BK potassium channel modulation by leucine-rich
Jiusheng Yan1,2and Richard W. Aldrich1
Section of Neurobiology, Center for Learning and Memory, University of Texas, Austin, TX 78712
Contributed by Richard W. Aldrich, April 2, 2012 (sent for review January 27, 2012)
Molecular diversity of ion channel structure and function underlies
variability in electrical signaling in nerve, muscle, and nonexcitable
cells. Regulation by variable auxiliary subunits is a major mecha-
nism to generate tissue- or cell-specific diversity of ion channel
function. Mammalian large-conductance, voltage- and calcium-ac-
tivated potassium channels (BK, KCa1.1) are ubiquitously expressed
the pore-forming, voltage- and Ca2+-sensing α-subunits (BKα), ei-
ther alone or together with the tissue-specific auxiliary β-subunits
(β1–β4). We recently identified a leucine-rich repeat (LRR)-contain-
ing membrane protein, LRRC26, as a BK channel auxiliary subunit,
which causes an unprecedented large negative shift (∼140 mV) in
voltage dependence of channel activation. Here we report a group
of LRRC26 paralogous proteins, LRRC52, LRRC55, and LRRC38 that
potentially function as LRRC26-type auxiliary subunits of BK chan-
nels. LRRC52, LRRC55, and LRRC38 produce a marked shift in the BK
channel’s voltage dependence of activation in the hyperpolarizing
direction by ∼100 mV, 50 mV, and 20 mV, respectively, in the ab-
sence of calcium. They along with LRRC26 show distinct expression
in different human tissues: LRRC26 and LRRC38 mainly in secretory
glands, LRRC52 in testis, and LRRC55 in brain. LRRC26 and its paral-
ogs are structurally and functionally distinct from the β-subunits
and we designate them as a γ family of the BK channel auxiliary
over a spectrum of different tissues or cell types.
accessory protein|patch clamp|signal peptide|cotranslational expression
in electrical properties among different cell types or states is
largely defined by expression and subunit composition of in-
dividual ion channel pore-forming principle subunits and often
tissue- or cell type-specific regulatory subunits (1, 2). The large
conductance, calcium- and voltage-activated potassium channel
(BK, also termed as BKCa, Maxi-K, KCa1.1, or Slo1) is a unique
by membrane depolarization and elevated intracellular free Ca2+
([Ca2+]i), playing a powerful integrative role in the regulation of
cellular excitability and calcium signaling (3). BK channels are
critically involved in diverse physiological processes such as neu-
ronal firing and neurotransmitter release (4), contractile tone of
smooth muscles (5, 6), frequency tuning of auditory hair cells (7),
and hormone secretion (8).
BK channels are homotetramers of the ubiquitously expressed,
pore-forming, Ca2+- and voltage-sensing α-subunits (BKα) either
alone or in association with tissue-specific regulatory β-subunits.
Thefourauxiliary β-subunits(β1–β4)areafamily of20-to30-kDa
expression patterns, e.g., β1 is mainly found in smooth muscle and
β4 in brain (9–11). We recently identified a leucine-rich repeat
(LRR)-containing membrane protein, LRRC26, as a BK channel
auxiliary subunit in lymph node carcinoma of the prostate
(LNCaP) cells (12). LRRC26 causes an unprecedented large
negative shift (∼ −140 mV) in voltage dependence of channel
activation, allowing activation near resting voltages and [Ca2+]iin
excitable or nonexcitable cells (12). LRRC26 is structurally and
on channels are membrane proteins responsible for electrical
signaling in nerve, muscle, and nonexcitable cells. The diversity
functionally distinct from the four β-subunits and thus represents
a different type of BK channel auxiliary subunit. Ion channel
auxiliary subunits, such as the BK or Kv channel β-subunits,
commonly exist in multiple paralogous forms with closely related
modulatory functions. Here we have comparatively examined the
modulatory effects and expression patterns of human LRRC26
and its paralogous proteins. Our results show that LRRC26 and
its three paralogs, LRRC52, LRRC55, and LRRC38, have dis-
tinct tissue-specific expression patterns and graded capabilities in
modulating the BK channel’s voltage dependence of activation.
We designate them as a γ family of the BK channel accessory
subunits, which potentially regulate the channel’s gating proper-
ties over a spectrum of different tissues or cell types.
