1 Center for Oral Biology, Aab Institute of Biomedical Sciences, and 2 Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642
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ABSTRACT |
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We used molecular biological and
patch-clamp techniques to identify the Ca2+-activated
K+ channel genes in mouse parotid acinar cells. Two types
of K+ channels were activated by intracellular
Ca2+ with single-channel conductance values of 22 and 140 pS (in 135 mM external K+), consistent with the
intermediate and maxi-K classes of Ca2+-activated
K+ channels, typified by the mIK1 (Kcnn4) and
mSlo (Kcnma1) genes, respectively. The presence of mIK1 mRNA
was established in acinar cells by in situ hybridization. The
electrophysiological and pharmacological properties of heterologously
expressed mIK1 channels matched those of the native current; thus the
native, smaller conductance channel is likely derived from the mIK1
gene. We found that parotid acinar cells express a single, uncommon
splice variant of the mSlo gene and that heterologously expressed
channels of this Slo variant had a single-channel conductance
indistinguishable from that of the native, large-conductance channel.
However, the sensitivity of this expressed Slo variant to the scorpion
toxin iberiotoxin was considerably different from that of the native
current. RT-PCR analysis revealed the presence of two mSlo -subunits
(Kcnmb1 and Kcnmb4) in parotid tissue. Comparison
of the iberiotoxin sensitivity of the native current with that of
parotid mSlo expressed with each
-subunit in isolation and
measurements of the iberiotoxin sensitivity of currents in cells from
1 knockout mice suggest that parotid acinar cells
contain approximately equal numbers of homotetrameric channel proteins
from the parotid variant of the Slo gene and heteromeric proteins
composed of the parotid Slo variant in combination with the
4-subunit.
secretory cells; fluid secretion; single channels; patch clamp
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INTRODUCTION |
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SALIVARY GLAND
SECRETION plays an extremely important role in maintaining the
health of oral tissues. The fluid secretion hydrates the oral cavity,
aiding in the mastication and swallowing of food. In addition, these
secretions neutralize acids and protect against the invasion of
potential pathogens. An increase in intracellular Ca2+,
usually associated with muscarinic receptor stimulation, triggers fluid
secretion by simultaneously activating apical Cl channels
and basolateral K+ channels. Indeed, nonselective
K+ channel blockers inhibit fluid secretion and
Cl
efflux (19, 21). The efflux of
Cl
and K+ across the apical and basolateral
membranes, respectively, produces a transepithelial potential
difference that draws Na+ through tight junctions into the
lumen. The resulting transepithelial osmotic gradient drives the
movement of water, creating a plasma-like primary secretion.
The presence of several types of Ca2+-activated cation channels in salivary acinar cells has been reported. These include a K+ channel with a large single-channel conductance near 160 pS in both rodents (22) and humans (23, 29), a 15-pS channel permeable to Na+ (22) in humans, a 30-pS Na+-permeable channel in rodents (22), a 30-pS K+-selective channel in humans, and a 40-pS K+ channel in mice (14). The variety of channel types in different tissues and species and the lack of any specific information on the genes that code for these channels complicate efforts to understand the roles of these various channels in fluid secretion.
As a first step toward an understanding of the role of K+
channels in salivary function, we designed an approach to determine the
molecular identities of the Ca2+-activated K+
channels in the mouse model system. Patch-clamp analysis of currents from mouse parotid acinar cells revealed two levels of single-channel current that were activated by increases in intracellular
Ca2+. On the basis of the expression profile of channel
mRNA in parotid acinar cells, in combination with the electrical and
pharmacological properties of native and recombinantly expressed
channels, the native, small-conductance channel is likely derived from
the mIK1 gene. The larger conductance channel likely reflects
expression of a single "insertless" splice variant of the mSlo
gene. Northern blot analysis revealed the expression of two
mSlo -subunits in mouse parotid tissue. We compared the iberiotoxin
sensitivity of the channels in native tissue with heterologously
expressed channels containing each
-subunit. These results and the
toxin sensitivity of channels from parotid acinar cells of a
1 knockout mouse suggest that ~50% of the native
maxi-K channels are homotetramers of the parotid mSlo splice variant
and that the remaining large-conductance channels are heteromeric
proteins from this mSlo variant complexed with the
4-subunit.
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MATERIALS AND METHODS |
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Western analysis.
