Ring of negative charge facilitates intracellular magnesium ions and polyamine block of BK channels through an electrostatic mechanism

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Physiology and Biophysics

First Committee Member

Karl L. Magleby - Committee Chair


Intracellular Mg2+ and natural polyamines block outward currents in BK channels in a voltage dependent manner. Here we investigate the contribution of the ring of eight negatively charged residues at the entrance to the inner vestibule of BK channels to this block. Channels with or without (E321N/E324N) the ring of negative charge were expressed in oocytes, and unitary currents recorded over a range of intracellular Mg2+ and polyamine concentrations. Removing the ring of charge decreased the block by Mg 2+ and polyamines, increasing Kdap(0 mV) for Mg2+ block from 48.3 +/- 3.0 to 121 +/- 8 mM and increasing Kdap(0) for spermine block from 8.0 +/- 1.0 to 721 +/- 9 mM (150 mM symmetrical KCl). Mg2+ and polyamines blocked with negative cooperativity with and without the ring of charge. Polyamines with fewer amine groups blocked less: putrescine < spermidine < spermine, and methylene based compounds of length similar to the polyamines but with no charge, did not block. With 3 M K+ i competitive block by Mg2+ was reduced but not abolished (Kdap(0) = 561 +/- 12 mM), the block by spermine became negligible, and the ring of charge no longer altered the block by either Mg2+ or polyamine. These observations suggest that the ring of negative charge facilitates block through an electrostatic attraction of Mg2+ and polyamine to the inner vestibule of the channel, increasing the Kdap(0) for Mg2+ block 2.5 fold and for spermine block 90 fold. The ring of charge would attract divalent Mg2+ preferentially over monovalent K+, and would stabilize polyamine in the vestibule. The observation of Mg2+ and polyamine block in the absence of the ring of charge indicates that these ions also have an additional site of action, presumably deeper in the vestibule.


Biophysics, General

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