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Tethered protein display identifies a novel Kir3.2 (GIRK2) regulator from protein scaffold libraries.
Bagriantsev SN
,
Chatelain FC
,
Clark KA
,
Alagem N
,
Reuveny E
,
Minor DL
.
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Use of randomized peptide libraries to evolve molecules with new functions provides a means for developing novel regulators of protein activity. Despite the demonstrated power of such approaches for soluble targets, application of this strategy to membrane systems, such as ion channels, remains challenging. Here, we have combined libraries of a tethered protein scaffold with functional selection in yeast to develop a novel activator of the G-protein-coupled mammalian inwardly rectifying potassium channel Kir3.2 (GIRK2). We show that the novel regulator, denoted N5, increases Kir3.2 (GIRK2) basal activity by inhibiting clearance of the channel from the cellular surface rather than affecting the core biophysical properties of the channel. These studies establish the tethered protein display strategy as a means to create new channel modulators and highlight the power of approaches that couple randomized libraries with direct selections for functional effects. Our results further underscore the possibility for the development of modulators that influence channel function by altering cell surface expression densities rather than by direct action on channel biophysical parameters. The use of tethered library selection strategies coupled with functional selection bypasses the need for a purified target and is likely to be applicable to a range of membrane protein systems.
Figure 1. (A) Schematic showing the library display
strategy used for identifying
Kir3.2 activators. (left) Representations of the random peptide, random
peptide (green) constrained by pZ (ribbons), and a pZ surface display
library (magenta positions on blue ribbon diagram) were linked to
the N- or C-terminal cytoplasmic ends of Kir3.2. (right) Cartoon showing
a single Kir3.2 subunit bearing a representative of the N- or C-terminal
pZ surface library. (B) Potassium transport complementation assay
shows the ability of N5-Kir3.2 to restore growth of the trk1Δtrk2Δ yeast on limiting potassium conditions
(1 mM KCl). (C) Sequence alignment of pZ and N5. pZ helical segments
are shown in large type. Positions randomized in the pZ surface library
and the N5 residues at the randomized positions are shown in blue
and orange for Helix 1 and Helix 2, respectively. (D) Exemplar two-electrode
voltage clamp recordings from Xenopus oocytes injected
with 3 ng of cRNA for Kir3.2, N5-Kir3.2, and pZ-Kir3.2. Lower right
panel shows voltage protocol used for recordings. Currents were evoked
by 1 s long step protocol from −110 to 40 mV, in 10 mV increments
from a holding potential of 0 mV in 90K. (E) Exemplar current–voltage
plots for the indicated constructs recorded in 90K with or without
5 mM BaCl2. (F) Quantification the effects of pZ and N5
on activity of Kir3.2, Kir3.1, Kv7.2, and TRPM8 currents. Kir3.2 were
evoked as in (D) and measured at −80 mV. Kv7.2 currents were
evoked by 2.5 s long step protocol from −110 to 40 mV in 10
mV increments from a holding potential of −80 mV and measured
at +30 mV at 2.4 s. TRPM8 currents were evoked in the presence of
250 μM menthol by a 900 ms long ramp from −110 to 40
mV, from a holding potential of −60 mV, and measured at 30
mV. Data shown as mean ± standard error of the mean (SEM) n ≥ 6). “nd” indicates not determined.
Figure 2. Exemplar recordings
(A) and quantification (B) of the effect of
3 μM carbachol on N5-Kir3.2 activity in Xenopus oocytes coinjected with 5 ng of mAChR. Data in (B) are mean ±
SD (n ≥ 6). (C) Quantification of surface
expression of HA-tagged Kir3.2 and N5-Kir3.2 as a function of injected
cRNA. Data are mean ± SEM (n ≥ 6). Inset
depicts cartoons of a single subunit of the HA-tagged Kir3.2 and N5-Kir3.2
constructs. (D) Immunoblot analysis of total lysates from oocytes
injected with different amounts of cRNA. (E) Quantification of surface
expression of N5-Kir3.2-HA in the presence of 15 μM brefeldin
A (BFA). Surface fluorescence (SF) measurements (mean ± SEM, n ≥ 6) were normalized to the initial fluorescence
values and fitted to the single exponential decay equation: SF = (1
– SFplateau) exp(−Kt) + SFplateau, where t is the time in
h, SFplateau is the minimal fluorescence, and K is the decay constant.
