Temperature dependence of proton permeation through a voltage-gated proton channel

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (Iratio) were determined. The use of Iratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q10s for the proton currents were evaluated from the Iratios. Q10 exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q10. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q10 was decomposed into Q10 of the channel and of the access resistances. Finally, the Q10 for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism

pH dependence and inhibition by extracellular calcium of proton currents via plasmalemmal vacuolar-type H+-ATPase in murine osteoclasts

The vacuolar-type H+-ATPase (V-ATPase) in the plasma membrane of a variety of cells serves as an acid-secreting pathway, and its activity is closely related to cellular functions. Massive proton secretion often leads to electrolyte disturbances in the vicinity of the cell and may in turn affect the activity of the V-ATPase. We characterized, for the first time, the proton currents mediated by plasmalemmal V-ATPase in murine osteoclast-like cells and investigated its activity over a wide range of pH gradients across the membrane (ΔpH = extracellular pH – intracellular pH). The V-ATPase currents were identified as outward H+ currents and were dependent on ATP and sensitive to the inhibitors bafilomycin A1 and N,N′-dicyclohexylcarbodiimide. Although H+ was transported uphill, the electrochemical gradient for H+ affected the current. The currents were increased by elevating ΔpH and depolarization, and were reduced by lowering ΔpH and hyperpolarization. Elevation of extracellular Ca2+ (5–40 mm) diminished the currents in a dose-dependent manner and made the voltage dependence more marked. Extracellular Mg2+ mimicked the inhibition. With 40 mm Ca2+, the currents decreased to < 40% at 0 mV and to < 10% at about −80 mV. Increases in the intracellular Ca2+ (0.5–5 μm) did not affect the current. The data suggest that acid secretion through the plasmalemmal V-ATPase is regulated by a combination of the pH gradient, the membrane potential and the extracellular divalent cations. In osteoclasts, the activity-dependent accumulation of acids and Ca2+ in the closed extracellular compartment might serve as negative feedback signals for regulating the V-ATPase

Efficacy and Safety of Bimagrumab in Sporadic Inclusion Body Myositis: Long-Term Extension of RESILIENT

Objective: To assess long-term (2 years) effects of bimagrumab in participants with sporadic inclusion body myositis. Methods: Participants (aged 36-85 years) who completed the core study (RESILIENT) were invited to join an extension study. Individuals continued on same treatment as in the core study (10 mg/kg, 3 mg/kg, 1 mg/kg bimagrumab, or matching placebo administered as intravenous infusions every 4 weeks). The co-primary outcome measures were 6-minute walk distance (6MWD) and safety. Results: Between November 2015 and February 2017, 211 participants entered double-blind placebo-controlled period of the extension study. Mean change in 6MWD from baseline was highly variable across treatment groups, but indicated progressive deterioration from weeks 24-104 in all treatment groups. Overall, 91.0% (n = 142) participants in the pooled bimagrumab group and 89.1% (n = 49) in the placebo group had 651 treatment-emergent adverse event (AE). Falls were slightly higher in the bimagrumab 3 mg/kg group vs 10 mg/kg, 1 mg/kg and placebo groups (69.2% [n = 36 of 52] vs 56.6% [n = 30 of 53], 58.8% [n = 30 of 51], and 61.8% [n = 34 of 55], respectively). The most frequently reported AEs in pooled bimagrumab group were diarrhea 14.7% (n = 23), involuntary muscle contractions 9.6% (n = 15), and rash 5.1% (n = 8). Incidence of serious AEs was comparable between the pooled bimagrumab and the placebo group (18.6% [n = 29] vs 14.5% [n = 8], respectively). Conclusion: Extended treatment with bimagrumab up to 2 years produced a good safety profile and was well-tolerated, but did not provide clinical benefits in terms of improvement in mobility. The extension study was terminated early due to core study not meeting its primary endpoint. Classification of evidence: This study provides Class IV evidence that for patients with sIBM, long-term treatment with bimagrumab was safe, well tolerated and did not provide meaningful functional benefit. The study is rated Class IV because of the open label design of Extension Treatment Period 2. Clinical trial registration: Registered at ClinicalTrials.gov: NCT02573467