Mouse brain and parotid homogenates were used to identify native maxi-K
channel protein. Tissues were prepared as previously described
(34). After dissection, the tissues were homogenized twice
by 10-s strokes at power level 5 with a Polytron homogenizer (Brinkmann
Instruments, Westbury, NY) in 5 ml of homogenization solution (10 mM
HEPES adjusted to pH 7.4 with Tris, 10% sucrose, 1 mM EDTA, and 1 mM
PMSF) per gram of tissue, with 1 tablet of Complete protease inhibitor
(Roche Applied Science, Indianapolis, IN) per 50 ml.
Homogenates were centrifuged at 2,500 g for 15 min at 4°C,
and the supernatants were saved. The pellets were resuspended in 5 ml
of homogenization buffer per gram of starting tissue and then
homogenized and recentrifuged as described above. The supernatants were
combined, and crude membrane proteins were precipitated by
centrifugation at 22,000 g for 20 min at 4°C. The
supernatants from this step were discarded, and the pellets were
resuspended in PBS containing 1 mM EDTA, 1 mM PMSF, and Complete protease inhibitor (1 tablet per 50 ml) and then passed once through a
25-gauge needle and once through a 30-gauge needle. Aliquots were
quickly frozen in liquid N2 and stored at 85°C until use.
Northern blot analysis, in situ hybridization, and RT-PCR.
Total RNA was prepared using Trizol reagent (Life Technologies, Grand
Island, NY) according to the manufacturer's instructions. mRNA was
further purified by chromatography on oligo(dT) resin. The RNA was
fractionated by electrophoresis in a formaldehyde-agarose gel and
transferred to Hybond-XL nylon membrane (Amersham). The blot was
hybridized with a 32P-labeled double-stranded probe
generated from nucleotides 96-519 of the mIK1 cDNA, nucleotides
1347-3591 of the mSlo cDNA, or the entire coding region of the
1 or
4 cDNAs in ExpressHyb solution (Clontech, Palo Alto, CA) as recommended by the manufacturer.
Cloning strategies. Parotid mSlo splice-site variation was analyzed by sequencing RT-PCR products amplified from mouse parotid gland first-strand cDNA. For clarity, all nucleotide designations refer back to the first published mSlo coding sequence (accession no. L16912; Ref. 8). Our analysis proceeded in three steps. First, we sequenced more than 20 clones from the COOH-terminal coding region containing nucleotides 1775-3591, where much of the splice-site variation occurs. Next, we used 5'-RACE (rapid amplification of cDNA ends) to determine the start codon, again sequencing multiple clones. Finally, we amplified the full-length open reading frame and examined the sequences coding for the transmembrane domains. This analysis suggested the presence of a single parotid mSlo variant (which we have called "parSlo" to unambiguously distinguish this from the common mSlo splice variant) that had no insertions at any of the characterized mSlo splice sites 1-6 (accession no. AF465344). Compared with mSlo, parSlo lacks 1) the first 81 bases, which results in the use of the "M1" alternative start codon; 2) nucleotides 1979-1987 at splice site 4, located between the eighth and ninth putative membrane-spanning segments; and 3) nucleotides 3408-3436 at splice site 6, which shifts the reading frame, resulting in the use of a cryptic stop codon and the formation of a "short" protein.
To clone parSlo for expression studies, we used PCR to amplify a region spanning nucleotides 776-3591 by using a NotI-tagged 3' primer and a mouse parotid cDNA template. An SphI-NotI fragment containing nucleotides 1347-3591 was then digested from the PCR product and cloned into the complementary sites in a vector containing an mSlo variant that is identical to parSlo from the start codon to nucleotide 1347 (8). The entire coding sequence was then digested from the resulting vector and subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) using KpnI and XbaI. TheCell transfection.
Chinese hamster ovary (CHO) cells and TSA cells [human embryonic
kidney (HEK)-293 cells stably transfected with the large T antigen,
provided by Ronald Li and Eduardo Marban, Johns Hopkins University,
Baltimore, MD] were cultured in DMEM plus 10% newborn calf serum and
antibiotics in a 5% CO2 incubator at 37°C. Before transfection, cells were plated onto 100-mm culture dishes and grown to
~70% confluency and then transfected with a total of 10 µg of DNA
per dish by using Superfect transfection reagent (Qiagen) following the
manufacturer's recommended protocol. The mIK construct was subcloned
into the pIRES2-EGFP vector, which allowed identification of
transfected cells with fluorescence microscopy. The parotid mSlo
variant (in pcDNA3.1) was expressed in a 1:1 ratio with the pIRES2-EGFP
vector. The 1- or
4-subunits (in
pIRES2-EGFP) were cotransfected with parSlo (pcDNA3.1) in a 4:1 ratio.
