We have probed internal and external accessibility of S4 residues to the membrane-impermeant thiol reagent methanethiosulfonate-ethyltrimethlammonium (MTSET) in both open and closed, cysteine-substituted Shaker K+ channels. Our results indicate that S4 traverses the membrane with no more than 5 amino acids in the closed state, and that the distribution of buried residues changes when channels open. This change argues for a displacement of S4 through the plane of the membrane in which an initially intracellular residue moves to within 3 amino acids of the extracellular solution. These results demonstrate that the putative voltage-sensing charges of S4 actually reside in the membrane and that they move outward when channels open. We consider constraints placed on channel structure by these results.

Voltage-gated potassium channels are comprised of four subunits, and each subunit has a pore domain and a voltage-sensing domain (VSD). The four pore domains assemble to form one single central pore, and the four individual VSDs control the gate of the pore. Recently, a family of voltage-gated proton channels, such as HV or voltage sensor only protein (VSOP), was discovered that contain a single VSD but no pore domain. It has been assumed that VSOP channels are monomeric and contain a single VSD that functions as both the VSD and the pore domain. It remains unclear, however, how a protein that contains only a VSD and no pore domain can conduct ions. Using fluorescence measurements and immunoprecipitation techniques, we show here that VSOP channels are expressed as multimeric channels. Further, FRET experiments on constructs with covalently linked subunits show that VSOP channels are dimers. Truncation of the cytoplasmic regions of VSOP reduced the dimerization, suggesting that the dimerization is caused mainly by cytoplasmic protein-protein interactions. However, these N terminus-and C terminus-deleted channels displayed large proton currents. Therefore, we conclude that even though VSOP channels are expressed mainly as dimers in the cell membrane, single VSOP subunits could function independently as proton channels.dimer ͉ FRET ͉ Hv ͉ voltage sensor ͉ voltage sensor only protein V oltage-gated proton channels (H V channels) have been found in many mammalian cells, including skeletal muscle, lungs, microglia, and blood (1). H V channels have also been shown to play a crucial role in the immune system: H V channels in macrophages are involved in the pathway for the generation of reactive oxygen species, which is critical to the process of phagocytosis and the destruction of foreign pathogens (1). H V channels are activated at depolarized voltages, and their activation can be blocked by Zn 2ϩ (2, 3).Recently, a family of voltage-gated proton channels, called H V or voltage sensor only protein (VSOP) channels, was cloned. VSOP channels were found to have four transmembrane domains, and these domains are homologous to the four transmembrane domains of the voltage-sensing domain (VSD) of voltage-gated potassium channels (2, 3). Voltage-gated potassium channels are comprised of four subunits, each of which has a pore domain and a VSD domain. The four pore domains come together to form one single central pore, and the four individual VSDs control the gate of the pore (4). It is not clear, however, how VSOP channels containing only a VSD and no pore domain can conduct ions. Recently, it was shown that the VSD in Na ϩ and K ϩ channels could function as a proton or cation pore independently of the centrally located pore (5-8). It was therefore suggested that a VSOP channel functions as a single, independent VSD and that this VSD makes up the entire proton channel (1, 9, 10). In the present work, we tested the hypothesis that Hv channels are expressed as monomers. We did this by examining whether HA-tagged mouse VSOP (mVSO...

Voltage-activated proton (HV) channels are essential components in the innate immune response. HV channels are dimeric proteins with one proton permeation pathway per subunit. It is not known how HV channels are activated by voltage and whether there is any cooperativity between subunits during voltage activation. Using cysteine accessibility measurements and voltage clamp fluorometry, we show data that are consistent with that the fourth transmembrane segment S4 functions as the voltage sensor in HV channels from Ciona intestinalis. Surprisingly, in a dimeric HV channel, S4 in both subunits have to move to activate the two proton permeation pathways. In contrast, if HV subunits are prevented from dimerizing, then the movement of a single S4 is sufficient to activate the proton permeation pathway in a subunit. These results suggest a strong cooperativity between subunits in dimeric HV channels.

Voltage-gated ion channels generate dynamic ionic currents that are vital to the physiological functions of many tissues. These proteins contain separate voltage-sensing domains, which detect changes in transmembrane voltage, and pore domains, which conduct ions. Coupling of voltage sensing and pore opening is critical to the channel function and has been modeled as a protein-protein interaction between the two domains. Here, we show that coupling in Kv7.1 channels requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP 2 . This result is due to loss of coupling because PIP 2 was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP 2 -dependent coupling at the interface between the voltage-sensing domain and the pore domain. This site is actually a conserved lipid-binding site among different K + channels, suggesting that lipids play an important role in coupling in many ion channels.V oltage-gated ion channels are integral membrane proteins that sense membrane voltage and respond by opening or closing a transmembrane pore. Ionic currents carried by voltage-gated ion channels control contraction in muscle, encode information in the nervous system, and trigger secretion in neurohormonal tissues. Voltage-gated ion channels contain four voltage-sensing domains (VSDs) and a central pore domain (PD) that are structurally distinct (1, 2). In voltage-gated potassium (Kv) channels, the first four transmembrane segments (S1-S4) of each α-subunit forms a VSD. In response to changes in transmembrane voltage, the VSD undergoes a conformational change, called activation, during which membrane depolarization moves the S4 segment outward (3). The PD is formed by the last two transmembrane segments (S5, S6) from four α-subunits and undergoes a mainly voltageindependent conformational change during which the intracellular ends of the S6 segments bend, opening the ionic pore (4, 5). Interestingly, the PD and VSD can exist in pore-only (6) and voltage sensor-only proteins, respectively, where they function independently (7,8). Confining sensitivity to voltage, or to other stimuli, within a domain diversifies the ion channel properties that can be achieved by partnering different pore and sensor domains. However, this modular architecture also raises a fundamental question as to how VSD activation is transmitted to the PD. Previous studies of this coupling process have revealed the importance of direct protein-protein interactions at the VSD-PD interface (9-13); however, the possible role of membrane lipids in VSD-PD coupling remains undetermined.The membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) modulates the activity of many ion channels, including some voltage-gated channels (14). Notably, all members of the Kv7 family (Kv7.1-Kv7.5), which play important physiological roles in the cardiac (15) or the nervous (16) systems, require PIP 2 to be opened by...

Polyunsaturated fatty acids (PUFAs) affect cardiac excitability. Kv7.1 and the β-subunit KCNE1 form the cardiac IKs channel that is central for cardiac repolarization. In this study, we explore the prospects of PUFAs as IKs channel modulators. We report that PUFAs open Kv7.1 via an electrostatic mechanism. Both the polyunsaturated acyl tail and the negatively charged carboxyl head group are required for PUFAs to open Kv7.1. We further show that KCNE1 coexpression abolishes the PUFA effect on Kv7.1 by promoting PUFA protonation. PUFA analogs with a decreased pKa value, to preserve their negative charge at neutral pH, restore the sensitivity to open IKs channels. PUFA analogs with a positively charged head group inhibit IKs channels. These different PUFA analogs could be developed into drugs to treat cardiac arrhythmias. In support of this possibility, we show that PUFA analogs act antiarrhythmically in embryonic rat cardiomyocytes and in isolated perfused hearts from guinea pig.