Workshop on Ion Channels, Mon, 08 Oct, 2007
Speaker: Peter Pohl
Abstract
Confinement of water due to pore geometry alters water’s physical characteristics. Changes in flow dynamics are partially due (i) to the loss of hydrogen bonds compared to the bulk fluid, and (ii) due to permeating molecules’ interaction with the channel wall. As channel diameter decreases and hydrophobicity increases, the probability for liquid vapour oscillations increases (1). The resulting intermittency of pore conductance supports the theory of hydrophobic gating. This theory explains how transmembrane pores which are incompletely occluded in their closed state prevent the movement of solutes and solvent molecules and makes the following testable predictions:
(i) The velocity of transport across the channel may increase beyond the diffusion limit:
(ii) The density of water molecules in the channel is lower than in bulk;
(iii) The lack of neighbouring water molecules affects the acid base equilibrium of permeating molecules.
Experimental evidence compatible with all three predictions was obtained in studies of water mobility in purified and reconstituted membrane channels. The diffusion coefficient of water molecules within the pore was derived from the single channel permeability coefficient pf. pf. was measured by imposing an osmotic gradient across reconstituted planar lipid bilayers and detecting the resulting shift in solute concentration close to the membrane surface. The bacterial potassium channel KcsA exhibited the highest pf. Water mobility inside the potassium channel exceeded bulk mobility 20-fold (2). In contrast, water mobility inside short peptidic nanopores was equal to bulk mobility. It decreased exponentially with increasing pore length. An increment of just one accommodation site for water resulted in a fourfold drop of pf (3). The non-linear dependence of pf on channel length indicated that several accommodation sites for water were unoccupied (3). In view of the vapour-like density of water, we tested whether water conducting channels may also destabilize NH4+ and provide a transport pathway for NH3. In line with the hypothesis, the water channel aquaporin-8 was found to transport neutral ammonia molecules as fast as it does convey water molecules (4).
After having confirmed predictions (i – iii), we checked whether the hydrophobic gating theory is applicable to the bacterial peptide translocation channel SecY. A point mutation in the hydrophobic pore ring, the narrow constriction zone in the center of the channel, confirmed that the ring is required for sealing the channel in its closed state. However, the pore ring alone did not prevent the passage of ions over a period of seconds or minutes, as shown by our electrophysiology experiments (5). Thus, SecY hydrophobic gating may operate on a shorter time scale, but for maintenance of the membrane barrier other gating mechanisms must be engaged.
Acknowledgement: Financial support was provided by the Austrian Science Fund (FWFW1201-N19, B19716-B05).
1. Beckstein, O. and M. S. P. Sansom. 2003. Liquid-vapor oscillations of water in hydrophobic nanopores. Proc. Natl. Acad. Sci. U. S. A. 100:7063-7068.
2. Saparov, S. M. and P. Pohl. 2004. Beyond the diffusion limit: Water flow through the empty bacterial potassium channel. Proc. Natl. Acad. Sci. U. S. A. 101:4805-4809.
3. Saparov, S. M., J. R. Pfeifer, L. Al-Momani, G. Portella, B. L. de Groot, U. Koert, and P. Pohl. 2006. Mobility of a one-dimensional confined file of water molecules as a function of file length. Phys. Rev. Lett. 96:148101.
4. Saparov, S. M., K. Liu, P. Agre, and P. Pohl. 2007. Fast and selective ammonia transport by aquaporin-8. J. Biol. Chem. 282:5296-5301.
5. Saparov, S. M., K. Erlandson, K. Cannon, J. Schaletzky, S. Schulman, T. A. Rapoport, and P. Pohl. 2007. Determining the Conductance of the SecY Protein Translocation Channel for Small Molecules. Mol. Cell 26:501-509.
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