Electrophysiological methods.
Single acinar cells were dissociated from mouse parotid glands with a
modification of the methods previously used for rats (1).
Briefly, glands were dissected from exsanguinated mice after
CO2 anesthesia. Unless otherwise indicated, the data
presented here are from BlackSwiss × 129/SvJ hybrid mice. In
addition, we also used 1 knockout mice (7),
kindly provided by R. Brenner and R. W. Aldrich (Department of
Molecular and Cellular Physiology, Stanford University School of
Medicine, Stanford, CA), and mice (C57BL/6J) that are the
wild-type "controls" for the
1 knockout animals.
Glands were minced in Ca2+-free minimum essential medium
(MEM; GIBCO BRL, Gaithersburg, MD) supplemented with 1% BSA (fraction
V; Sigma Chemical, St. Louis, MO). The tissue was treated for 20 min
(37°C) with a 0.02% trypsin solution (MEM-Ca2+ free + 1 mM EDTA + 2 mM glutamine + 1% BSA). Digestion was
stopped with 2 mg/ml soybean trypsin inhibitor (Sigma Chemical), and
the tissue was further dispersed by two sequential 60-min treatments with collagenase (100 U/ml type CLSPA; Worthington Biochemical, Freehold, NJ) in MEM-Ca2+ free plus 2 mM glutamine plus 1%
BSA. The dispersed cells were centrifuged and washed with basal medium
Eagle (BME) (GIBCO)/BSA free. The final pellet was resuspended in
BME/BSA free plus 2 mM glutamine, and cells were plated onto
poly-L-lysine-coated glass coverslips for
electrophysiological recordings.
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RESULTS |
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Fluid secretion in parotid acinar cells is stimulated by
muscarinic agonists that mobilize intracellular Ca2+. This
process involves the activation of both Ca2+-activated
Cl and Ca2+-activated K+
channels. We found significant activity of only two types of single
K+ channel currents in on-cell patches of mouse parotid
acinar cells stimulated by 5 µM of the secretagogue carbachol.
Examples of these currents are shown in Fig.
1, top inset. When the
currents from on-cell patches were recorded with an extracellular
K+ concentration of 135 mM, the two levels were near 22 and
140 pS (Fig. 1, filled squares). These same two levels of conductance were observed from patches excised into Ca2+-containing
intracellular solutions (Fig. 1, open squares). Of eight patches, five
contained both channel types, two contained openings only of the
small-conductance level, and one had openings of only the
large-conductance channel. The amplitude histograms (see
Electrophysiological methods) showed no signs of
subconductance states.
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These conductance levels suggest that the
Ca2+-activated K+ channels in mouse parotid
acinar cells are members of the intermediate and maxi-K families,
respectively. Because the intermediate-conductance K+
channels are not voltage or time dependent (15, 20, 36, 37) and maxi-K channels are voltage and time dependent
(13), the presence of both classes of these channels in
parotid acinar cells predicts that the whole cell currents would have
two Ca2+-dependent components: one that is time and voltage
independent and one that is time and voltage dependent. Figure
2 shows that parotid acinar cells do
indeed have both these components.
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Figure 2A, inset, shows whole cell currents
recorded from a parotid acinar cell patched with an internal
Ca2+ concentration of 160 nM. Shown are currents in
response to steps of membrane voltage to 110,
30, +10, and +50 mV
from a holding potential of
70 mV. Steps to negative voltages
elicited currents with little time dependence (after the capacitative
current transient), whereas strong depolarizations produced currents in
a time- and voltage-dependent manner. The voltage dependence of the
current measured at the end of the 40-ms pulses is shown in the main
part of Fig. 2A. The current-voltage relationship can be
seen to contain two components: a linear one at potentials more
negative than about
50 mV, and an outward rectifying component at
positive potentials. The broken line represents a linear fit to the
current at the four most negative potentials. The interpolated
zero-current potential was
78 mV, which is close to the expected
K+ equilibrium potential of
82 mV for the solutions used
in this experiment. Thus the Ca2+-activated current in
mouse parotid acinar cells is consistent with the expression of both
intermediate-conductance and maxi-K channels.
