Is the Activation Gate Closed in Kv Channel Closed-State Inactivation?


Sunday, February 3, 2013 127a
650-Pos Board B419
Access Constraints and Binding Energetics of a Potassium Channel
Inactivation Gate
Gaurav Venkataraman, Deepa Srikumar, Miguel Holmgren.
NIH, Bethesda, MD, USA.
Voltage-activated potassium (Kv) channels open an intracellular gate in re-
sponse to changes in transmembrane voltage, allowing K

þ
to permeate at rates

near diffusion. In some Kv channels, permeation can be interrupted by the
N-terminus of the protein acting as a blocking particle, binding at the intracel-
lular cavity of the channel and physically obstructing the permeation pathway.
Despite being tetramers, Kv channels require only one bound N-terminus to
inactivate. In nature, inactivation is targeted by RNA editing, which converts
an isoleucine of the intracellular cavity into a valine. The functional conse-
quence is a large increase in unbinding kinetics.
How does each ‘‘wall’’ of the cavity contribute to the increase in unbinding
kinetics of a single bound N-terminus? To address this question we made con-
catenated Shaker heterotetramers consisting of only one free N-terminus and
a single isoleucine to valine mutation at position 470 (I470V), mimicking the
RNA editing site in humans and rodents. Surprisingly, the rate of unbinding
was the same for individual I470V mutations in each wall; each corresponded
to a decrease in binding energy of about 0.4 RT. In fact, the binding energy de-
creased linearly as a function of the number of I470V mutated subunits. These
results suggest that despite being tethered to a particular subunit, the single in-
activation particle has sufficient freedom to interact with position 470 of each
subunit with roughly equal probability. Where does this freedom come from?
It is expected that the inactivation gates enter the cavity through lateral
‘windows’, similarly to K

þ
. Can our single tethered inactivation gate enter

through each of the four windows of the protein? We will address this question
by making individual cysteine mutations at each window and testing the inac-
tivation gate’s binding kinetics after chemical modification.

651-Pos Board B420
Gating Interactions between the Cytoplasmic T1 Domain and Pore of a Kv
Channel
Brian Urbani, Manuel Covarrubias.
Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA,
USA.
Whereas the gating roles of the voltage-sensing and pore domains of Kv
channels have been extensively investigated, much less is known about the
mechanisms governing the regulation of gating by the cytoplasmic T1 domain.
Previous work from this lab strongly suggests that the Kv4.1 T1 domain un-
dergoes conformational changes directly linked to gating (J. Gen. Physiol.,
127:391-400, 2006); however, the structural basis of the communication
between the activation gate and the T1 domain has remained elusive. To
tackle this problem, we applied cysteine scanning mutagenesis, voltage-
clamp electrophysiology, double mutant cycle analysis (DMCA) and oxidizing
reagents to probe specific interactions between the cytoplasmic T1 domain L4
helix, the S4-S5 linker and S6 tail. The energetic impact of the mutations on
activation gating is apparently small. By comparison, the energetic impact
of the mutations on inactivation gating was significantly greater. DMCA of
K141C(L4)þA422C(S6) revealed a significant interaction energy (4 kcal/
mol), which suggests an interaction between K141 in the L4 helix and A422
in S6. Supporting this possibility, the currents induced by K141C(L4)þ
A422C(S6) are inhibited (~65% of control) by an oxidizing agent (tBuHO2),
whereas the single mutants are not; and the effect is reversed by the reducing
agent DTT. These results indicate the formation of a disulfide bond between
the interacting side chains, which is consistent with physical interaction
between K141(L4) and A422(S6). Overall, we conclude that regulation of
inactivation gating by the T1 domain involves a physical interaction between
the T1 L4 helix and the distal segment of S6. Supported by NIH grant R01
NS032337 (MC).

652-Pos Board B421
Is the Activation Gate Closed in Kv Channel Closed-State Inactivation?
Jeffrey D. Fineberg, Manuel Covarrubias.
Thomas Jefferson University, Philadelphia, PA, USA.
Kv4.x channel complexes including auxiliary subunits such as DPP6-S and
KChIP1 undergo preferential closed-state inactivation (CSI; Baehring and
Covarrubias, 2011, J Physiol, 589.3:461-479; Fineberg et al., 2012, J Gen
Physiol, in press). However, the molecular basis of CSI in Kv4.x channels
remains poorly understood. A key unanswered question of this problem is
whether the channel’s activation is actually closed, as CSI implies. We have
previously proposed that CSI results when the activated channel fails to open
or simply prefers to close. This hypothesis specifically suggests that the intra-
cellular activation gate may adopt a stable closed conformation, even under
depolarized conditions that would initially favor the activated ‘‘up’’ state of
the voltage sensors. To test this hypothesis, we are exploiting the trapping
paradigm previously investigated by Holmgren et al. to test the state of the
intracellular activation gate (1997, J Gen Physiol, 109:527-535). Essentially,
if the activation gate closes behind a quaternary ammonium (QA) blocker
inside the pore, the blocker cannot exit the pore; effectively, it is trapped.
We are expressing the ternary Kv4.1/I400C channels (this mutation favors trap-
ping) including DPP6-S and KChIP1 in Xenopus oocytes. Then, in the inside-
out patch-clamp configuration, we are using a computer-controlled perfusion
system to deliver the QA derivative decyltrimethylammonium bromide (C10)
to the bath and probe the ability of the channel’s activation gate to trap C10
in the closed, open and inactivated states. Preliminary results show that the
trapping paradigm may yield an answer to the central question of this study.
If the activation gate is indeed closed during CSI, C10 trapping will only occur
when the drug is applied to the conducting channel, but no trapping of C10 will
be observed in the closed and inactivated states. Supported by NIH grant R01
NS032337 (MC).

