S1 Constrains S4 in the Voltage Sensor Domain of Kv7.1 K+ Channels

Voltage-gated K+ channels comprise a central pore enclosed by four voltage-sensing domains (VSDs). While movement of the S4 helix is known to couple to channel gate opening and closing, the nature of S4 motion is unclear. Here, we substituted S4 residues of Kv7.1 channels by cysteine and recorded whole-cell mutant channel currents in Xenopus oocytes using the two-electrode voltage-clamp technique. In the closed state, disulfide and metal bridges constrain residue S225 (S4) nearby C136 (S1) within the same VSD. In the open state, two neighboring I227 (S4) are constrained at proximity while residue R228 (S4) is confined close to C136 (S1) of an adjacent VSD. Structural modeling predicts that in the closed to open transition, an axial rotation (∼190°) and outward translation of S4 (∼12 Å) is accompanied by VSD rocking. This large sensor motion changes the intra-VSD S1–S4 interaction to an inter-VSD S1–S4 interaction. These constraints provide a ground for cooperative subunit interactions and suggest a key role of the S1 segment in steering S4 motion during Kv7.1 gating

Non-Native R1 Substitution in the S4 Domain Uniquely Alters Kv4.3 Channel Gating

The S4 transmembrane domain in Shaker (Kv1) voltage-sensitive potassium channels has four basic residues (R1–R4) that are responsible for carrying the majority of gating charge. In Kv4 channels, however, R1 is replaced by a neutral valine at position 287. Among other differences, Kv4 channels display prominent closed state inactivation, a mechanism which is minimal in Shaker. To determine if the absence of R1 is responsible for important variation in gating characteristics between the two channel types, we introduced the V287R mutant into Kv4.3 and analyzed its effects on several voltage sensitive gating transitions. We found that the mutant increased the voltage sensitivity of steady-state activation and altered the kinetics of activation and deactivation processes. Although the kinetics of macroscopic inactivation were minimally affected, the characteristics of closed-state inactivation and recovery from open and closed inactivated states were significantly altered. The absence of R1 can only partially account for differences in the effective voltage sensitivity of gating between Shaker and Kv4.3. These results suggest that the S4 domain serves an important functional role in Kv4 channel activation and deactivation processes, and also those of closed-state inactivation and recovery

Lipid-dependent gating of a voltage-gated potassium channel

Recent studies hypothesized that phospholipids stabilize two voltage-sensing arginine residues of certain voltage-gated potassium channels in activated conformations. It remains unclear how lipids directly affect these channels. Here, by examining the conformations of the KvAP in different lipids, we showed that without voltage change, the voltage-sensor domains switched from the activated to the resting state when their surrounding lipids were changed from phospholipids to nonphospholipids. Such lipid-determined conformational change was coupled to the ion-conducting pore, suggesting that parallel to voltage gating, the channel is gated by its annular lipids. Our measurements recognized that the energetic cost of lipid-dependent gating approaches that of voltage gating, but kinetically it appears much slower. Our data support that a channel and its surrounding lipids together constitute a functional unit, and natural nonphospholipids such as cholesterol should exert strong effects on voltage-gated channels. Our first observation of lipid-dependent gating may have general implications to other membrane proteins

Artificial Modulation of the Gating Behavior of a K+ Channel in a KvAP-DNA Chimera

We present experiments where the gating behavior of a voltage-gated ion channel is modulated by artificial ligand binding. We construct a channel-DNA chimera with the KvAP potassium channel reconstituted in an artificial membrane. The channel is functional and the single channel ion conductivity unperturbed by the presence of the DNA. However, the channel opening probability vs. bias voltage, i.e., the gating, can be shifted considerably by the electrostatic force between the charges on the DNA and the voltage sensing domain of the protein. Different hybridization states of the chimera DNA thus lead to different response curves of the channel

Voltage-Dependent Gating in a “Voltage Sensor-Less” Ion Channel

An unusual mechanism of ion channel regulation generates voltage-dependent gating in the absence of a canonical voltage-sensing domain

Continuum Molecular Simulation of Large Conformational Changes during Ion–Channel Gating

A modeling framework was developed to simulate large and gradual conformational changes within a macromolecule (protein) when its low amplitude high frequency vibrations are not concerned. Governing equations were derived as alternative to Langevin and Smoluchowski equations and used to simulate gating conformational changes of the Kv7.1 ion-channel over the time scale of its gating process (tens of milliseconds). The alternative equations predict the statistical properties of the motion trajectories with good accuracy and do not require the force field to be constant over the diffusion length, as assumed in Langevin equation. The open probability of the ion–channel was determined considering cooperativity of four subunits and solving their concerted transition to the open state analytically. The simulated open probabilities for a series of voltage clamp tests produced current traces that were similar to experimentally recorded currents

Channelopathies in Cav1.1, Cav1.3, and Cav1.4 voltage-gated L-type Ca2+ channels

Voltage-gated Ca2+ channels couple membrane depolarization to Ca2+-dependent intracellular signaling events. This is achieved by mediating Ca2+ ion influx or by direct conformational coupling to intracellular Ca2+ release channels. The family of Cav1 channels, also termed L-type Ca2+ channels (LTCCs), is uniquely sensitive to organic Ca2+ channel blockers and expressed in many electrically excitable tissues. In this review, we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within their pore-forming α1 subunits causing hypokalemic periodic paralysis and malignant hyperthermia sensitivity (Cav1.1 α1), incomplete congenital stationary night blindness (CSNB2; Cav1.4 α1), and Timothy syndrome (Cav1.2 α1; reviewed separately in this issue). Cav1.3 α1 mutations have not been reported yet in humans, but channel loss of function would likely affect sinoatrial node function and hearing. Studies in mice revealed that LTCCs indirectly also contribute to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Cav2.1 α1 in tottering mice. Ca2+ channelopathies provide exciting disease-related molecular detail that led to important novel insight not only into disease pathophysiology but also to mechanisms of channel function

Sphingomyelin Functions as a Novel Receptor for Helicobacter pylori VacA

The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells

Lifestyle and socio-demographic factors associated with high-risk HPV infection in UK women

The world age-standardised prevalence of high-risk HPV (hrHPV) infection among 5038 UK women aged 20–59 years, with a low-grade smear during 1999–2002, assessed for eligibility for TOMBOLA (Trial Of Management of Borderline and Other Low-grade Abnormal smears) was 34.2%. High-risk HPV prevalence decreased with increasing age, from 61% at ages 20–24 years to 14–15% in those over 50 years. The age-standardised prevalence was 15.1, 30.7 and 52.7%, respectively, in women with a current normal, borderline nuclear abnormalities (BNA) and mild smear. In overall multivariate analyses, tertiary education, previous pregnancy and childbirth were associated with reduced hrHPV infection risk. Risk of infection was increased in non-white women, women not married/cohabiting, hormonal contraceptives users and current smokers. In stratified analyses, current smear status and age remained associated with hrHPV infection. Data of this type are relevant to the debate on human papillomavirus (HPV) testing in screening and development of HPV vaccination programmes