Intermediate-conductance K+ channels have a high sensitivity to the antifungal agent clotrimazole (15, 20, 36, 37). Maxi-K channels are generally resistant to this agent (39). Thus, if these two types of channels are expressed in parotid acinar cells, the linear, time-independent component should be inhibited by clotrimazole with little or no effect on the nonlinear, time-dependent current. Figure 2B shows that the addition of 300 nM clotrimazole to the cell shown in Fig. 2A inhibited the linear component by ~70% with no clear effect on the time-dependent, nonlinear current. The average inhibition of the linear component by 300 nM clotrimazole was 78 ± 2.8% (mean ± SE, n = 4). Current block in several similar experiments with a range of clotrimazole concentrations was consistent with an EC50 value of 30 ± 5.5 nM.
Thus mIK1 represents a reasonable candidate for the time- and
voltage-insensitive Ca2+-activated K+ channel
in parotid acinar cells. The human IK isoform, hIK1, has been shown via
a multiple tissue RNA dot blot to be highly expressed in salivary
glands as well as in several other nonexcitable tissues
(17). Northern blot analysis of mouse tissues with the use
of a probe complementary to the 5' portion of mIK1 detected a single
transcript of ~2.2 kb in both colon and parotid gland, consistent
with the expected size of the mIK1 message (Fig.
3A). Subsequent in situ
hybridization of semi-thin parotid gland sections with an antisense
cRNA suggested that mIK1 expression is limited to the acinar cells and
does not occur in cells of the salivary ducts (Fig. 3B).
Hybridization with a sense control produced only a weak and nonspecific
signal (data not shown).
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To solidify the identification of mIK1 as the time- and voltage-independent Ca2+-activated K+ channel in parotid acinar cells, we compared several properties of mIK1 expressed in HEK cells with the currents in the native cells. An example of single-channel currents in an excised inside-out patch from a HEK cell transfected with mIK1 is shown in Fig. 1, bottom inset. The current-voltage relationship of single, expressed mIK1 channels (Fig. 1, open triangles) was indistinguishable from the native, small-conductance channels. Figure 2C, inset, shows that expressed mIK1 had little voltage and time dependence, and the main part of Fig. 2C shows that expressed mIK1 channels were quite sensitive to 300 nM clotrimazole. Several similar experiments with a range of clotrimazole concentrations were consistent with inhibition of recombinantly expressed mIK1 channel by this compound with an EC50 of 18 ± 5 nM, a value a bit smaller than that for the linear component of native cells.
We also compared the tetraethylammonium (TEA) sensitivity of the linear component of native cell current to that of heterologously expressed mIK1 channels. The native current was inhibited by TEA with an EC50 value of 6 ± 1 mM. Current through expressed mIK1 channels was inhibited with a similar EC50 value of 8.7 ± 1 mM (data not shown).
Thus we found mIK1 to be robustly expressed in mouse parotid acinar cells, and many properties of expressed mIK1 channels were quite similar to those of the linear, Ca2+-activated K+ current in native cells. We used a similar approach to test the possibility that a product of the Slo gene underlies the time- and voltage-dependent Ca2+-activated current in mouse parotid acinar cells.
The Slo1 channel was originally cloned from Drosophila
(2) and subsequently from mouse and human sources
(8, 33). Alternative splicing of the Slo1 gene product in
mice (Kcnma1 gene) and humans (KCNMA1 gene)
results in multiple variants with distinct expression patterns and
calcium sensitivities (4, 8, 18, 31). Furthermore, two
additional Slo isoforms are encoded by separate genes. Slo2 was first
isolated from C. elegans and is sensitive to chloride as
well as calcium (40), whereas Slo3 expression is
restricted to the testes in mice and humans and is insensitive to
calcium but sensitive to intracellular pH (30). Both Slo2
and Slo3 homologs can be found in the human genome database of
predicted coding regions and in the mouse EST database. RT-PCR analysis
of Slo isoforms in mouse parotid gland revealed that expression was
limited to the mSlo1 isoform; the amplification of products of the
predicted size from kidney and testes cDNA confirmed the efficacy of
the mSlo 2 and 3 primer pairs, respectively (Fig.