Figure 3. (A) Coomassie-stained SDS-PAGE of purified recombinant
pZ and N5.
(B) Comparison of pZ and N5 CD spectra at 4 °C. (C) Thermal denaturation
of pZ and N5, monitored by CD at 222 nm. CD data were measured in
a buffer of 150 mM KCl, 4 mM β-mercaptoethanol, 10 mM phosphate,
pH 7.4.
Figure 4. Quantification of activity
(A) , and surface expression (B) of
N5-Kir3.2 mutants measured in Xenopus oocytes injected
with 3 ng of cRNA of each construct. Currents were evoked by a 1 s
long step protocol from −110 to 40 mV, in 10 mV increments,
from a holding potential of 0 mV in 90K. Surface expression was measured
by labeling of the surface of the oocytes with an α-HA antibody.
Helix 1 and Helix 2 are indicated by blue and orange, respectively.
Data are mean ± SEM (n ≥ 6). (C) Immunoblot
analysis of total lysates from oocytes injected with 3 ng of cRNA
of each of the indicated constructs.
Figure 5. (A) Sequence alignment of wild-type and mutant N5 proteins.
Quantification
of activity (B) and surface expression (C) of indicated N5-Kir3.2
and mutants measured in Xenopus oocytes injected
with 3 ng of cRNA of each construct. Currents were evoked by a 1 s
long step protocol from −110 to 40 mV, in 10 mV increments,
from a holding potential of 0 mV in 90K. Surface expression was measured
by labeling of the surface of the oocytes with an α-HA antibody.
Data are shown as mean ± SEM (n ≥ 6).
(D) Immunoblot analysis of total lysates from oocytes injected with
3 ng of cRNA for the indicated constructs.
Figure 6. (A) Exemplar
current–voltage plots recorded in oocytes injected
with 3 ng of cRNA for the indicated constructs. Currents were evoked
by a 1 s long step protocol from −110 to 40 mV, in 10 mV increments,
from a holding potential of 0 mV in 90K. (B) Quantification of activity
and surface expression of N5mut-Kir3.2-HA channels in the
oocytes injected with 3 ng of cRNA for each construct. Surface expression
was measured by labeling of the surface of the oocytes with an α-HA
antibody. Data are mean ± SEM (n ≥ 6)
normalized to N5-Kir3.2-HA. (C) Immunoblot analysis of total lysates
from oocytes injected with 3 ng of cRNA for the indicated constructs.
Figure 7. (A) Exemplar
two-electrode voltage clamp recordings from Xenopus oocytes injected with 3 ng of cRNA for HA-tagged
Kir3.2 having N5 or pZ attached to the C-terminus (Kir3.2-HA-N5 and
Kir3.2-HA-pZ, respectively). Currents were evoked by a 1 s long step
protocol from −100 to 50 mV, in 10 mV increments from a holding
potential of 0 mV in 90K. (B) Current–voltage plots recorded
in 90K for Kir3.2-HA-pZ and for Kir3.2-HA-N5 with or without the addition
of 5 mM BaCl2. (C) Quantification of activity and (D) surface
expression of indicated Kir3.2-HA constructs measured in Xenopus oocytes injected with 3 ng of cRNA of each construct. Surface expression
was measured by labeling of the surface of the oocytes with an anti-HA
antibody. Data from at least two experiments were normalized to N5-Kir3.2-HA
and shown as mean ± SEM (n = 8–25). (E)
Immunoblot analysis of total lysates from oocytes injected with 3
ng of cRNA for the indicated constructs.
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