Polymeric maleimido–quaternary ammonium (QA) compounds have been shown to function as molecular tape measures when covalently tethered to external cysteine residues of a Shaker K+ channel (Blaustein R.O., P.A. Cole, C. Williams, and C. Miller. 2000. Nat. Struct. Biol. 7:309–311). For sufficiently long compounds, the cysteine–maleimide tethering reaction creates a high concentration, at the channel's pore, of a TEA-like moiety that irreversibly blocks current. This paper investigates a striking feature of the maleimide–cysteine tethering kinetics. Strong blockers—those that induce substantial levels (>80%) of irreversible inhibition of current—react with channel cysteines much more rapidly than weak blockers and, when delivered to channels with four cysteine targets, react with multiexponential kinetics. This behavior is shown to arise from the ability of a strong blocker to concentrate its maleimide end near a channel's cysteine target by exploiting the reversible pore-blocking affinity of its QA headgroup.
Kinetics of Tethering Quaternary Ammonium Compounds to K+ Channels
This increase in concentration with increasing length may not seem intuitive; however, the length dependence of tethered QA concentration at the pore involves a complex interplay between the number of configurational states a tethered compound can block in, and the volume element that the compound sweeps out. Polymer statistical theory predicts that a length will eventually be reached for which the increase in volume element overcomes the increase in the number of states. At this point the length dependence will reverse, and longer compounds will actually lead to a lower concentration of QA at the pore. I have not yet seen this occur with the range of compounds that I have tried (up to 45 Å in length).
The peak current minus the steady-state current underestimates the true inactivating component because channels begin inactivating immediately after depolarization. This error is magnified when the inactivating fraction becomes blocked. Although simulations (not depicted) reveal that this does not significantly affect kinetic measurements, it does preclude accurate calculation of the extent of the tethering reaction.
Robert O. Blaustein; Kinetics of Tethering Quaternary Ammonium Compounds to K+ Channels . J Gen Physiol 1 August 2002; 120 (2): 203–216. doi: https://doi.org/10.1085/jgp.20028613
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