224 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS
to each other. Actually, these experiments and others place the N - terminal end
of one subunit ’ s S4 helix in close proximity to the C - terminal end of S5 in the
adjacent subunit of the tetrameric complex. The reference 28 authors believe
that this foregoing evidence confi rms their placement of S4 in its position
between the pore and helices S1 – S3.
Accessibility measurements have placed residues on voltage sensor helices
S1 – S4 in the following varying positions: (1) close to the intracellular side of
the membrane or in the cytoplasm; (2) close to the extracellular side of the
membrane or outside the membrane extracellularly; (3) being wholly within
the membrane. This type of experiment, carried out by the MacKinnon group,
has already been described in the discussion of the KvAP structure (1ORQ,
1ORS) previously. (See the discussion of reference 25 above.) Laine, Papazian,
and Roux^28 describe further fl uorescence resonance energy transfer (FRET)
experiments in which cysteine residues are introduced into S1 – S3 sites by site -
directed mutagenesis.^34 The cysteines then are labeled with tretramethylrho-
damine maleimide, a fl uorescent molecule, to probe the position ’ s accessibility
extracellularly. Since the fl uorescent reagent cannot pass through the mem-
brane, voltage - dependent changes in fl uorescence relative to wild - type K +
channel indicates whether the residue is accessible to solvent. Residues that
showed important fl uorescent changes were observed at the C - terminal end
of S1 and the N - terminal end of S2 (the S1 – S2 linker) and the C - terminal side
of S3 (S3b) (S3 – S4 linker), indicating that these residues were exposed to the
external solution. No signifi cant fl uorescent changes were seen for helical seg-
ments within the membrane (235 – 246 for S1 and 281 – 329 in S2 – S3). Bezanilla
and co - workers used histidine scanning mutagenesis of the S4 helix to position
important S4 residues through their accessibility from the external or internal
side of the membrane.^35 Their experiments showed that Shaker S4 residues
arg365, arg368, and arg371 traverse the membrane from the internal side at
hyperpolarized potentials (membrane negative inside) to the external side at
depolarized potentials (membrane negative outside). These results are similar
to those found by the MacKinnon team for KvAP using the biotinylation
experiments described previously. In these experiments, with the channel in
its activated state (pore open, depolarized), extracellular accessible positions
ranged from gly101 (Shaker phe324) in helix S3b to leu122 (Shaker val367)
approaching the center of the S4 helix but closer to its N - terminal end. Leu121
and leu122 were also accessible from the internal side with the channel in its
resting state (pore closed) in the MacKinnon group experiments.
Conclusions from the reference 28 review article are as follows: (1) Several
models backed by experiment place S4 near the groove between adjacent
subunits, while the MacKinnon group model places S4 near the periphery of
the protein; (2) lanthanide - based resonance energy transfer (LRET) places
two S4 residues in segments across the tetramer from each other at a distance
of 45 Å ; (3) a method based on tethered quaternary ammonium pore blockers
places the extracellular ends of the S1 and S3 helices further away from the
ion conduction pore than the S3 – S4 linker, arguing that the S4 helix resides