A LRR domain comprises repeating 20–29 residue leucine-rich
sequence stretches, which contain a consensus sequence of
LxxLxLxxN (where x can be any amino acid), and often two cys-
teine-rich sequences of variable length that cap the N- and
C-terminalsides ofthetandem LRRrepeat units (13,14).Among
∼400 LRR-containing proteins in the human protein database
(UniProt), three LRRC26 paralogous proteins of unknown
function, LRRC38, LRRC52, and LRRC55 (Fig. 1) have a pro-
tein size of ∼35 kDa and a predicted extracellular LRR domain
structure and single transmembrane topology that are most
closely related to LRRC26. Their amino acid sequence similarity
of 30–40% is comparable to the sequence similarities among the
BK channel β-subunits. As discussed later, we designate them as
a group of BK channel γ-subunits. The amino acid sequences of
the structurally determinant residues of their LRR domains and
become moredivergent in thenon-LRR regions(Fig.1). TheirN-
terminal LRR domains comprise six LRR units in the middle and
two cysteine-rich regions called LRRNT and LRRCT flanking on
the N- and C-terminal sides, respectively.
LRRC26 and its paralogs all have a short N-terminal sequence
containing a hydrophobic segment (HS) preceding the LRR do-
main (Fig. 1). These N-terminal regions are predicted to be signal
peptides that are cleaved in the mature proteins and essential for
extracellular translocation of the LRR domain, as demonstrated
the N-terminal sequence can function as a cleavable signal pep-
tide, a FLAG epitope tag was introduced at the N- or C-terminal
side of the LRRC26’s predicted N-signal peptide sequence. Im-
munoblot with an anti-FLAG antibody was performed to de-
termine the retention or loss of the FLAG tag in the mature
proteins (Fig. 2A). We observed that an N-terminally tagged
Author contributions: J.Y. and R.W.A. designed research; J.Y. performed research; J.Y.
analyzed data; and J.Y. and R.W.A. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or raldrich@
2Present address: Department of Anesthesiology and Perioperative Medicine, University of
Texas MD Anderson Cancer Center, Houston, TX 77030.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| May 15, 2012
| vol. 109
| no. 20
FLAG epitope was absent, whereas a C-terminally tagged FLAG
behind the predicted cleavage site was retained in the mature
LRRC26 protein when heterologously expressed in HEK-293
cells. Disruption of the signal peptide function by deletion of the
hydrophobic segment (ΔHS) was able to block the cleavage and
retain the N-terminally tagged FLAG in the expressed protein
(Fig. 2A). Thus, the LRRC26’s N-terminal sequence fully func-
tions as an N-signal peptide that is cleaved in the mature protein.
Glycosylation is a commonly occurring posttranslational mod-
ification for extracellularly secreted proteins. LRRC26 and its
paralogs’ LRR domains all contain consensus N-glycosylation site
(s), Asn–Xaa–Ser/Thr, where Xaa is not a proline. LRRC26 is
predicted to be N-glycosylated at Asn147 in the middle of
the LRR domain. We observed that the transiently expressed
LRRC26 protein in HEK-293 cells can migrate as two molecular
mass bands, a dominant upper band and a minor or barely visible
lower band, in SDS/PAGE (Fig. 2B). Both the N147Q mutation
and enzymatic removal of the N-linked glycan by PNGase F
resulted in a disappearance of the upper glycosylated-mass band
(Fig. 2B). This result is consistent with an extracellular location of
the LRR domain in LRRC26.
An X-ray structure of LRRC26 or its paralogous proteins is not
available. However, their LRR units are mostly canonical LRR
and LRR C-terminal (LRRCT) regions’ cysteine-rich sequences
are similarly present in many other LRR-containing proteins.
Thus,theLRRdomain structuresofLRRC26and itsparalogs can
be modeled from known X-ray structures of other LRR-contain-
ing proteins, hagfish variable lymphocyte receptor B (16) for the
LRRNT and LRR regions and mouse TLR4 (17) for the LRRCT
region, on the basis of the amino acid sequence similarities in the
corresponding regions (Fig. S1). Given the difference in the as-
signment of the start residue of the first LRR unit, the LRRC26’s
UniProt database and our previous report (12) or a structurally
more appropriate separation into six similar LRR units (Figs. 1
and 2C).In the modeled structure of theLRRC26’s LRR domain,
the six stacked LRR units form a curved parallel β-sheet lining the
concave face and small helices/turns flanking the convex circum-
ference. The hydrophobic core of the LRR domain is tightly
packed by the parallel inward-pointing leucine residues, shielded
by the LRRCT and LRRNT caps on the N- and C-terminal ends.