4). The presence of mSlo was tested for
directly by performing Northern and Western blot analyses of parotid
gland mRNA and membrane proteins, respectively (Fig. 4, B
and C). A single transcript of ~5.3 kb was recognized by an mSlo-specific cDNA probe (Fig. 4B), whereas a
commercially available anti-mSlo antibody reacted with a protein with
an apparent molecular mass of 115 kDa in both brain and parotid
membrane preparations (Fig. 4C). The brain mSlo protein
appeared to be slightly larger than the parotid version, consistent
with the splice variation that we observed in the parotid gland (see
below). Preincubation of the antibody with a purified peptide
consisting of the mSlo recognition site resulted in a loss of
mSlo-specific signal. Finally, in situ analysis of parotid sections
failed to detect mSlo (data not shown), suggesting that the message may
be expressed at levels below the detection sensitivity of this method.
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Alternative splicing of the Slo channel transcript results in a
molecular diversity that can alter the voltage and calcium sensitivity
of the protein in many species (4, 8, 18, 26, 31). To
date, at least six splice sites have been characterized in the coding
region of the mouse transcript, and the beginning of the transcript,
including the start of the open reading frame, has been shown to be
highly diverse (Ref. 8; Fig.
5A). To assess the
distribution of mSlo1 splice variants in the parotid gland, the coding
region for the COOH terminus of the protein, where most of the
identified splice sites occur, was amplified using RT-PCR and multiple
clones were sequenced. All of the clones were found to be identical and
insertless at each of the characterized splice sites. Additional
5'-RACE and RT-PCR of the full-length clone suggested that a
single mSlo1 transcript was expressed in the parotid gland (data not
shown). This transcript corresponded to a short version of the protein,
resulting from an in-frame stop codon following the final splice site.
This splice variant is highly homologous to the human Slo1 mRNA
(accession no. NM_002247) and differs primarily in the last eight
conceptually translated amino acids, as shown in Fig. 5B.
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We compared several properties of the mouse parotid mSlo variant (parSlo) expressed in CHO and HEK cells with the time- and voltage-dependent current in native parotid cells. Figure 1, bottom inset, contains an example of single-channel currents in an inside-out patch from a HEK cell transfected with the parSlo. The single-channel current-voltage relationship of parSlo channels expressed in HEK (Fig. 1, open circles) and CHO cells (Fig. 1, open inverted triangles) was similar to that of the large-conductance channels in the native cells.
Maxi-K channels are sensitive to the scorpion toxins CTX and IBX
(13). Expressed IK1 channels are also sensitive to CTX but
are relatively insensitive to IBX (15, 36). Thus IBX is a
specific pharmacological tool for maxi-K channels, so we examined IBX
block of native currents and recombinantly-expressed mSlo channels.
Figure 6A, inset,
contains an example of the time- and voltage-dependent currents of
native parotid acinar cells recorded in the presence of 300 nM
clotrimazole to inhibit the linear mIK1 currents. The main part of Fig.
6A shows that application of 500 nM IBX had a modest effect
on the current recorded at +50 mV. In this example, 500 nM IBX blocked
~33% of the current. The average amount of block of native current
by this concentration was 38 ± 2.1% (n = 6).
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Heterologously expressed parSlo produced time- and voltage-dependent currents (Fig. 6B, inset) that were generally similar to the currents in native cells. However, though not investigated in detail, the time course of activation of expressed parSlo was somewhat faster than the current in the native cells. Significantly, expressed parSlo channels were considerably more sensitive to IBX than the native current. The main part of Fig. 6B shows that 100 nM IBX blocked 95% of the current (at +50 mV) through parSlo channels heterologously expressed in CHO cells. Similar data with 2-100 nM IBX were fit by a standard binding isotherm with an EC50 value of 6.1 ± 2 nM for IBX block of homomeric parSlo channels expressed in HEK cells.
The good agreement between the single-channel conductance coupled with
the discrepancy between the IBX sensitivity of the native current and
expressed parSlo raises the possibility that the native channels may
contain an auxiliary subunit in addition to the product of the parSlo
gene. Four -subunits that can assemble with Slo proteins have been
identified (3, 6, 9, 10, 24, 35), and the presence of
these
-subunits alters the scorpion toxin sensitivity of the
heteromeric channels (3, 10, 24).
To test for the presence of these -subunits in the mouse parotid
gland, we designed primer pairs for RT-PCR analysis based on multiple
sequence alignments of the published
-subunit gene products (human
KCNMB1-4; mouse Kcnmb1 and 4).
