| ObjectiveTo study the inhibition mechanisms of the Methanobacterium thermoautotro-phicum potassium channel (MthK) by the quaternary ammonium (QA) salts. MthKis a calcium-gated prokaryotic potassium channel. Although the high resolutioncrystal structure of MthK is used as a structure model for pharmacology ofeukaryotic potassium channel, but there are not much progress of the inhibitionmechanism of MthK channel. Quaternary ammonium drugs (QAs) is a group ofclassical potassium channel blockers, which could inhibit hERG channel, TREK-1,TASK, Shaker potassium channel, and etc.,(Grissmer.1989; Paula L Piechotta.2011;Kee-Hyun Choi.2011.). In2011, TEA was approved by the U.S. FDA as a drugtreating myasthenia gravis. However, the binding sites of quaternary ammoniumsalts on potassium ion channels still lack the three-dimensional structure informationand the actions of QAs on single channel level also need further investigation.Therefore, we aim to clarify the actions of QAs on MthK channel by analyzing thethree-dimensional structure and single channel electrophysiological currents ofMthK potassium channel. To disclosure the inhibition mechanism of MthK channelwill provide experimental evidence and theoretical guidance to the pharmacology ofeukaryotic potassium channels.Method1.In order to get the protein of wild type MthK and mutant protein E96Q, wecarry out the molecular biology, protein expression and purification experiments.2.And then, we synthesize a group of QAs (TPeA，DTMA，TBA，DTMA，TEA and so on).3.Next, we used plane lipid bilayer experiment to recording the MthK currents.The last, we analyzed the action of QAs on the single channel activities of MthKcurrents.Result 1. QAs are effective to inhibit the wild type MthK. The length of side chain ofQAs has a positive correlation with the inhibition effects on MthK channels. TheIC50of TPeA is0.2μM, which have five-carbon atom side chain. And TBA hasfour–carbon atom side chain, with IC50of is29.8μM. Then with three-carbonatom side chain, TPrA is less effect with IC50of5.3mM. The TEA is least effectivethan others, with IC50of10.6mM. While the side chain length is larger than fivealkyl groups, the inhibition effect became weak, which means that steric hindranceand hydrophobicity of QAs play a critical role for the inhibition of MthK channels.2. Single channel analysis of MthK currents reveals that there are two mode ofQAs blockage: the small molecular side-chain QAs (TEA) block the conductance ofMthK channel. Otherwise, big molecular side-chain QAs mainly inhibit the openprobability (Po) of MthK. The middle size molecular side-chain QAs inhibit bothconductance and open probability of the channels. This implied that the smallmolecule QAs (TEA) with a strong positive charge, may bind to the channel byelectrostatic interaction. And the decrease of MthK conductance may be due to thefast binding and dissociation. And the hydrophobic large QAs may bind to the deepinner pore of transmembrane domain through the hydrophobic interaction.3. Glu96is an important site for the fast inhibition of MthK currents.Theglutamate acid E96forms a weak positive center at the cross bundle area oftransmembrane pore. Electrophysiological tests show that: the conduction inhibitionis typical effect of TEA on wild MthK currents, but the neutral mutants E96Qabolished the conductance inhibition of TEA. E96Q mutant also partial reduced theinhibition of current amplitude by calcium ions. These results suggest that E96siteof MthK channels is a newly discovered binding site for TEA and other positivelycharged inhibitors.ConclusionQuaternary ammonium (QA) could inhibit MthK channel from intracellular sidethrough both hydrophobic interactions and electrostatic interactions. The size of... |
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