Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inward open state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inward open state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.W ater molecules in transmembrane protein pores participate in the transport of protons across the membrane bilayer. This process has been extensively studied experimentally and through computational simulations, particularly in small channels such as gramicidin A and influenza A M2. The movement of ions through channels is coupled to the diffusion of water through the pore, but protons are transported at a rate that is faster than the diffusion of H 3 O + (1, 2). Instead of diffusing through channels, protons move concertedly across networks of hydrogen-bonded waters through what is known as the Grotthuss mechanism (3-5). This mechanism of proton transport was initially discovered by the behavior of water in solution, and it has also been proposed to occur within membrane proteins containing water-filled pores (6-9). The matrix 2 (M2) protein of influenza A is one of the smallest proton-selective channels found in nature. This makes it an ideal system for studying the involvement of water in the selective transport of protons across the membrane. The M2 protein of influenza A is a tetramer made up of four 97-residue-long monomers. M2 is multifunctional, with different functions lying in different regions of the sequence. Residues 1-22 make up a conserved N-terminal domain that assists the incorporation of M2 into the virion (10) and is absent in influenza B viruses. The transmembrane domain of M2 (residues 22-46) is necessary for tetramerization (11) and forms a proton-selective channel (12-15) that is the target of the adamantane class of drugs, amantadine and rimantadine (11,(16)(17)(18). The transme...

1. Electrical and pharmacologic properties of ATP-induced current in outer hair cells isolated from guinea pig cochlea were investigated in the whole-cell recording mode by the use of a conventional patch-clamp technique. 2. Under current-clamp conditions, rapid application of ATP depolarized the outer hair cells resulting in an increase in conductance. The ATP-induced response did not show any desensitization during a continuous application. 3. At a holding potential of -70 mV, the ATP-induced inward current increased in a sigmoidal fashion over the concentration range between 3 microM and 1 mM. The half-maximum concentration (EC50) was 12 microM and the Hill coefficient was 0.93. 4. The ATP-induced current had a reversal potential near 6 mV, which was close to the theoretical value (1 mV) calculated from the Goldman-Hodgkin-Katz equation for permeable intra- and extracellular cations. 5. In the current-voltage (I-V) relationship for the ATP response, a slight inward-going rectification was observed at more positive potentials than the reversal potential. 6. The substitution of extracellular Na+ by equimolar choline+ shifted the reversal potential of the ATP-induced current to more negative values. The substitution of Cs+ in the internal solution by N-methyl-D-glucamine+ (NMG+) shifted it in the positive direction. The reversal potential of ATP-induced current was also shifted to positive values with increasing extracellular Ca2+ concentration. A decrease of intracellular Cl- by gluconate- did not affect the reversal potential, thereby indicating that the ATP-induced current is carried through a large cation channel.(ABSTRACT TRUNCATED AT 250 WORDS)

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