220 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS
voltage - sensor paddles remain inaccessible as long as the channel is held
closed by a negative membrane voltage and that the Fab binding site on two
helical turns of S3b and one turn of S4 (the epitope) becomes exposed to the
membrane ’ s external side on depolarization.
Researchers then used a sophisticated biochemical test to place individual
amino acids into positions within the channel membrane under conditions of
polarization and depolarization — that is, under conditions when the pore
would be closed (positive charge on the membrane ’ s exterior, negative on the
interior) or open (negative charge on the membrane ’ s exterior, positive on
the interior). Finkelstein and co - workers had used this test earlier.^26 The pro-
cedure is as follows: (1) Using site - directed mutagenesis, place cysteine resi-
dues one at a time at specifi c amino acid locations of interest within the
channel protein (G112C, I127C, and L103C, for instance); (2) react cysteine
residues with biotin (biotinylation) — see Figure 5.9 ; (3) reconstitute the K +
channel into planar lipid membranes; and (4) determine whether avidin, a rigid
glycoprotein molecule incapable of entering the membrane but binding to
biotin with strong affi nity, binds from the external or internal side of the mem-
brane under depolarization conditions. Taking into account the length of the
biotin molecule and where it binds to avidin (that cannot enter the mem-
brane), one can place the cysteine α - carbons within 10 Å of the bulk aqueous
solution on either side of the membrane. The system functions as a molecular
ruler.
Wild - type K + channels are not affected or inhibited by external avidin,
showing a small reduction in current when avidin is applied internally. The
G112C mutant, positioned at the end of membrane segment S3b on the
voltage - sensor paddle, was inhibited completely by external avidin but not
internal avidin. Mutant I127C, located near the center of membrane segment
S4 on the voltage - sensor paddle and next to important gating - charge mover
arg126, was inhibited completely by internal avidin but not by external avidin.
Mutant L102C, located near the N - terminal end of segment S3b, showed a
different behavior. External avidin reduces the current but does not abolish it
Figure 5.9 The biotin molecule.SHN NH
H HCOOHOBiotinThe carboxyl group
is attached through a
linker to a cysteine
residue on the protein
chain.