The delayed rectifier I Ks potassium channel, formed by coassembly of α-(KCNQ1) and β-(KCNE1) subunits, is essential for cardiac function. Although KCNE1 is necessary to reproduce the functional properties of the native I Ks channel, the mechanism(s) through which KCNE1 modulates KCNQ1 is unknown. Here we report measurements of voltage sensor movements in KCNQ1 and KCNQ1/KCNE1 channels using voltage clamp fluorometry. KCNQ1 channels exhibit indistinguishable voltage dependence of fluorescence and current signals, suggesting a one-to-one relationship between voltage sensor movement and channel opening. KCNE1 coexpression dramatically separates the voltage dependence of KCNQ1/KCNE1 current and fluorescence, suggesting an imposed requirement for movements of multiple voltage sensors before KCNQ1/KCNE1 channel opening. This work provides insight into the mechanism by which KCNE1 modulates the I Ks channel and presents a mechanism for distinct β-subunit regulation of ion channel proteins.

Voltage-gated proton (Hv1) channels play important roles in the respiratory burst, in pH regulation, in spermatozoa, in apoptosis, and in cancer metastasis. Unlike other voltage-gated cation channels, the Hv1 channel lacks a centrally located pore formed by the assembly of subunits. Instead, the proton permeation pathway in the Hv1 channel is within the voltage-sensing domain of each subunit. The gating mechanism of this pathway is still unclear. Mutagenic and fluorescence studies suggest that the fourth transmembrane (TM) segment (S4) functions as a voltage sensor and that there is an outward movement of S4 during channel activation. Using thermodynamic mutant cycle analysis, we find that the conserved positively charged residues in S4 are stabilized by countercharges in the other TM segments both in the closed and open states. We constructed models of both the closed and open states of Hv1 channels that are consistent with the mutant cycle analysis. These structural models suggest that electrostatic interactions between TM segments in the closed state pull hydrophobic residues together to form a hydrophobic plug in the center of the voltage-sensing domain. Outward S4 movement during channel activation induces conformational changes that remove this hydrophobic plug and instead insert protonatable residues in the center of the channel that, together with water molecules, can form a hydrogen bond chain across the channel for proton permeation. This suggests that salt bridge networks and the hydrophobic plug function as the gate in Hv1 channels and that outward movement of S4 leads to the opening of this gate.voltage gating | mutagenesis cycle | molecular dynamics | VSOP | blocker T he best-studied function of voltage-gated proton (Hv1) channels is in the immune system, where the activity of Hv1 channels has been shown to play a key role in charge compensation for the electron extrusion by NADPH oxidase during the respiratory burst in phagocytes (1, 2). In addition, this channel is also found in many other cell types including neurons, sperm, and lung airway epithelia cells, where it has been implicated in acid extrusion, male fertility, and the pathology of asthma, respectively (3, 4). During stroke, the activity of Hv1 channels exacerbates neuronal death (5). In 2006, two independent groups identified the genes coding for the Hv1 channel in humans (hHv1) (6) and mice and Ciona intestinalis (voltage-sensing only protein; we call it "Ci-Hv1" in this paper) (7). The sequences of Hv1/(VSOP) are homologous to the sequences of the voltagesensing domain (VSD) of other voltage-gated ion channels, such as voltage-gated sodium, potassium, and calcium channels (Nav, Kv, and Cav channels), and the voltage sensor-containing phosphatase VSP (6, 7). Hydropathy analysis of the Hv1 sequence suggests the existence of four transmembrane (TM) segments (Fig. 1A), designated S1 to S4, similar to the four TM segments of the VSD of Kv channels (Fig. S1). However, Hv1 channels lack the last two TM segments of Kv channels that make up t...

The functional properties of KCNQ1 channels are highly dependent on associated KCNE β subunits. Mutations in KCNQ1 or KCNE subunits can cause congenital channelopathies, such as deafness, cardiac arrhythmias, and epilepsy. The mechanism by which KCNE1 beta subunits slow the kinetics of KCNQ1 channels is a matter of current controversy. Here we show that KCNQ1/KCNE1 channel activation occurs in two steps: first, mutually independent voltage sensor movements in the four KCNQ1 subunits generate the main gating charge movement and underlie the initial delay in the activation time course of KCNQ1/KCNE1 currents. Second, a slower and concerted conformational change of all four voltage sensors and the gate, which opens the KCNQ1/KCNE1 channel. Our data show that KCNE1 divides the voltage sensor movement into two steps with widely different voltage dependences and kinetics. The two voltage sensor steps in KCNQ1/KCNE1 channels can be pharmacologically isolated and further separated by a disease-causing mutation.

