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The thiol reagent N-ethylmaleimide (NEM) completely inhibits the proton pump activity of the H+-ATPase in chromaffin granule 'ghosts' at concentrations which only partly (approximately 20%) inhibit the Mg2+-dependent ATP hydrolysis. Half-maximal inhibition was obtained at approximately 13 microM NEM as compared to 18 microM for the classical proton channel inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), and the apparent stoichiometry of the inhibitors at complete inhibition was NEM : DCCD congruent to 1 : 2. HIgh concentrations of NEM (greater than 100 microM) induce a dissipation of the transmembrane potential generated by MgATP. These findings establish NEM as a valuable proton channel inhibitor in chromaffin granules and explain the rather complex effect of NEM previously reported for catecholamine accumulation in this organelle.

Brown adipose tissue is a thermogenic organ that effectively participates in the maintenance of body temperature in newborn mammals and in adult hibernating animals. The high triglyceride content of brown adipose tissue cells, the high oxidative capacity and specific mitochondrial proton channel which enables energy dissipation in the form of heat are involved in the protection of the animal body against cold (Lindberg 1970; Janský 1973; Nichols 1979; Cannon and Needergard 1985).

Publisher Summary This chapter discusses the preparation, dissociation into subunits, and reconstitution of an active complex for the proton-conducting portion (F 0 ) from Escherichia coli ATP synthase. The F 0 part of the ATP synthase complex (F 1 F 0 ) of E. coli is composed of three different polypeptides and the complex serves as a proton channel. After reconstitution into liposomes a functional F 0 complex can be tested either by measuring passive proton translocation or by adding back F 1 . Passive proton translocation through F 0 is measured using K+-loaded liposomes. ATPase activity of reconstituted F 1 F 0 complexes is measured by determining the liberated orthophosphate. The chapter outlines the procedure for preparation of F 0 by hydrophobic interaction chromatography. Reconstitution of a functional F 0 complex is only achieved by incorporation of all three kinds of subunits into phospholipid vesicles.

I wish to make a general comment upon the nature of mechanical changes within proteins. The comment applies equally to receptors which must transmit information across a considerable distance, e.g. across a membrane, to the initiation of activity by remote control such as the interaction of ion-binding with ATP formation or hydrolysis, and even to the opening out of a large molecule so that it can be read, e.g. the loosening of histones from DNA. The motion must be directed and functional.

The mechanism of the process leading to cell-cell fusion induced by enveloped viruses at a mildly acidic pH is as yet unknown. In this report we demonstrate that the fusion events induced by three viruses of different families, namely Semliki Forest (togavirus), vesicular stomatitis (rhabdovirus) and influenza (orthomyxovirus), share common features. In all three systems a sudden drop of the intracellular pH—below the critical eextracellular pH required to trigger “fusion from within” (FFWI)—is observed. This influx of protons is specific and not due to a general leakiness of the plasma membrane, and therefore might be caused by the opening of a proton channel.

Effect of azide on proton transfer of bacteriorhodopsin (bR) mutants that lack the proton donor Asp-96 and/or proton acceptor Asp-85 was investigated by a photoelectrochemical method. In the presence of azide, D96N completely recovered the response profile of the wild type and D96N/D85N gave a very small current with the same amplitude as in D85N. On the contrary, D85N showed no azide effect. These results indicate that azide affects the process of shuttling protons from D96 to the Schiff base rather than does the proton transfer from the Schiff base to D85.

The effect of the charge exchange (p, n) scattering in the proton channel is explicitly included in a DWBA expression for the (d, p) stripping reaction. The expression obtained predicts anomalies in the energy dependence of the cross section.

A lower angular limit to a classical orbital description of proton channel, when the continuos string model is assumed, is derived.

The voltage-gated proton channel (Hv1) of leukocytes, basophils, airway epithelium, and spermatozoa is extremely selective. Hv1 presents structural and functional similarities to the voltage-sensor domain (VSD) of voltage-gated potassium (Kv) and sodium (Nav) channels. Hv1 and the VSDs of Kv and Nav sense changes in membrane potentials and contain four α-helical segments as well as conserved arginine residues on the fourth helix. Although the electrophysiological features of Hv1 are well characterized, the molecular mechanism of proton conduction is unknown. This is largely because an experimentally-determined structure of Hv1 is not available. An alternative source of structural information is homology modeling, whereby a model of Hv1 can be constructed using the atomic structures of the VSDs of Kv and Nav as templates. However, since the sequence identity between Hv1 and templates is below 20%, a critical assessment of generated models is essential. Here we present a homology modeling-simulation strategy using alternative sequence alignments to construct, assess, and validate alternative homology models. Comparison of a range of structural properties between the models and templates is used to guide the selection of an optimal model for Hv1. This method represents a generalized strategy that can be applied to other membrane proteins that lack high sequence identity to their templates.

Voltage-gated proton channels mediate proton extrusion in a large variety of cells. Hv1, the first and so far only cloned voltage-gated proton channel, is composed of the first four transmembrane segments (S1-S4) that compose voltage-sensing domains (VSD), but lacks the pore domain of "classic" voltage-gated channels. Hv1 channels associate as dimers, but each subunit contains its own pore, suggesting that the pore and the gate are contained within each VSD. Earlier work showed that, as in "classic" voltage-gated channels, the arginine-rich S4 transmembrane segment is the voltage sensor in Hv1. However, the molecular mechanism of opening is still ill-defined. In this study, we identify the S1 segment as the gate of Hv1 channels. Observed changes in the accessibility of the internal and external ends of S1 indicate a voltage-driven rearrangement of this segment. Moreover, the conformational change of S1 precisely tracks channel opening in voltage dependence and kinetics. Finally, we show that gating of Hv1 involves a close interaction between the S1 and S4 segments that depends on S4's third arginine (R3) and an Hv1 specific aspartate on S1 (D1), two residues that have been previously proposed to interact in the open state to form the proton selectivity filter. Overall, our findings suggest that the S1 segment has specialized as the determinant for gating and proton conduction in Hv1.

Background Proton currents are required for optimal respiratory burst in phagocytes. Recently, HVCN1 was identified as the molecule required for the voltage-gated proton channel activity associated with the respiratory burst in neutrophils. Although there are similarities between eosinophils and neutrophils regarding their mechanism for respiratory burst, the role of proton channels in eosinophil functions has not been fully understood. Results In the present study, we first identified the expression of the proton channel HVCN1 in mouse eosinophils. Furthermore, using HVCN1-deficient eosinophils, we demonstrated important cell-specific effector functions for HVCN1. Similar to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils produced significantly less reactive oxygen species (ROS) upon phorbol myristate acetate (PMA) stimulation compared with WT eosinophils. In contrast to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils did not show impaired calcium mobilization or migration ability compared with wild-type (WT) cells. Uniquely, HVCN1-deficient eosinophils underwent significantly increased cell death induced by PMA stimulation compared with WT eosinophils. The increased cell death was dependent on NADPH oxidase activation, and correlated with the failure of HVCN1-deficient cells to maintain membrane polarization and intracellular pH in the physiological range upon activation. Conclusions Eosinophils require proton channel HVCN1 for optimal ROS generation and prevention of activation-induced cell death.