Molecular Physical Mechanics Research On Structure-Function Correlation Of The NaK Ion Channel

Ion channels are pore-forming membrane proteins which regulate the specific species of ionconducting across the plasma membrane down their electrochemical gradient. They implement thegating (opening or closed) by responding to nerve impulses such as the change of membrane potential,ligand concentration and the pressure, playing a fundamental role in the electrophysiological process.Since the crystal structure of a potassium channel from bacteria was firstly determined in1998, aseries of different potassium channels have been resolved subsequently, which means the research ofion channels has entered into the ear of structural biology. The molecular mechanism of ion channelgating and the selective ion conduction has been studied on the atomic level. However, the presenttheoretical research about ion channels is limited primarily to the potassium channels due to the lackof crystal structures of other channels. Recently, the crystal structure of a non-selective tetramericcation channel (NaK channel) has been determined from Bacillus cereus, which can conduct both K+and Na+. The selectivity filter of the NaK channel shares high sequence homology with the cyclicnucleotide-gated channels (CNG channels), which are significant in visual and olfactory transduction.In this thesis, using molecular dynamics simulation as well as statistical mechanics calculation, wesystematically study the structural stability, non-selective ion conduction and the coupling gatingmechanism of the NaK channel to explore its structure-function correlation. The findings are brieflyconcluded below:(1) The structural characteristic of the selectivity filter in NaK channel and the ion bindingproperties. Comparing with the conserved sequence (TVGYG) of the selectivity filter in potassiumchannels, the tyrosine (Y) is replaced by aspartic acid (D) in NaK channel, which cause the significantstructural differences of the selectivity filter between the two channels. In NaK channel, the particularresidue configuration form a vestibule in the region corresponding to the first two binding sites in theselectivity filter of potassium channel KcsA. Analysis of the simulation results indicates that K+ionsprefer to bind within the sites formed by two adjacent planes of oxygen atoms from the selectivityfilter, while Na+ions are inclined to bind to a single plane of four oxygen atoms. For K+ions, S1andS3are the stable sites, while Na+ions have a special preference to sit at site S01and S23. The absenceof ions at its corresponding sites above will destabilize the selectivity filter. External Ca2+blockage isone of important structural properties of CNG channels, which is also found in the NaK channel. Ourcalculation results show that Ca2+blockage stablelizes the selectivity filter, meanwhile both K+andNa+ions can diffuse in the vestibule, accompanying with movements of the water molecules, which results the appropriate coordination number of the ions and the loss of selectivity of NaK.(2) The mechanism of non-selective ion conduction in NaK channel. We find four smallgrottos connecting with the vestibule of the NaK selectivity filter, which form a vestibule-grottocomplex (V-G complex) perpendicular to the filter pore with a few water molecules within it. Oursimulations show the the similar water supplying mechanism that bulk water can penetrate fromtheextracellularwater pits into the grottos by reorientation of certain residues. In a resting channel, thewater molecules in the V-G complex are nearly in a static state due to the shrinkage in the vestibule.When the ions flow out the channel driven by an electric field, the expansive vestibule will facilitatethe fast water exchange within the complex. Water molecules inside the vestibule can coordinate theion instead of backbone carbonyl oxygen atoms. Interestingly, two or more of the water molecules inthe vestibule can hydrate and convey the K+or Na+ion by reducing the ion-carbonyl interactions,while having only one water molecule in the vestibule will cause difficulty in ion conduction.(3) The structure-function correlated mechanism of the peripheral channel in NaK. Similarto KcsA, it is found that there are four lateral orifices outstretching from the cavity in NaK, and eachorifice is formed by the inner helices of two adjacent subunits and the pore helix. We also find fourside pores surrounding the central pore at the intracellular side, which combine with the orifices toform four peripheral pathways leading from the cytoplasm to the central cavity. Water molecules canenter into the peripheral pathways and wet them. Our simulation results show that in the closed NaK,both K+and Na+can reach the cavity through the hydrated peripheral pathways, and the energybarriers are significant lower than the intracellular gate of the central pore. Through the analysis ofsequence, crystal structure and cysteine modification results, we suggest that the activation gate islocated at the bundle crossing, formed by the four inner helices, beneath the central cavity, and sealthe intracellular part of the central pore in the closed state. In some other channels, ions or smallmolecules would also enter into the cavity through the hydrated peripheral pathways by experiencingcertain energy barriers.(4) The coupling between activation and C-type inactivation gating of NaK. The couplingbetween activation and C-type inactivation gating of channels underlies the ion conduction, and isassociated with steric interactions of residues in the inner helix and the selectivity filter. Experimentsshow that when potassium channels are activated via external stimuli, there is a large hinge-bendingmotion around the inner helix bundle to permit the ion conduction through the central pore; whereasC-type inactivation occurs with the opening of the inner helix, leading the selectivity filter into acollapsed or non-conductive state. In contrast to KcsA and Kv channels, our simulation results showthat the selectivity filter is more stable in the opening NaK than in the closed. At the same time, however, we find another non-conductive structure of the selectivity filter in the open NaK channel,involving a reorientation of the side chain of Thr63towards the conduction pathway. This typetransition, together with the pinching at Gly67, would lead to the flicker of the single-channel tracesof NaK.(5) The structure-function correlation in mutant NaK. When the residues of the selectivityfilter in NaK have been mutated, this part of the structure resembles the potassium channels, whichremains stable under the occupancy of Na+ions. It is found that both Na+and K+ions can occupy thebinding sites stably in the selectivity filter throughout the simulations. As we revealed in the wild typeof NaK, the stable binding sites of K+ions are formed by two adjacent planes of oxygen atoms,suggesting the configuration of [S1, S3] or [S2, S4]; Na+ions prefer to bind to a single plane of fouroxygen atoms, and [S12, S34] is the preferential configuration. The hydrogen-bonded networkbetween the selectivity filter and the pore helix ensures the structral stability of the selectivity filter inmutant NaK.