Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening

Background: hERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening. Methods and Findings: Tetramethylrhodamine-5-maleimide (TMRM) fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449) in the S1–S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the VK of activation to 227.562.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1–S2 linker cysteines with valines allowed unobstructed recording of S3–S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-VON, with VK,1 = 237.861.7 mV, and VK,2 = 43.567.9 mV. The first phase, VK,1, was,20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-VK = 218.361.2 mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (V K = 234.461.5 mV), suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarizatio

The selectivity, voltage-dependence and acid sensitivity of the tandem pore potassium channel TASK-1 : contributions of the pore domains

We have investigated the contribution to ionic selectivity of residues in the selectivity filter and pore helices of the P1 and P2 domains in the acid sensitive potassium channel TASK-1. We used site directed mutagenesis and electrophysiological studies, assisted by structural models built through computational methods. We have measured selectivity in channels expressed in Xenopus oocytes, using voltage clamp to measure shifts in reversal potential and current amplitudes when Rb+ or Na+ replaced extracellular K+. Both P1 and P2 contribute to selectivity, and most mutations, including mutation of residues in the triplets GYG and GFG in P1 and P2, made channels nonselective. We interpret the effects of these—and of other mutations—in terms of the way the pore is likely to be stabilised structurally. We show also that residues in the outer pore mouth contribute to selectivity in TASK-1. Mutations resulting in loss of selectivity (e.g. I94S, G95A) were associated with slowing of the response of channels to depolarisation. More important physiologically, pH sensitivity is also lost or altered by such mutations. Mutations that retained selectivity (e.g. I94L, I94V) also retained their response to acidification. It is likely that responses both to voltage and pH changes involve gating at the selectivity filter

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

Transcutaneous electrical nerve stimulation reduces exercise-induced perceived pain and improves endurance exercise performance

Purpose. Muscle pain is a natural consequence of intense and prolonged exercise and has been suggested to be a limiter of performance. Transcutaneous electrical nerve stimulation (TENS) and interferential current (IFC) have been shown to reduce both chronic and acute pain in a variety of conditions. This study sought to ascertain whether TENS and IFC could reduce exercise-induced pain (EIP) and whether this would affect exercise performance. It was hypothesised that TENS and IFC would reduce EIP and result in an improved exercise performance. Methods. In two parts, 18 (Part I) and 22 (Part II) healthy male and female participants completed an isometric contraction of the dominant bicep until exhaustion (Part I) and a 16.1 km cycling time trial as quickly as they could (Part II) whilst receiving TENS, IFC and a SHAM placebo in a repeated measures, randomized cross-over, and placebo controlled design. Perceived EIP was recorded in both tasks using a validated subjective scale. Results. In Part I, TENS significantly reduced perceived EIP (mean reduction of 12%) during the isometric contraction (P = 0.006) and significantly improved participants’ time to exhaustion by a mean of 38% (P = 0.02). In Part II, TENS significantly improved (P = 0.003) participants’ time trial completion time (~2% improvement) through an increased mean power output. Conclusion. These findings demonstrate that TENS can attenuate perceived EIP in a healthy population and that doing so significantly improves endurance performance in both submaximal isometric single limb exercise and whole-body dynamic exercise

Genomics, molecular epidemiology and diagnostics of infectious hypodermal and hematopoietic necrosis virus

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimps worldwide, which has resulted in severe mortalities of up to 90 % in cultured Penaeus (Litopenaeus) stylirostris from Hawaii and hence designated Penaeus stylirostris densovirus (PstDNV). IHHNV is distributed in shrimp culture facilities worldwide. It causes large economic loss to the shrimp farming industry. Our knowledge about the natural reservoirs of IHHNV is still scarce. Recent studies suggest that there is sufficient sequence variation among the isolates from different locations in Asia, suggesting multiple geographical strains of the virus. Four complete genomes and several partial sequences of the virus are available in the GenBank. Complete genome information would be useful for assessing the specificity of diagnostics for viruses from different geographical areas. Comparisons of complete genome sequences will help us gain insights into point mutations that can affect virulence of the virus. In addition, because of unavailability of shrimp cell lines for culturing IHHNV in vitro, quantification of virus is difficult. The recent progress in research regarding clinical signs, geographical distribution, complete genome sequence and genetic variation, transmission has made it possible to obtain information on IHHNV. A comprehensive understanding of IHHNV infection process, pathogenesis, structural proteins and replication is essential for developing prevention measures. To date, no effective prophylactic measure for IHHNV infection is available for shrimp to reduce its impact. This review provides an overview of key issues regarding IHHNV infection and disease in commercially important shrimp species