Voltage-gated proton (Hv) channels play an essential role in phagocytic cells by generating a hyperpolarizing proton current that electrically compensates for the depolarizing current generated by the NADPH oxidase during the respiratory burst, thereby ensuring a sustained production of reactive oxygen species by the NADPH oxidase in phagocytes to neutralize engulfed bacteria. Despite the importance of the voltage-dependent Hv current, it is at present unclear which residues in Hv channels are responsible for the voltage activation. Here we show that individual neutralizations of three charged residues in the fourth transmembrane domain, S4, all reduce the voltage dependence of activation. In addition, we show that the middle S4 charged residue moves from a position accessible from the cytosolic solution to a position accessible from the extracellular solution, suggesting that this residue moves across most of the membrane electric field during voltage activation of Hv channels. Our results show for the first time that the charge movement of these three S4 charges accounts for almost all of the measured gating charge in Hv channels.

Summary Voltage-gated proton (Hv1) channels are dimers, where each subunit has a separate permeation pathway. However, opening of the two pathways is highly cooperative. It is unclear how Hv1 channels open their permeation pathways, because Hv1 channels lack a classic pore domain. Using voltage clamp fluorometry, we here detect two conformational changes reported by a fluorophore attached to the voltage sensor S4 in Hv1 channels. The first is voltage dependent and precedes channel opening, with properties consistent with reporting on independent S4 charge movements in the two subunits. The second is less voltage dependent and closely correlates with channel opening. Mutations that reduce dimerization or alter the intersubunit interface affect both the second conformational change and channel opening. These observations suggest that, following an initial S4 charge movement in the two subunits, there is a second, cooperative conformational change, involving interactions between subunits, that opens both pathways in Hv1 channels.

The β1a subunit, one of the auxiliary subunits of CaV1.1 channels, was expressed in COS‐1 cells, purified by electroelution and electrodialysis techniques and identified by Western blot using monoclonal antibodies. The purified β1a subunit strongly interacted in vitro with the alpha interaction domain (AID) of CaV1.1 channels. The actions of the purified β1a subunit on CaV1.1 channel currents were assessed in whole cell voltage clamp experiments performed in vesicles derived from frog and mouse adult skeletal muscle plasma membranes. L‐type inward currents were recorded in solutions containing Ba2+ (IBa). Values of peak IBa were doubled by the β1a subunit in frog and mouse muscle vesicles and the amplitude of the slow component of tail currents was greatly increased. The actions of the β1a subunit on CaV1.1 channel currents reached a steady state within 20 min. The β1a subunit had no effect on the time courses of activation or inactivation of IBa or shifted the current‐voltage relation. Non‐linear capacitive currents were recorded in solutions that contained mostly impermeant ions. Charge movement depended on voltage with average Boltzmann parameters: Qmax+ 28.0 ± 6.6 nC μF−1, V+−58.0 ± 2.0 mV and k+ 15.3± 1.1 mV (n= 24). In the presence of the β1a subunit, these parameters remained unchanged: Qmax+ 29.8 ± 3.5 nC μF−1, V+−54.5 ± 2.2 mV and k+ 16.4± 1.3 mV (n= 21). Overall, the work describes a novel preparation to explore in situ the role of the β1a subunit on the function of adult CaV1.1 channels.

Voltage-gated proton (Hv) channels have long been found with electrophysiological tools in many different cell types. However, the molecular identity of Hv channels was not discovered until 2006, when two labs independently demonstrated that the gene HVCN1 that codes for a protein called Hv1 or VSOP generates voltage-gated proton currents when expressed in heterologous cells. Surprisingly, the sequence of Hv1/VSOP was found to be homologous to the sequence of the voltage-sensing domain of voltage-gated K + channels. Recent studies have led to our present understanding of the structure of Hv1/VSOP channels and the structural underpinnings of some of the functional properties of Hv1/VSOP channels, including voltage sensing, subunit cooperativity, permeation and selectivity, pH dependence, zinc inhibition and modulation by PKC of Hv1/VSOP channels. The cloned Hv1/VSOP channels display most of the properties of native voltage-gated proton channels. However, the molecular mechanisms underlying some of these properties are still unknown.

Reports on phase coexistence regimes and directions of tie lines of ternary lipid mixtures are often controversial. The origin of these controversies is typically the experimental window of the applied experimental techniques. Additional complications arrive due to putative influences of labels on the phase behavior. Therefore, we combined small-and wide-angle x-ray scattering, differential scanning calorimetry and attenuated total reflection-Fourier transform infrared spectroscopy to probe the stability and physical properties of coexisting domains under label-free conditions. The capabilities of this combination are demonstrated on a model system composed of palmitoyl oleoyl phosphatidylcholine, sphingomyelin and ceramide. This mixture mimics sphingomyelinase activity on biological membranes. We found compositional fluctuations (unstable microscopic domains) in the absence of ceramide and macroscopically separated fluid and gel phases upon the addition of ceramide. Additionally, we observed broad phase transitional regions in the presence of ceramide, where also phase fluctuations occurred. Results are compared to a previously reported phase diagram and discussed in relation to the biological activity of sphingomyelinase. Our study demonstrates the necessity of applying a mix of experimental techniques to probe local/global structural, as well as fast/slow motional properties in complex lipid mixtures.