir) channels are transmembrane proteins that regulate membrane electrical excitability and K+ transport in many cell types generating and controlling the membrane resting potential. The function of this channel although simple is primordial for physiological processes such as the creation and the propagation of the neuronal action's potential, the regulation of cellular volume, the muscular contraction and the cardiac pulse. Their physiological importance is highlighted by the fact that genetically inherited defects in Kir channels are responsible for a wide-range of channelopathies including Andersen's syndrome a pathology that can cause periodic paralysis or serious heart problems. To date unfortunately, this disease does not have any effective treatment. The goal of these studies is to understand how the channel gates and what are the conformational changes required. Our work focused on the KirBac3.1 channel, homologous to Kir2.1 human. We started with known crystallographic data from the KirBac3.1WT and two mutants S129R and W46R. We explored conformational changes using Molecular Dynamics using Excited Normal Modes (MDeNM), a new method which gives access to vibrational motions (Molecular Dynamics) and global motions (Normal Modes) at the same time. MDeNM successfully opened the closed state of the protein giving the opportunity to identify the key motions involved in the gating such as the role of the cytoplasmic domain, the slide-helix as well as the transmembrane helices during the opening of the channel. In parallel, HDX-MS Spectrometry studies had been performed and gave important information on the flexibility of the protein. Experimental and theoretical studies were compared for validation. In addition, human Kir2.1 was purified and imaged with a Titan Krios microcope. The structure will help to understand the gating mechanism of this human channel.