The following goals applied: first, to flank homologous segments of the
-subunit coding regions; second, to prevent cross-hybridization; and
third, to end the primers on codons that represent highly conserved
amino acids. This strategy was expected to yield primer pairs for
2- and
3-subunits that would recognize
both the human and mouse homologs, because the mouse cDNA sequences had
yet to be reported. Our results indicated that
1- and
4-subunits were most prominently expressed in mouse
parotid gland, whereas the
2 and
3
amplimer sets could be validated for use in mice by using mouse kidney and trachea cDNA templates, respectively (Fig.
7). Furthermore, successive rounds of
amplification failed to result in the accumulation of specific PCR
products for
2- or
3-subunits from
parotid cDNA, suggesting that their expression levels are below the
detection limits of our assay (data not shown).
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Expression of the 1- and
4-subunit mRNA
in parotid gland was demonstrated directly by Northern blot analysis
(Fig. 7B). We used a commercially available
anti-
1 antibody but were unable to detect a specific
signal in a preparation of parotid gland membranes by Western blot
analysis (data not shown). Similarly, attempts to localize the signal
to specific cells in the parotid gland through immunohistochemistry
were unsuccessful. Thus molecular studies alone cannot determine which
of the two
-subunits, if either, are expressed in parotid acinar
cells. To answer this question, we compared the electrophysiological
and pharmacological characteristics of the parSlo channel coexpressed
with either
1 or
4 to the endogenous
parotid acinar cell current.
Heteromeric parSlo+1 channels expressed voltage- and
time-dependent currents (Fig. 6C, inset)
generally similar to the currents in native cells. These heteromeric
channels had a single-channel conductance indistinguishable from that
of homomeric parSlo channels (not shown) consistent with results with
hSlo and the human
1-subunit (27). However,
the addition of the
1-subunit substantially reduced the
IBX sensitivity of the channels, as shown in the main part of Fig.
6C. Even 500 nM IBX had only a modest effect on heteromeric parSlo+
1: an average block at +50 mV of 17.5 ± 7%
(n = 5). This degree of block is consistent with an
EC50 value of 2,400 nM, and in agreement with this
value, 2,500 nM blocked 54 and 47% of the current in two independent
experiments. Heteromeric parSlo+
1 channels remained
sensitive to external TEA, as shown in Fig. 6C.
Coexpression of parSlo and 4 also produced time- and
voltage-dependent currents resembling those in the native cells (Fig. 6D, inset) with the same single-channel
conductance as the native channels and homomeric parSlo channels
expressed in HEK and CHO cells (data not shown). As shown in the main
part of Fig. 6D, heteromeric parSlo+
4
channels were entirely insensitive to 500 nM IBX. This concentration of
toxin reduced the current (at +50 mV) through the heteromeric channels
expressed in CHO cells by 1.9 ± 1.6% (n = 3).
Application of 2,500 nM for >10 min (Fig. 6D) had no more
effect than 500 nM (P > 0.1, n = 3).
As shown, these heteromeric parSlo+
4 channels remained
sensitive to external TEA.
Thus the voltage- and time-dependent current in mouse parotid acinar
cells did not have the IBX sensitivity of homomeric parSlo channels or
that of heteromeric parSlo+1 or
4
channels, raising the possibility that the native current could include
two populations of channels: one that is relatively sensitive to the
toxin (like homomeric parSlo channels), and one that is toxin resistant
(like heteromeric parSlo+
1 or
4
channels). The data in Fig. 8A
show that only ~39% of the native current (in BlackSwiss × 129/SvJ mice) was sensitive to IBX. This IBX-sensitive component had an EC50 value of ~31 ± 14 nM. About 61% of the
voltage- and time-dependent current in mouse parotid acinar cells was
insensitive to IBX up to concentrations as large as 2,500 nM. We also
examined the CTX sensitivity of the channels in native cells and found
that 55 ± 2% of the current was sensitive to this toxin with an
EC50 value of 13 ± 2 nM (data not shown); ~45% of
the current was insensitive to CTX up to levels as high as 500 nM.