LRRC26 and its paralogs all contain four pairs of fully conserved
cysteine residues in their LRRNT and LRRCT regions that po-
tentially form four disulfide linkages in the favorable oxidizing
extracellular environment (Figs. 1 and 2C).
We previously observed that the modulatory effect of LRRC26
on BK channels cannot be reliably or reproducibly measured in
HEK-293 cells when the cDNA constructs of LRRC26 and BKα
were simply mixed and cotransfected. This can be explained by
during DNA transfection or insufficient plasma membrane ex-
pression of the LRRC26 protein in a heterologous expression
system. We found that the LRRC26’s N-signal sequence can be
used as an internal cleavable signal peptide to allow efficient
cotranslational protein synthesis and assembly of the stoichio-
metrically complexed BKα/LRRC26 channels when LRRC26 was
segments and potential cleavage sites in the N-terminal signal peptide sequences are indicated. Potential N-glycosylation sites of Asn residues are shown in red.
Cysteine pairs (four in total) for potential disulfide formation are indicated. Key residues of the consensus sequence (LxxLxLxxN) in each LRR unit are marked by
a filled square at the bottom. The sequence similarities between LRRC26 and its paralogs are 38.2% (LRRC38), 33.4% (LRRC52), and 37.4% (LRRC55).
Protein sequence alignment of human LRRC26 and its paralogs. Conserved residues are shaded at three levels (100, 80, and 50%). The hydrophobic
age of an N-terminally tagged FLAG epitope in mature protein as detected
by immunoblots. (B) LRRC26 is glycosylated at an Asn147 site as detected by
a loss of the PNGase treatment-induced mobility shift in N147Q mutant. (C)
LRRC26’s predicted membrane topology and LRR domain structure on the
endoplasmic reticulum (ER) membrane. The LRR domain is N-glycosylated in
the lumen of the ER and then exported to the extracellular side of plasma
membrane. The sulfur atoms of the four cysteine pairs for potential disulfide
formations are shown as bonded yellow balls. The nitrogen atom of Asn147
for N-glycosylation linkage is shown by a green sphere.
Structural features of LRRC26. (A) Signal-peptide dependent cleav-
| www.pnas.org/cgi/doi/10.1073/pnas.1205435109Yan and Aldrich
fused to the C terminus of BKα through its N terminus (12). Such
an internal cleavage event was significantly blocked when the
function of the signal peptide was disrupted by deletion of its
hydrophobic segment (ΔHS) in the fusion construct of BKα and
LRRC26. Consequently, the fused LRRC26 mutant (ΔHS) was
unable to modulate BK channels presumably due to improper
membrane topology (12). The signal peptide region, particularly
its hydrophobic segment, in the precursor BKα–LRRC26 fusion
protein is predicted to be cleaved from the mature BKα and
LRRC26 proteins by signal peptidase and thus exerts minimal
additional effect on the mature BK channel’s gating properties.
With this cotranslational expression method, we were able to
obtain reproducible measurement of the electrophysiological
properties of the LRRC26-complexed BK channels in HEK-293
cells (12). We found that the N-signal sequences of LRRC38,
LRRC52, and LRRC55 can similarly function as fully cleavable
internal signal peptides. Fusion constructs of BKα and LRRC26
paralogs expressed mature BKα and LRR proteins in similar
molecular mass sizes as if they were individually expressed (Fig.
S2). The BKα–LRRC55 fusion construct expressed a very low
level of mature BKα protein, which generated a low level of BK
channel current in some transfected cells but was not detectable
by Western blot with anti-BKα antibody in our tested condition.
Because the mature LRRC55 protein was still well expressed, this
low level of BKα expression is likely due to degradation, resulting
from C-terminal tagging of the LRRC55’s long N-terminal frag-
ment after signal peptidase cleavage. With these fusion/cotrans-
lational expression constructs, we were able to measure repro-
ducibly the modulatory effects of LRRC38, LRRC52, and
LRRC55 on the BK channel’s gating properties in HEK-293 cells
by patch-clamp recording.
