From the Departments of Medicine and Pharmacological
and Physiological Sciences, the University of Chicago,
Chicago, Illinois 60637 and the § Department of Medicine,
the University of Wisconsin, Madison, Wisconsin 53792
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ABSTRACT |
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ATP-sensitive potassium channels
(KATP) are implicated in a diverse array of
physiological functions. Previous work has shown that alternative usage
of exons 14, 39, and 40 of the muscle-specific KATP channel
regulatory subunit, sur2, occurs in tissue-specific patterns. Here, we show that exon 17 of the first nucleotide binding fold of sur2 is also alternatively spliced. RNase
protection demonstrates that SUR2( ATP-sensitive potassium channels
(KATP),1 first
described in cardiac myocytes (1, 2), are weak inwardly rectifying
K+ channels, which are inhibited by intracellular ATP and
activated by Mg-ADP (3, 4). KATP are found in a diversity
of tissues, including cardiac, skeletal, and smooth muscles, brain, and
the Current evidence indicates that KATP are hetero-octameric
complexes (17, 18) consisting of at least two types of subunits as
follows: a member of the small inward rectifier potassium channel family, Kir6.1 (ubiquitous KATP) or Kir6.2 ( To date, five species of sur mRNA transcribed from two
different sur genes have been isolated and shown to yield
functionally distinct KATP when expressed with Kir6.2.
sur1 (14) confers typical Exon 17 encodes the 13 amino acids next to the Walker A motif
containing exon 16 of the first nucleotide binding fold (NBF). The
Walker A motif is highly conserved in many ATP-binding proteins (21,
24) and is required for the functional integrity of the NBF. In
sur1, mutations of the conserved lysine residue (Lys-719 in
sur1) in the Walker A motif of NBF1 produce a decrease in
ATP binding to the sur (25). This invariant lysine residue
has been associated with facilitation of ATP hydrolysis (26-28).
Electrophysiological experiments also implicate NBF1 of sur1
in the ATP sensitivity to ATP block (29). Given the close proximity of
exon 17 to the Walker A motif within the NBF, we hypothesized that the
alternate use of exon 17 would affect the nucleotide sensitivity of
sur2/Kir6.2 recombinant KATP channels. Indeed,
we describe here that exon 17 influences the ATP sensitivity by
changing the bursting pattern of the recombinant channel.
RT-PCR Analysis of Exon 17
Total RNA was isolated from whole organ mouse tissues by TRIzol
extraction (Life Technologies, Inc.). The RNA was primed with the
sur2 gene-specific primer (5'-CTGACC CCCACTCAGGTTGATGCC-3'), or with oligo(dT), and reverse-transcribed with Superscript II (Life
Technologies, Inc.) following the manufacturer's instruction. Single-stranded cDNA (1 µl) was used as template for polymerase chain reaction amplification using Klentaq polymerase
(CLONTECH) under the following conditions: 35 cycles of 96 °C × 30 s melting, 58 °C × 30 s annealing, 72 °C × 1 min extension. The primers are as
follows: forward (in exon 16) 5'-CA TCTCTTCTCCTTGCCATCCTT GG-3'; reverse (in exon 21) 5'- CCTCTCTCCAATTTC AGTTTGGTCTCC-3'.
Long RT-PCR for Detection of Midcoding Region Splicing and
Alternative CTD Usage
Exon 14/Exon 39-40 Relationship--
Primers (forward, 5'-GAA
GCTGGCGGAGGCTCAGAAGAG-3'; reverse, 5'- GATCGGGCCCACTTTTCGAG TGTGGACAGA
TCGTA-3) were designed to reside 500 bp 5' to exon 14 and 3' of exon
40. These primers were used to amplify cDNA using Klentaq
polymerase under the following conditions: 36 cycles of 96 °C × 30 s melting, 68 °C × 3 min annealing/extension. The
products were purified by Qiaprep PCR purification (Qiagen) and
digested with HaeII (New England Biolabs). The digested
products were resolved on 0.8% SeaKem agarose gels (FMC), and the
corresponding sur2 fragments with and without exon 14 were
gel-purified using Qiaex II gel purification (Qiagen). The purified DNA
fragments were digested with XbaI (New England Biolabs) and
then resolved on 2.0% SeaKem agarose gels.
