Activation-Dependent Subconductance Levels in the drk1 K Channel Suggest a Subunit Basis for Ion Permeation and Gating

Abstract

Ion permeation and channel opening are two fundamental properties of ion channels, the molecular bases of which are poorly understood. Channels can exist in two permeability states, open and closed. The relative amount of time a channel spends in the open conformation depends on the state of activation. In voltage-gated ion channels, activation involves movement of a charged voltage sensor, which is required for channel opening. Single-channel recordings of drk1 K channels expressed in Xenopus oocytes suggested that intermediate current levels (sublevels) may be associated with transitions between the closed and open states. Because K channels are formed by four identical subunits, each contributing to the lining of the pore, it was hypothesized that these sublevels resulted from heteromeric pore conformations. A formal model based on this hypothesis predicted that sublevels should be more frequently observed in partially activated channels, in which some but not all subunits have undergone voltage-dependent conformational changes required for channel opening. Experiments using the drk1 K channel, as well as drk1 channels with mutations in the pore and in the voltage sensor, showed that the probability of visiting a sublevel correlated with voltage- and time-dependent changes in activation. A subunit basis is proposed for channel opening and permeation in which these processes are coupled.

Single-channel basis for the slow activation of the repolarizing cardiac potassium current, I Ks

“…A channel can be seen to dwell in multiple levels for prolonged periods during its initial activation (Fig. 4A), with sublevels visited briefly and interspersed by rapid switching between open and closed levels, as previously seen in Kv channels (11). The mean time to reach the main amplitude (level 4) from the start of each sublevel opening burst was 209 ± 46 ms (n = 21 bursts), excluding two bursts where the channel opened to levels >4 for brief periods and then closed for ∼750 and 1,460 ms, respectively, before reopening and subsequently attaining the main conductance level.…”

“…The highest prevalence of sublevels was observed at voltages expected to activate one set of subunits but not the other, and so it was concluded that subconductance levels arise from heteromeric pore conformations. Such a mechanism for subconductance is consistent with the observation of sublevels coupled to activation in the Shaker (10, 18) and K v 2.1 channel (11,17). Therefore, the presence of sublevels in I Ks may have important implications for the gating of this channel complex.…”

“…A possible explanation for this persistence is that I Ks subunits exhibit relatively low cooperativity, so that activation of one subunit only weakly promotes activation of other subunits, hence the equal rates in the model for the substate transitions. The final steps of the I Ks activation pathway then are slow enough to reveal stable sublevels, as was the case in a slow mutant of K v 2.1 (11). If so, KCNE1 may slow I Ks activation by promoting sublevel stability through as yet undetermined mechanisms.…”

“…Recent fluorescence studies of I Ks voltage sensor movement have suggested that multiple voltage sensors must move before the KCNQ1/KCNE1 channel complex can conduct (9) and that this requirement both differentiates the complex from the pore-forming KCNQ1 subunit kinetics when expressed alone (9) and provides at least a partial explanation for the very slow activation of I Ks currents. Singlechannel studies on mutant Shaker (10) and K v channels suggest that subconductance occupancy arising from heteromeric pore conformations also can slow the overall activation time course as a result of only partial opening of the pore (11). Preliminary evidence indicates that subconductance states play a role in the opening behavior of I Ks channels, but the details and significance of these states are unknown (12).…”

Molecular Physiology of Low-Voltage-Activated T-type Calcium Channels

“…Channels can also open partially, leading to a subconductance state that has a smaller current (27,64,104). In contrast to other ion channels (66), the gating behavior of this subconductance state was found to be similar to the main conductance state (64).…”