Potassium channels catalyze the selective transfer of potassium across the cell membrane and are essential for setting the resting potential in cells, controlling heart rate and modulating the firing pattern in neurons. Tetraethylammonium (TEA) blocks ion conduction through potassium channels in a voltage-dependent manner from both sides of the membrane. Here we show the structural basis of TEA blockade by cocrystallizing the prokaryotic potassium channel KcsA with two selective TEA analogs. TEA binding at both sites alters ion occupancy in the selectivity filter; these findings underlie the mutual destabilization and voltage-dependence of TEA blockade. We propose that TEA blocks potassium channels by acting as a potassium analog at the dehydration transition step during permeation.

Resonance Raman and FTIR spectra are reported for the fully reduced carbon monoxy derivative of the quinol aa 3-600 oxidase from Bacillus subtilis. The resonance Raman spectra display two isotope-dependent vibrational modes at 520 and 575 cm-1. The FTIR spectrum displays a single vibrational mode at 1963 cm-1. We assign the band at 520 cm-1 to the Fe−CO stretching mode, the band at 575 cm-1 to the Fe−C−O bending mode, and the band at 1963 cm-1 to the C−O stretching mode. The frequencies of these modes are similar to those that have been reported for the CO-bound mammalian cytochrome c oxidase. Despite the fact that two different heme−protein conformations that affect the iron−his bond strength are present in the ferrous ligand-free form of aa 3-600, the CO-bound adduct has a single conformation in which the His−Fe−CO CuB moiety has the same structure as the α form found in the mammalian cytochrome c oxidase. The present and previous data on the vibrational frequencies of ferrous ligand-free and ferrous CO-bound forms of terminal oxidases show that an inverse linear relationship exists between the frequencies of the Fe−his and Fe−CO stretching modes. We suggest that the frequencies of both the Fe−CO and C−O modes found in heme−CuB oxidases are affected by the proximal His376, which is H-bonded to the peptide carbonyl of Gly351, and by distal effects on the heme a 3-bound CO exerted by CuB.

A four-pulse electron paramagnetic resonance experiment was used to measure long-range inter-subunit distances in reconstituted KvAP, a voltage-dependent potassium (Kv) channel. The measurements have allowed us to reach the following five conclusions about the native structure of the voltage sensor of KvAP. First, the S1 helix of the voltage sensor engages in a helix packing interaction with the pore domain. Second, the crystallographically observed antiparallel helix-turn-helix motif of the voltagesensing paddle is retained in the membrane-embedded voltage sensor. Third, the paddle is oriented in such a way as to expose one face to the pore domain and the opposite face to the membrane. Fourth, the paddle and the pore domain appear to be separated by a gap that is sufficiently wide for lipids to penetrate between the two domains. Fifth, the critical voltage-sensing arginine residues on the paddle appear to be lipid exposed. These results demonstrate the importance of the membrane for the native structure of Kv channels, suggest that lipids are an integral part of their native structure, and place the voltage-sensing machinery into a complex lipid environment near the pore domain.

Resonance Raman and FTIR spectra are reported for the oxidized azide-bound derivative of the quinol cytochrome bo 3 oxidase from Escherichia coli. The resonance Raman spectra display three isotope-dependent vibrational modes at 419, 2040, and 2061 cm-1. The FTIR spectra display two isotope-dependent bands at 2040 and 2061 cm-1. We assign the band at 419 cm-1 to ν(Fe−N3−CuB) and the bands at 2040 and 2061 cm-1 to νas(N3). The observation of two νas(N3) modes suggests that the azide ion binds to two different enzyme conformations, both forming bridging complexes with the binuclear center. Comparison of the FTIR data of the azide-bound cytochrome bo 3 and cytochrome a a 3 complexes reveal that there are quantitative differences in the structure of the heme o 3−CuB and heme a 3−CuB binuclear pockets upon azide binding. The present data on the vibrational frequencies of the azide-bound cytochrome bo 3 complex do not support the recent proposal that azide binds as a terminal ligand to CuB (Little, R. H.; Cheesman, M. R.; Thomson, A. J.; Greenwood, C.; Watmough, N. J. Biochemistry 1996, 35, 13780−13787) but are more reasonably interpreted to conclude that azide functions as a bridge between heme o 3 and CuB.

Resonance Raman spectroscopy has been employed to investigate the reduced cyano complexes of cytochrome aa(3) from bovine heart and Rhodobacter sphaeroides and of cytochrome bo(3) from E. coli. In the aa(3)-type oxidases, the frequency of the Fe-CN stretching mode is located at 468 cm(-1), and the bending Fe-C-N vibration, at 500 cm(-1). The fully reduced cytochrome bo(3)-CN complex gives rise to a stretching vibration at 468 cm(-1), a bending vibration at 491 cm(-1), and a stretching C-N vibration at 2037 cm(-1). The observed differences between aa(3) and bo(3) oxidases in the frequencies of the Fe-C-N group suggest a quantitative difference in the structure of the His-heme a(3)(2+)/Cu(B)(1+) and His-heme o(3)(2+)/Cu(B)(1+) binuclear pockets upon CN- binding.