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Even though only 40-45% of the native time-, voltage-, and Ca2+-dependent current was sensitive to even very high IBX and CTX concentrations, much, if not all, of the current could be blocked by external TEA, as shown in Fig. 6, C and D. In addition, essentially all the current could be inhibited by paxilline. This diterpene mycotoxin is a very potent nonpetidyl inhibitor of maxi-K channels (reviewed in Ref. 13). In the example in Fig. 8B, 1 µM paxilline blocked 96% of the time- and voltage-dependent current in the presence of 160 nM intracellular Ca2+ (and 300 nM clotrimazole to inhibit current through mIK1 channels). The average inhibition in five such experiments was 94 ± 1.9%. This result and those with external TEA indicate that 100% of the current recorded with these conditions is through maxi-K channels even though not all the current can be blocked by even very high concentrations of the scorpion toxins.
The moderate sensitivity of heteromeric parSlo+1
channels to IBX (see Fig. 6C and related text above)
suggests that these channels do not contribute significantly to the
total maxi-K current in native parotid acinar cells. However, the slow
onset and small block of the heterologously expressed heteromeric
channels diminishes the strength of any such conclusion. An alternate
method to test the idea that heteromeric parSlo+
1
channels do not contribute significantly to the maxi-K current in
native cells is to determine the IBX sensitivity of the current in
cells from mice in which the
1 gene has been disrupted
(7). The results of such a test are presented in Fig.
9.
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Figure 9 shows the amount of IBX block of maxi-K channels in
1 knockout mice (filled squares) along with similar data
from their wild-type counterparts (open circles). There appears to be
little difference between the IBX block of wild-type and
1 knockout animals, and all the data are consistent with
56 ± 2% of the current sensitive to IBX with an EC50
value of 22 ± 6 nM; that is, there is ~50% of the maxi-K
current in mouse parotid that is insensitive to IBX whether or not the
1 gene is active, confirming the suggestion that
heteromeric parSlo+
1 channels do not significantly
contribute to the maxi-K current of mouse parotid acinar cells.
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DISCUSSION |
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There have been multiple studies of K+ channels in several types of salivary glands from different species (12, 14, 16, 22, 29). The results of these various studies suggest that the K+ channel expression pattern may be specific to particular glands and particular species. Therefore, a complete understanding of the physiological role of K+ channels in salivary glands requires a systematic approach that includes identifying the relevant genes in a single model system as well as a quantitative comparison of the electrophysiological properties of the native channels with those of expressed, candidate genes.
Developments in targeted gene ablation technology have made the mouse an attractive system in which to examine the physiological role of ion transport proteins in transmembrane fluid dynamics (25). We found that mouse parotid acinar cells express significant amount of only two types of Ca2+-activated K+ channels: 1) a relatively time- and voltage-independent K-selective channel that has a single-channel conductance (in 135 mM external K+) of ~22 pS, and 2) a voltage- and time-dependent K+ channel with a single-channel conductance of ~140 pS. These properties suggested Kcnn4 (mIK1) and Kcnma (mSlo) as candidate genes coding for these channels. Thus we designed experiments to determine whether these genes were expressed in mouse parotid acinar cells and compared several properties of the native channels with those of the heterologously expressed candidate genes.
Northern blot and in situ analyses showed that mIK1 was abundantly expressed in acinar but not ductal cells of the mouse parotid gland, consistent with a primary role in secretion in this tissue. This finding is consistent with the observation that salivary glands express the highest detectable levels of mIK1 transcript among all of the organs surveyed (16). Other sources of mIK1 include nonexcitable tissues such as lung, trachea, kidney, and stomach. The distribution of mIK1 in tissues rich in epithelia supports a significant role for mIK1 in secretion or electrolyte reabsorption.
Heterologously expressed mIK1 channels have several properties in common with the voltage- and time-independent K+ current in mouse parotid acinar cells: 1) a single-channel conductance indistinguishable from that of the native current, 2) a similar lack of voltage and time dependence, 3) a similar sensitivity to clotrimazole, and 4) a nearly identical sensitivity to TEA.
Because the voltage- and time-dependent component of K+ current in the native cells appeared to have the properties of a maxi-K channel, we tested for the expression of genes known to code for these types of channels. The gene family to which Slo1, the original maxi-K channel, belongs is composed of two additional members, Slo2 and Slo3. As expected on the basis of their published distribution (mSlo3 is testes specific) and activities (mSlo2 is chloride dependent; mSlo3 is pH sensitive/Ca2+ insensitive), the results of RT-PCR analysis suggest that mSlo1 is the most prominently expressed member of this gene family in the parotid gland.