653-Pos Board B422
Water at the Potassium Channel Gate: Quantum Calculations Show an
Oscillation in Water Structure
Alisher M. Kariev, Michael E. Green.
City College of the City University of NY, New York, NY, USA.
Quantum calculations on the open gate and the immediately surrounding re-
gion of the Kv1.2 voltage gated potassium channel show that water adopts
two conformations there, one when the ion is present, and another with no
ion present. An energy minimum at the gate with no ion allows an ion to enter
from the solution; from the gate it repels an ion in the channel pore cavity into
the selectivity filter (essentially the ‘‘knock-on’’ mechanism). The pore cavity
minimum can then be occupied by the ion moving from the gate minimum,
leaving it available for anion from bulk, so the cycle continues. The arrange-
ment of water, and the ion hydration, defines the gate energy minimum, and
this in turn depends on the highly conserved prolines, especially P407
(2A79 pdb numbering), in the PVPV sequence in the gate. Absent an energy
minimum at the gate an ion entering from solution would be repelled back
into solution by the ion in the cavity. Furthermore, the ion remains hydrated
by 8 water molecules, at least 1 more than in bulk, as it passes through the
gate, based on optimizations with the ion at three different positions: 1) at
the gate position nearest P407 2) 2Å above 3) 2Å below, this position. The
open gate maintains very nearly the same protein atom positions throughout.
Additional calculations will be required to determine the exact interaction en-
ergy with an ion in the cavity, and the amount of water that is present in the
cavity. Optimizations of the system configuration were done at HF/4-22GSP
level, with 692 atoms (693 with the ion). Energy was determined using both
B3LYP/6-31G* and bvp86/6-31G*, but the system was too large for free en-
ergy determination.

654-Pos Board B423
Water Structure at the Potassium Channel Gate: The Importance of the
Prolines in the PVPV Sequence, as Shown by Computational Mutations,
and the Role of a Histidine in Gating
Alisher M. Kariev, Michael E. Green.
City College of the City University of NY, New York, NY, USA.
Conservation of the PVPV sequence at the potassium channel gate has not been
well understood. Quantum computations (optimization at HF/4-22GSP level)
of the gate of the Kv1.2 channel that are presented here show that the
proline-proline distances (ring atoms) are preserved as the ion moves through
the gate. This calculation is supplemented by mutating one of the prolines to
valine (P407V, 2A79 pdb numbering for all residues in this abstract) in the
computation, producing a PVVV sequence. Water structure changed dramati-
cally, as the protein backbone moved inward nearly 3Å, effectively closing
the channel; the proline ring N-N distance between diagonally opposite pro-
lines dropped from 14.3 Å (distance from optimization: X-ray distance =
15.6Å) to 11.4 Å, too small to allow passage of a hydrated K

þ
. This suggests

that the prolines play a key structural role, modulating both protein and water
structure. Furthermore, protonation of H418 may close the channel. Protonation
of this histidine shows it moving toward the gate by about 3 Å, which should
close the gate electrostatically, while deprotonation opens the gate. Protonation
was calculated with two added protons, the other being accepted by E142 of the
T1 intracellular moiety. When deprotonated, the histidine does not move as
much as 1Å from the X-ray position in the optimization, and is linked to
E142; this accounts for the known importance of the T1 segment in gating.
E136, also of T1, could accept a third proton. There is a salt bridge structure
including these residues similar to the one occupying a key position in the pro-
ton pathway in the Hv1 channel.


	Access Constraints and Binding Energetics of a Potassium Channel Inactivation Gate
	Gating Interactions between the Cytoplasmic T1 Domain and Pore of a Kv Channel
	Is the Activation Gate Closed in Kv Channel Closed-State Inactivation?
	Water at the Potassium Channel Gate: Quantum Calculations Show an Oscillation in Water Structure
	Water Structure at the Potassium Channel Gate: The Importance of the Prolines in the PVPV Sequence, as Shown by Computation ...