To evaluate whether they function as BK channel regulatory
BKα in HEK-293 cells using the above cotranslational expression
method. We previously reported that LRRC26 modified the BK
channel’s voltage dependence of activation by an ∼ −140 mV shift
of the V1/2(voltage of half maximal activation). We found that
LRRC52, LRRC55, and LRRC38 are also able to modify the BK
channel’s voltage dependence of activation toward more negative
voltages, but to different extents (Table S1). In the virtual absence
of [Ca2+]i, LRRC52 and LRRC55 caused a large negative shift of
the V1/2by −101 ± 4 mV and −51 ± 2 mV, respectively, and
LRRC38 produced a smaller but reproducible shift of V1/2by
that the V1/2shifts caused by LRRC52 and LRRC55 in the pres-
ence of different concentrations of [Ca2+]i(0.76–26 μM) are on
average ∼36 mV smaller than in the absence of [Ca2+]i(Fig. 3 A–
C), suggesting a modification in the apparent calciumsensitivity of
BK channels by these two regulatory LRR proteins. According to
the commonly used voltage and calcium-dependent allosteric
gating model of BK channel activation, the apparent calcium
sensitivity, which is defined as the slope in the V1/2vs. log [Ca2+]i
sensitivity (binding or allosteric coupling) or a modification in the
voltage-related gating parameters (18, 19). Because the latter may
result in a change in the shape of the normalized conductance vs.
to estimate whether the BK channel’s microscopic calcium sensi-
tivity is affected by these LRRC26 paralogs. Both V1/2and Qapp
(apparent gating charge) are determined from a single Boltzmann
function fit of the G–V curve (Table S1). A nonlinear relationship
between the QappV1/2vs. log [Ca2+]iwas observed when all [Ca2+]i
conditions are included for plotting. As shown in Fig. 3D,
LRRC26’s three paralogs all cause slight changes in the relation-
ship between QappV1/2and log [Ca2+]i, e.g., a slope change of
∼20% by LRRC52 and LRRC38 and of ∼40% by LRRC55 from
0.76 to 4.2 μM [Ca2+]i, suggesting some effects on the microscopic
calcium sensitivity of BK channel activation. However, further
detailed investigation in the context of the allosteric gating
mechanism of BK channel activation by voltage and Ca2+will be
needed to separate the effects on Ca2+- and voltage-activation
pathways.Consistent with thedifferent capabilitiesin inducing G–
V shifts, LRRC26 and its paralogs modify the BK channel’s K+
current kinetics to different extents (Fig. S3). LRRC26 and its
three paralogs all greatly increase the activation rates of BK
channel currents. Additionally, LRRC26, LRRC52, and LRRC55
(at positive voltages) significantly decelerate channel deactiva-
tion, whereas a slight acceleration effect was also observed for
LRRC38 and LRRC55 (at negative voltages).
2+] μ M[Ca
2+] μ M
80120 160 200 240
+ LRRC26 (γ1)
+ LRRC52 (γ2)
+ LRRC55 (γ3)
+ LRRC38 (γ4)
-120 -80 -40040 80 120 160 200
(normalized at 0 Ca2+)
1.9 μM [Ca2+]i
BKα alone or the BKα together with LRRC26, LRRC52, LRRC55, or LRRC38 in the virtual absence of [Ca2+]i(A) or in the presence of 1.9 μM [Ca2+]i(B). (C) Plot of
V1/2vs. [Ca2+]i(log scale after break). (D) Plot of QappV1/2vs. [Ca2+]inormalized at 0 [Ca2+]i.
Modulatory effects of LRRC26 and its paralogs on BK channels in HEK-293 cells. (A and B) Voltage dependence of the BK channel activation for the
Yan and AldrichPNAS
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From the above results, we consider LRRC52, LRRC55, and
LRRC38, together with previously reported LRRC26, a family of
in shifting the BK channel’s voltage dependence of activation to-
ward more negative voltages. Auxiliary subunits of ion channels, e.
