Abstract
Ba2+ is frequently used infunctional studies of the topology of potassium-selective pores because it is a permeant blocker. Consequently, the rate and extent of current block, or unblock, can be quantified to assess changes of the pore structure in different recording conditions and in various channel states. Here we describe experiments in which external Ba2+ was applied to Kv4.2 channels to determine the properties and state-dependence of Ba2+ block. In the standard protocol, [K+]o was 3.5 mM and 10 ms pulses to 80 mV were applied at 1 Hz from a Vh of −80 to −100 mV. τblock, derived from a monoexponential fit of a diary plot of the peak current amplitudes, decreased as the [Ba2+]o increased, however the value for τblock saturated with higher (20-40 mM) concentrations. This suggests that, as in Kv1 channels, there are serial binding sites in the resting conformation of the channel. Increasing [K+] from 3.5 to 40 mM decreased the steady-state level of Ba2+ block with 1 Hz pulses, as expected for a competitive interaction involving one or more binding sites in the pore. Decreasing the test pulse frequency decreased the extent of the Ba2+ block and τunblock was markedly decreased by increasing [K+]o; both features are the opposite of what is observed in Kv1 channels. A test pulse applied following a 10 s exposure to 20 mM Ba2+ revealed roughly 3-fold greater block of inactivated channels (Vh=0 mV) compared to the block of resting channels (Vh =-80 mV). This observation supports the widely-held notion that the outer pore mouth of inactivated Kv4 channels is not constricted, i.e., not P/C-type inactivated, but nonetheless implies that the outer pore does undergo a substantial structural change in the transition from the resting to the inactivated state.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have