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Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca2+. The only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue ‘C-linker’. To determine the linker’s role in human BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combining atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.

Voltage-gated ion channels feature voltage sensor domains (VSDs) that exist in three distinct conformations during activation: resting, intermediate, and activated. Experimental determination of the structure of a potassium channel VSD in the intermediate state has previously proven elusive. Here, we report and validate the experimental three-dimensional structure of the human KCNQ1 voltage-gated potassium channel VSD in the intermediate state. We also used mutagenesis and electrophysiology in Xenopus laevisoocytes to functionally map the determinants of S4 helix motion during voltage-dependent transition from the intermediate to the activated state. Finally, the physiological relevance of the intermediate state KCNQ1 conductance is demonstrated using voltage-clamp fluorometry. This work illuminates the structure of the VSD intermediate state and demonstrates that intermediate state conductivity contributes to the unusual versatility of KCNQ1, which can function either as the slow delayed rectifier current (IKs) of the cardiac action potential or as a constitutively active epithelial leak current.

Upon membrane depolarization, the KCNQ1 potassium channel opens at the intermediate (IO) and activated (AO) states of the stepwise voltage-sensing domain (VSD) activation. In the heart, KCNQ1 associates with KCNE1 subunits to form IKs channels that regulate heart rhythm. KCNE1 suppresses the IO state so that the IKs channel opens only to the AO state. Here, we tested modulations of human KCNQ1 channels by an activator ML277 in Xenopus oocytes. It exclusively changes the pore opening properties of the AO state without altering the IO state, but does not affect VSD activation. These observations support a distinctive mechanism responsible for the VSD-pore coupling at the AO state that is sensitive to ML277 modulation. ML277 provides insights and a tool to investigate the gating mechanism of KCNQ1 channels, and our study reveals a new strategy for treating long QT syndrome by specifically enhancing the AO state of native IKs currents.

Voltage-gated ion channels generate electrical currents that control muscle contraction, encode neuronal information, and trigger hormonal release. Tissue-specific expression of accessory (β) subunits causes these channels to generate currents with distinct properties. In the heart, KCNQ1 voltage-gated potassium channels coassemble with KCNE1 β-subunits to generate the IKs current (Barhanin et al., 1996; Sanguinetti et al., 1996), an important current for maintenance of stable heart rhythms. KCNE1 significantly modulates the gating, permeation, and pharmacology of KCNQ1 (Wrobel et al., 2012; Sun et al., 2012; Abbott, 2014). These changes are essential for the physiological role of IKs (Silva and Rudy, 2005); however, after 18 years of study, no coherent mechanism explaining how KCNE1 affects KCNQ1 has emerged. Here we provide evidence of such a mechanism, whereby, KCNE1 alters the state-dependent interactions that functionally couple the voltage-sensing domains (VSDs) to the pore. DOI: http://dx.doi.org/10.7554/eLife.03606.001, eLife digest Cells are surrounded by a membrane that prevents charged molecules from flowing directly into or out of the cell. Instead ions move through channel proteins within the cell membrane. Most ion channel proteins are selective and only allow one or a few types of ion to cross. Ion channels can also be ‘gated’, and have a central pore that can open or close to allow or stop the flow of selected ions. This gating can be affected by the channel sensing changes in conditions, such as changes in the voltage across the cell membrane. Research conducted more than half a century ago—before the discovery of channel proteins—led to a mathematical model of the flow of potassium ions across a membrane in response to changes in voltage. This model made a number of assumptions, many of which are still widely accepted. However, Zaydman et al. have now called into question some of the assumptions of this model. Based on the original model, it has been long assumed that the voltage-sensing domains that open or close the central pore in response to changes in voltage must be fully activated to allow the channel to open. It had also been assumed that the voltage-sensing domains do not affect the flow of ions once the channel is open. Zaydman et al. have now shown that these assumptions are not valid for a specific voltage-gated potassium channel called KCNQ1. Instead, this ion channel opens when its voltage-sensing domains are either partially or fully activated. Zaydman found that the intermediate-open and activated-open states had different preferences for passing various types of ion; therefore, the gating of the channel and the flow of ions through the open channel are both dependent on the state of the voltage-sensing domains. This is in direct contrast to what had previously been assumed. The original model cannot reproduce the gating of KCNQ1, nor can any other established model. Therefore, Zaydman et al. devised a new model to understand how the interactions between different states of the voltage-sensing domains and the pore lead to gating. Zaydman et al. then used their model to address how another protein called KCNE1 is able to alter properties of the KCNQ1 channel. KCNE1 is a protein that is expressed in the heart muscle cell and mutations affecting KCNQ1 or KCNE1 have been associated with potentially fatal heart conditions. Based on the assumptions of the original model, it had been difficult to understand how KCNE1 was able to affect different properties of the KCNQ1 channel. Thus, for nearly 20 years it has been debated whether KCNE1 primarily affects the activation of the voltage-sensing domains or the opening of the pore. Zaydman et al. found instead that KCNE1 alters the interactions between the voltage-sensing domains and the pore, which prevented the intermediate-open state and modified the properties of the activated-open state. This mechanism provides one of the most complete explanations for the action of the KCNE1 protein. DOI: http://dx.doi.org/10.7554/eLife.03606.002