g., the BK channel β-subunits, commonly have tissue-specific ex-
pression patterns that fit different functional requirements of dif-
ferent cell types. To help estimate their physiological relevance in
BK channel regulation, we quantitatively determined the expres-
sion profiles of LRRC26 and its three paralogs at the mRNA level
in 20 different human tissues using the quantitative real-time PCR
(qPCR) method. We observed that LRRC26 is highly expressed in
normal human tissues of salivary gland, prostate, and trachea, and
a moderate level of expression was found in thyroid gland, thymus,
colon, aorta, and fetal brain (Fig. 4B). A low level of LRRC26
expression was also detected in lung, testis, adult brain, and cere-
bellum. LRRC52 is dominantly expressed in testis and skeletal
muscle. A much lower level of LRRC52 expression was also
detected in several other tissues including placenta, kidney, lung,
and some glands.LRRC55 expression appears to be specific to the
nervous system,which is most abundantinfetal brain and secondly
in adult brain. LRRC38 is mainly expressed in skeletal muscle,
adrenal gland and thymus, and a low level expression was also
detected in brain and cerebellum.
Among known accessory subunits of the voltage-gated ion
channels, LRRC26 has a characteristic LRR domain structure
and exerts a profound influence on voltage dependence of
a voltage-gated ion channel. The LRR domain in many proteins
is known to provide a structural framework for protein–protein
interactions, typically through the concave β-sheet side (13).
LRRC26 and its three paralogs, LRRC52, LRRC55, and
LRRC38 thus represent a unique type of ion channel auxiliary
protein family. These proteins cause marked and graded modu-
lation in the BK channels’ voltage dependence of activation and
exhibit tissue-specific expression patterns. They are distinct from
the four BK channel β-subunits in both structures and modula-
tory effects. For convenience, we designate them as a family of
BK channel γ-subunits, e.g., LRRC26 as γ1, LRRC52 as γ2,
LRRC55 as γ3, and LRRC38 as γ4, in the order of magnitude of
their modulatory effects on BK channel gating.
LRRC26 and its three paralogs belong to a previously grouped
“Elron” subfamily of the “extracelluar LRR”-containing proteins,
which also contain two additional proteins, LRTM1 and LRTM2
considered to be “LRR_only” (absence of other classified protein
domain) proteins of low stringency (20), which are mainly present
in mammals and defined as paralogs in the Ensembl database.
However, LRTM1 and LRTM2 contain an additional extracellu-
lar LRR-unrelated segment of ∼50 residues located between the
LRRCT and the transmembrane (TM) regions, which is fully ab-
sent in LRRC26, LRRC38, LRRC52,and LRRC55(Fig. S1).Our
other four members, have no significant modulatory effect on the
BK channel’s gating properties when they were coexpressed with
BKα in HEK-293 cells (Fig. S4). LRTM1 and LRTM2 are hereby
tentatively not considered as functional paralogs of LRRC26 in
BK channel modulation.
The γ-subunits are similarly related to each other in overall
protein sequence. However, they display very different capa-
bilities in shifting the BK channel’s V1/2in the absence of [Ca2+]i
by ∼ −140 mV (γ1 or LRRC26), −100 mV (γ2 or LRRC52),
−50 mV (γ3 or LRRC55), and −20 mV (γ4 or LRRC38). Our
Relative mRNA abundance
Relative mRNA abundance
Relative mRNA abundance
Relative mRNA abundance
LRRC26 (γ1)LRRC52 (γ2)
LRRC55 (γ3)LRRC38 (γ4)
detected by quantitative TaqMan real-time PCR. RPLPO (large ribosomal protein) was used as a reference gene for internal control. The relative mRNA
abundance is plotted in log scale after break.
Relative expression levels of LRRC26 and its paralogs in different human tissues. The relative expression level of each individual LRR protein was
| www.pnas.org/cgi/doi/10.1073/pnas.1205435109 Yan and Aldrich
previous mutagenesis studies indicated that most parts of the γ1
protein are necessary for its modulatory function, suggesting that
γ1 might work as a whole in BK channel modulation (12). Thus,
it is difficult to predict from the protein sequences how γ1 and its
three paralogs are different in BK channel modulation. The
observed smaller shifts in voltage dependence of BK channel
activation caused by γ2 (LRRC52) and γ4 (LRRC38) than γ1
(LRRC26) might be partly explained by their lower expression
level relative to the BKα protein in the cotranslational/fusion
expression condition, which can be caused by degradation and is
expected to result in variability in the bound γ-subunits per BK
channel complex and consequently decreased apparent gating
charges or slopes of the observed G–V curves. However, γ2 is
notably different from γ1 in its effects on the rates of BK channel
deactivation at very negative voltages, e.g., −120 mV (Fig. S3).