The mSlo1 gene has been shown to have alternatively spliced products, and many of the splice variants exhibit distinct electrophysiological characteristics and greatly differing Ca2+ sensitivities (4, 8, 18, 31). We assayed the most common splice sites in the NH2 and COOH termini of the protein and found that a single homogenous insertless variant was present in the parotid gland. Although this variant has previously been described in mice (31), it has not been assayed electrophysiologically. Instead, a separate variant, mbr5 (8), was expressed for patch-clamp analysis under the assumption that it was insertless (31). However, the mbr5 variant contains a three-amino acid insertion (IYF) at the second splice site in the COOH terminus and, in addition, codes for the "long" form of the protein. The true insertless form of mSlo ends prematurely because of a cryptic stop codon brought into frame by the splicing of nucleotides 3408-3436 from the mSlo coding region. Interestingly, the insertless mSlo variant found in the parotid most closely resembles one of the published human Slo sequence variants (11, 27, 33).
The mouse parotid mSlo variant (parSlo) expressed in HEK and CHO
cells had a single-channel conductance that was indistinguishable from
the large-conductance channel in the native cells. However, heterologously expressed parSlo channels were substantially more sensitive to IBX than was the current in the native cells. Thus we
tested for the presence of four mSlo -subunits known to alter scorpion toxin sensitivity. We found that the mouse parotid expressed only the
1- and
4-subunits, and we
examined the IBX sensitivity of parSlo coexpressed with each of these subunits.
We found that both the mouse 1- and
4-subunits reduce the IBX sensitivity of the mouse
parotid mSlo variant parSlo, with a larger effect produced by the
4-subunit: heteromeric parSlo+
4 channels
were insensitive to IBX concentrations as large as 2,500 nM. This is
the first examination of the actions of IBX on this particular mSlo
splice variant in heteromeric channels with the mouse
-subunits, so
no direct comparison to existing data is possible. However, our results
are qualitatively similar to those obtained with human Slo (hSlo)
channels coexpressed with human
1- and
4-subunits (3, 10, 24), although the
coassembly of the mouse parotid mSlo variant with the
-subunits
appears to more substantially decrease IBX sensitivity.
We found that the IBX and CTX dose-response relationships of the native
channels (Figs. 8A and 9) were consistent with two populations of maxi-K type channels in the native cells: one (~50% of the total) with a reasonably high affinity for toxin, and another population that is essentially insensitive to the toxin (to 500 nM CTX
and 2,500 nM IBX). Because we found heteromeric parSlo+1 channels to be moderately sensitive to IBX and hSlo+
1
are quite CTX sensitive (3, 24), it seems reasonable to
consider that the maxi-K, Ca2+-activated K+
current in native parotid is composed of approximately equal numbers of
homotetrameric parSlo channels and heteromeric parSlo+
4 channels. This suggestion is strengthened by our results showing that
~50% of the maxi-K current in parotid acinar cells from
1 knockout mice remained insensitive to 2,500 nM IBX.
Although the
1 gene ablation results could be
compromised by an exact compensation by
4 expression,
the most parsimonious explanation that accounts for all the data is
that mouse parotid acinar cells express equal numbers of homotetrameric
parSlo channels and heteromeric parSlo+
4 channels.
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ACKNOWLEDGEMENTS |
---|
We thank Keerang Park for cloning the mIK1 from mouse parotid tissue and Paul D. Kingsley for assistance with the in situ analysis. We are grateful to J. E. Melvin for considerable discussions of this work and for critically reading the manuscript. We thank J. Thompson for assistance with the electrophysiological experiments and for critically reading the manuscript and Jodi Pilato and Pamela McPherson for the preparation of parotid acinar cells.
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FOOTNOTES |
---|
* K. Nehrke and C. C. Quinn contributed equally to this work.
This work was supported by National Institute of Dental Research Grants DE-13539 (T. Begenisich) and DE-14119 (K. Nehrke).
Address for reprint requests and other correspondence: T. Begenisich, Dept. of Pharmacology and Physiology, Box 711, Univ. of Rochester Medical Center, Rochester, NY 14642 (E-mail: ted_begenisich{at}urmc.rochester.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 16, 2002;10.1152/ajpcell.00044.2002
Received 29 January 2002; accepted in final form 15 October 2002.
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