Voltage-gated ion channels are crucial for electrical activity and chemical signaling in a variety of cell types. Structure-activity studies involving electrophysiological characterization of mutants are widely used and allow us to quickly realize the energetic effects of a mutation by measuring macroscopic currents and fitting the observed voltage dependence of conductance to a Boltzmann equation. However, such an approach is somewhat limiting, principally because of the inherent assumption that the channel activation is a two-state process. In this analysis, we show that the area delineated by the gating charge displacement curve and its ordinate axis is related to the free energy of activation of a voltage-gated ion channel. We derive a parameter, the median voltage of charge transfer (Vm), which is proportional to this area, and prove that the chemical component of free energy change of a system can be obtained from the knowledge of Vm and the maximum number of charges transferred. Our method is not constrained by the number or connectivity of intermediate states and is applicable to instances in which the observed responses show a multiphasic behavior. We consider various models of ion channel gating with voltage-dependent steps, latent charge movement, inactivation, etc. and discuss the applicability of this approach in each case. Notably, our method estimates a net free energy change of approximately −14 kcal/mol associated with the full-scale activation of the Shaker potassium channel, in contrast to −2 to −3 kcal/mol estimated from a single Boltzmann fit. Our estimate of the net free energy change in the system is consistent with those derived from detailed kinetic models (Zagotta et al. 1994. J. Gen. Physiol. doi:10.1085/jgp.103.2.321). The median voltage method can reliably quantify the magnitude of free energy change associated with activation of a voltage-dependent system from macroscopic equilibrium measurements. This will be particularly useful in scanning mutagenesis experiments.

Summary Perception of heat or cold in higher organisms is mediated by specialized ion channels whose gating is exquisitely sensitive to temperature. The physicochemical underpinnings of this temperature-sensitive gating have proven difficult to parse. Here, we took a bottom-up protein design approach, and rationally engineered ion channels to activate in response to thermal stimuli. By varying amino acid polarities at sites undergoing state-dependent changes in solvation, we were able to systematically confer temperature-sensitivity to a canonical voltage-gated ion channel. Our results imply that the specific heat capacity change during channel gating is a major determinant of thermo-sensitive gating. We also show that reduction of gating charges amplifies temperature-sensitivity of designer channels which accounts for low voltage-sensitivity in all known temperature-gated ion channels. These emerging principles suggest a plausible molecular mechanism for temperature-dependent gating that reconcile how ion channels with an overall conserved transmembrane architecture may exhibit a wide range of temperature-sensing phenotypes.

Plasmids containing 23S rRNA randomized at positions 2057−2063 and 2502−2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the preexisting wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3′-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analogue to recognize dipeptides. Several clones inhibited by the puromycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analogue (4) and a fluorescent oxazole (5) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues.

In a voltage-dependent sodium channel, the activation of voltage-sensors upon depolarization leads to the opening of pore gates. To elucidate the principles underlying this conformational coupling, we investigated a putative gating interface in domain III of the sodium channel using voltage-clamp fluorimetry and tryptophan-scanning mutagenesis. Most mutations have similar energetic effects on voltage-sensor activation and pore opening. However, several mutations stabilized the activated voltage-sensor while concurrently destabilizing the open pore. Mapping of these mutations onto a homology model of the sodium channel showed that most localize to hinge regions of the gating interface. Our analysis shows that these residues are involved in energetic coupling of the voltage-sensor and the pore when both are in the resting or activated conformations, supporting the notion that electromechanical coupling in a voltage-dependent ion channel involves the movement of rigid segments connected by elastic hinges.