Additionally, a similar extent of BK channel modulation was
observed when γ-subunits were coexpressed with BKα separately
(non-cotranslationally) in HEK-293 cells (Fig. S5). It remains to
be determined whether the difference in glycosylation status
among these LRR proteins contributes to their difference in BK
The γ-subunitsare distinct from the four BK channel β-subunits
in their modulatory effects. The β-subunits drastically decrease
both the activation and deactivation rates of the BK channel
in the absence of [Ca2+]i, although β1 can exceptionally cause an
μM) conditions through an enhanced calcium sensitivity (10, 11,
21, 22). The γ-subunits rather markedly shift the BK channel’s
voltage dependenceofchannel activationin thenegative direction
all contain a short stretch of acidic residues,282EEDEDE287(γ2
or LRRC52),307EEEE310(γ3 or LRRC55), and286EDEDED291
(γ4 or LRRC38), in their intracellular C terminus (Fig. 1), re-
sembling the “Ca2+-sensing” acidic clusters found in the BKα
These acidic residue clusters might potentially affect the Ca2+
sensitivity of channel activation through a direct Ca2+association
or by interactions with the BKα’s Ca2+-docking sites.
can functionally compete with γ1 (LRRC26), suggesting some
commonly shared mechanism in physical association or modula-
tory action between these two distinct types of BK channel reg-
ulatory proteins (12). Like the β-subunits, the TM domain in γ1 is
essential for its modulatory function and physical association with
the BKα (12). It remains unclear whether the β- and γ-subunits’
the BK channel complex.
Similar to tissue-specific expression of the four BK channel
β-subunits (10), the four γ-subunits show tissue-specific expression
patterns in the 20 different human normal tissues tested. Consis-
tent with its abundant expression in human salivary gland as
detected in this study, γ1 (LRRC26) in mouse parotid acinar cells
potent activator mallotoxin (25). In secretory glands or epithelial
cells, γ1 activates BK channels and thus facilitates the channel’s
function in fluid and electrolyte secretion. γ1 may additionally
account for the low-voltage–activated BK channels observed in
breast cancer cells (26), inner ear hair cells (27), and arterial
smooth muscle cells (28–31). LRRC26 was recently reported to
suppress tumor growth and metastasis (32), agreeing with its high
expression in the early stage prostate cancer LNCaP cells and di-
minished expression in the very late stage prostate cancer PC-3
cells (12, 33). γ2 (LRRC52) predominantly expresses in testis and
skeletal muscle. Similar to the observed modulatory effect of the
BK channel β-subunits on the closely related Slo3 channels, a re-
cent study indicates that γ2 (LRRC52) is also a candidate regu-
latory subunit of the Slo3 channels in sperm cells (34). Expression
of BKα is highest in the premeiotic germ cells but lowest in the
postmeiotic germ cells (35). γ2 (LRRC52) in germ cells may
modulate both BK and Slo3 channels in a cell-stage–dependent
manner. Among all tested human tissues, γ3 (LRRC55) was most
strongly expressed in the brain. According to the γ3 gene expres-
sion map obtained with EGFP BAC transgenic mice, GFP protein
under the control of endogenous γ3 transcriptional regulatory
elements waswellexpressedin many differentregions ortissues of
fetal and adult mouse brains, including the mitral cell layer of
olfactory bulb, medial habenular nuclei of thalamus, ventral teg-
mental area (VTA), substantia nigra, and cortex (http://www.
gensat.org). The specific expression of γ3 in the nervous system
may provide a mechanism of BK channel activation that is more
responsive to membrane depolarization preceding [Ca2+]ieleva-
tion. The greatly diminished activating effect of γ3 upon [Ca2+]i
increase may prevent BK channels from being overactive during
an action potential in excitable cells.
Materials and Methods
Expression of BKα and LRRC26 or Its Paralogs in HEK-293 Cells. Recombinant
cDNA constructs of human BKα and LRRC26 and its paralogs were used
for heterologous expression in HEK-293 cells. Cloned or synthetic cDNA
sequences of human LRRC26, LRRC38, LRRC52, and LRRC55 were subcloned
into the mammalian expression vector of pCDNA6, with FLAG and V5 tags
attached at their C termini. Fusion cDNA constructs, which encode precursor
fusion proteins of human BKα and C-terminally tagged LRR proteins, were
generated with pCDNA6 vector and used to facilitate cotranslational as-
sembly of the BKα/LRR protein complexes after endogenous cleavage by
peptidases at the linker (signal peptide) region in the mature proteins. HEK-
293 cells were obtained from ATCC and transfected with the designed
plasmid(s) using Lipofectamine 2000 (Invitrogen), and used within 16–72 h
for electrophysiological assays or at ∼48 h for biochemical assays.
