C. M. Venkatachalam

Temperature dependence of single channel currents and the peptide libration mechanism for ion transport through the gramicidin A transmembrane channel

SummaryA study of the temperature dependence of gramicidin A conductance of K+ in diphytanoyllecithin/n-decane membranes shows the plot of In (single channel conductance) as a function of reciprocal temperature to be nonlinear for the most probable set of conductance, states. These results are considered in terms of a series of barriers, of the dynamics of channel conformation,vis-a-vis the peptide libration mechanism, and of the effect of lipid viscosity on side chain motions again as affecting the energetics of peptide libration.

[15] Ion interactions at membranous polypeptide sites using nuclear magnetic resonance: Determining rate and binding constants and site locations

Publisher Summary This chapter describes the determination of rate and binding constants and site locations ion interactions at membranous regions of polypeptide sites using nuclear magnetic resonance (NMR). Monitoring a site within the membrane is made difficult by the slow reorientation of the membrane. For interpreting ion correlation times, it is helpful when the reorientation correlation time of the site is long compared to the lifetime of the ion in the channel. The membranous gramicidin channel, the ion correlation time, derived from resonance linewidth analyses, can be interpreted as the ion occupancy time in the channel. The reciprocal of the ion occupancy time becomes the important off-rate constant for an ion leaving the channel. It is suggested that gramicidin channel is a particularly favorable membranous peptide that can function as a proving ground for these NMR approaches to the characterization of ion interactions. The process of substantiating or validating the interpretation or analyses of NMR data on ion interaction with membranous polypeptide is facilitated by the particular characteristics of the gramicidin channel transport and structure.

Potassium-39 NMR of K+ interaction with the gramicidin channel and NMR-derived conductance ratios for Na+, K+ and Rb+

SummaryA potassium-39 NMR study of potassium ion interaction with the gramicidin transmembrane channel in phospholipid bilayers at high ion activity is reported which allows determination of a weak binding constant, Kbw ≃8.3/m, and an off-rate constant for the weak site,koffw≃2.6×107/sec. These values are interpreted with the aid of additional NMR data as the binding constant for formation of the doubly occupied channel state and the rate constant for an ion leaving the doubly occupied state. Considering the singly occupied channel state for the potassium ion to be “electrically silent” at 1 molar ion activity, as with the sodium ion, the single-channel conductance for 100 mV and 30°C calculated to be 29 pS, and using the same approximation with previous NMR results on the sodium and rubidium ions, reasonable conductance ratios were calculated. Further experimental estimates of the other three constants with the experimental location of binding sites and Eyring rate theory to introduce voltage dependence allowed a more complete calculation of the two-site channel. The single-channel conductance for potassium ion is calculated to be 24 pS at 1m activity and 26 pS at 0.6m activity, which compares for diphytanoyl phosphatidylcholine membranes to an experimental most probable single-channel conductance of 25 pS and a mean channel conductance of 20 pS. The calculated conductance ratios using NMR-derived constants were γ(K)/γ(Na)=2.0 and γ(Rb)/γ(Na)=4.3. These results are close to the experimental values and provide further basis for the use of NMR of quadrupolar ions to provide information on the ionic mechanism of channel transport.

Shortened analog of the gramicidin a channel argues for the doubly occupied channel as the dominant conducting state

A shortened analog of the gramicidin A transmembrane channel has been synthesized and its transport characterized in planar lipid bilayer membranes. General considerations of a shorter diffusional length and a shorter distance over which the voltage drop occurs (i.e., an increased electric field) would contribute to an increase in single-channel conductance. The finding of a decreased single-channel conductance supports the perspective that the dominant conducting state is the doubly occupied channel wherein distance-dependent repulsion due to the first ion in the channel impedes entry of the second ion in the shorter channel.

Cesium-133 NMR longitudinal relaxation study of ion binding to the Gramicidin transmembrane channel

Abstract Cesium ion interaction with the Gramicidin transmembrane channel is studied using cesium-133 NMR. The channel system provides unusual insight into the use of spin - 7 2 nuclei to obtain ion interaction information. In spite of the small quadrupole moment for 133Cs, the absolute magnitude of which is 1 40 that of 23Na, the change in longitudinal relaxation rate for 133Cs interaction with channels is twice that observed for 23Na under identical conditions. Also, only small positive ion resonance chemical shifts are experimentally observed which are of opposite sign to those observed for the other alkali metal ions. The possibility of the cesium ion pairing with chloride ion in the channel binding site is explored as explanation for the small positive chemical shift and large T1 changes. The large T1 changes provide values for both tight and weak binding constants, i.e., Kbt ≈ 60/M and Kbw ≈ 4/M. In the null region of the partially relaxed spectra two narrow lines (separated by 20 Hz at 13 MHz) and a broad line were observed and the longitudinal relaxation rates were faster for the narrow components than for the broad component. Evidence is presented indicating that all resolvable lines were derived from a single equilibrated pool of spin - 7 2 nuclei. The null region demonstrates that a single, intense, narrow resonance line dominates the longitudinal relaxation in accordance with the analyses of Bull, Forsen, and Turner.

Dispersity of des-L·Val7-D·Val8-gramicidin A single channel conductances argues for different side chain orientations as basis

Planar bilayer studies are reported on the channel activity of des-L X Val7-D X Val8-Gramicidin A. This analog is designed to provide more long-lived side chain distributions involving the Trp residues than occur with Gramicidin A. The carbonyls of these residues coordinate the permeant cation and the energetics of the coordination, which is proposed to depend on side chain orientation, determines the free energies of the rate limiting entrance-exit barriers and the binding sites. The finding of an increased dispersity of single channel conductance for the analog supports the perspective that dispersity derives from different side chain distributions on the same backbone conformation. Channel mechanism is not understood until dispersity is explained.