Structural Modeling. A structural model of the LRRC26’s LRR domain was built
by homology modeling with SWISS MODEL and SWISS-pdb viewer (36). The
crystal structure of hagfish variable lymphocyte receptor B (16) [Protein Data
Bank (PDB) ID code 2O6S; amino acids 24–187] was used as a template for
the homology modeling of the LRRNT and the six LRR units of LRRC26. The
structure of the LRRC26’s LRRCT region was modeled with the crystal
structure of mouse TLR4 (17) (PDB ID code 2Z64; amino acids 583–625).
Electrophysiology. The BK channel’s gating properties including voltage and
Ca2+dependence, and K+current kinetics were determined by patch-clamp
recording in excised inside-out patches of HEK-293 cells with symmetric
solutions of 136 mM KMeSO3, 4 mM KCl, and 20 mM Hepes (pH 7.20)
supplemented with 2 mM MgCl2for external solution and a certain amount
of CaCl2buffered by 5 mM HEDTA or nitrilotriacetic acid for internal solu-
tion. The free Ca2+concentration in the internal solution ([Ca2+]i) was
measured with a Ca2+-sensitive electrode (Orion Research). The steady-state
activation was expressed as G/Gmaxcalculated from the relative amplitude
of the tail currents (deactivation at −120 mV). The voltage of half-maximal
activation (V1/2) and the equivalent gating charge (z) were obtained by
fitting the relations of G/Gmaxvs. voltage with single Boltzmann function G/
Gmax= 1/(1 + e-zF(V-V1/2)/RT). SEM was used to plot error bars for variation in
Quantitative Expression Analyses. Total RNA samples of 20 different human
tissues (Clontech, human total RNA master panel) were used for expression
analyses of LRRC26 and its three paralogs by quantitative real-time PCR. The
fist-strand cDNA was synthesized from a template of total RNA using reverse
transcriptase with primer of oligo(dT). TaqMan real-time PCR was performed
to quantitate the amount of synthesized cDNA of a target gene. LRRC26 and
its paralogs are all encoded by two exons. To ensure specificity, the probes
were designed to encompass the connecting sites of the two exons. The
following forward and reverse primers and probes were used: LRRC26,
5′-CGCGTCAGAGGCCGAG-3′, 5′-TGGCTAAAGGCGGCGTC-3′, and 5′-6FAM-AC-
GCCTGACGCTCAGCCCCC-TAM-3′; LRRC52, 5′-TCCTGGACTTCGCCATCTTC-3′,
5′-TCAGCTCTGTGGGCTCCAC-3′, and 5′-6FAM-CATATGGACCCCTCAGATGA-
TCTAAATGCC-TAM-3′; LRRC55, 5′-TGGCAATCCCTGGGTGTG-3′, 5′-AGCCAGC-
Yan and AldrichPNAS
| May 15, 2012
| vol. 109
| no. 20
TGAGAATCTGCTGTAC-3′, and 5′-6FAM-CTGCTGAAGTGGCTGCGAAACCG-T- Download full-text
AM-3′; LRRC38, 5′-TGGATCCAGGAGAACGCATC-3′, 5′-TATCCTCCTGCTCTCC-
ATGGG-3′, and 5′-6FAM-AAGGCCTTGATGAAATCCAGTGCTCCC-TAM-3′. The
efficiency of target amplification with the designed primers and probes
were validated with templates of serially diluted plasmid DNA. Human
RPLPO (large ribosomal protein) endogenous control (primer and FAM/MGB
probe; Invitrogen) was used as an internal control for comparision among
different RNA samples.
ACKNOWLEDGMENTS. We thank Chris Lingle and Chengtao Yang for
discussion. Work was supported in part by National Institutes of Health
Grant NS075118 (to J.Y.).
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| www.pnas.org/cgi/doi/10.1073/pnas.1205435109 Yan and Aldrich