Exon 17/Exon 39-40 Relationship--
Primers were generated to
reside entirely within exon 17 and exon 39 or were designed to exclude
the two exons leaving 3-4 bp overhanging the splice junction at the 3'
end of the primer (3' overhangs are underlined). The primers are
as follows: exon 17, 5'-GTAAATGA ATCTGAGCCTTCTTTTGAAGCAACCCGAAG-3';
straddle 17, 5'-CCCTGGAAGG AAAAGTTTACTGGAACAACAG-3'; exon
39, 5'-TGGAGCAGGTTTGGACCAGTA TCAC-3'; straddle 39, 5'-CTGCAGTCAGAATGGTGTGAACCCGATGAG-3'. PCR conditions
and agarose gel electrophoresis are as stated above.
RNase Protection Assay and S1 Nuclease Assay
Probe Generation (RNase)--
A 375-bp cDNA probe
corresponding to the sur2 coding region bp 1879-2254 was
generated by PCR with Pfu polymerase (Stratagene) and
subcloned into Bluescript KS+ (Stratagene). Sequence integrity and
orientation was confirmed by dye termination sequencing (Applied Biosystems, Perkin-Elmer). This template was used to generate labeled
antisense RNA transcripts by in vitro transcription in the
presence of 800 Ci/mmol Probe Generation (S1 Nuclease)--
A full-length
sur2 cDNA with exon 39 CTD was subcloned into a
pcDNA3 vector. The construct was digested with Xba and
primed with an antisense oligonucleotide directed to exon 39, 5'-TGGAGCAGGTTTGGACCAGTATAC-3'. Run-off primer extension was performed
in the presence of 3000 Ci/mmol Solution Hybridization--
40 µg of total RNA, isolated by
TRIzol extraction, was coprecipitated with 5 × 105
cpm labeled probe, resuspended in hybridization solution (Ambion), and
allowed to hybridize for 18 h at 44 °C. The hybridized RNA were
digested with RNase A and T1 for 30 min (RNase protection assay) or
with S1 nuclease at 37 °C for 30 min, and the protected products
were resolved on a standard denaturing sequencing gel. The gel was
autoradiographically exposed to Biomax MR film (Kodak) overnight at
Cell Culture and Transfection
Full-length sur2 cDNA coding regions (bp 1-4644)
were subcloned into a mammalian expression vector (pcDNA3,
Invitrogen) using standard molecular biological techniques (30). COS-1
and COS-7 cells (ATCC), grown in Dulbecco's modified Eagle's
medium/high glucose + 10% fetal calf serum (Life Technologies, Inc.),
were plated at a density of 1 × 106 cells onto scored
glass coverslips in 60-mm plates, 12-18 h prior to transfection. Each
plate was transfected with 5 µg of total plasmid DNA, with a molar
ratio of 4:4:1 SUR2-pcDNA3:m Electrophysiology--
Cells were selected for recording based on
their green fluorescence at 525 nm when excited at 480 nm due to the
coexpression of green fluorescent protein. Pipettes (1-3 megohms) were
filled with "pipette solution" containing (mM) 140 KCl,
1 CaCl2, 0.22 MgCl2, 5 HEPES-KOH or HEPES-NaOH
at pH 7.4. In some experiments, 130 NaCl + 10 KCl was iso-osmotically
substituted for 140 NaCl as indicated in the text. Transfected cells
were placed in a chamber bath perfused with "internal solution"
containing (mM) 140 KCl, 0.2 MgCl2, 2 EGTA, 5 HEPES-KOH, pH 7.3; ATP, tolbutamide, and glyburide were added to the
internal solutions as indicated in the text. The solutions superfusing
the patch membrane were switched within 1 s using a fast solution
switching system, DAD12 (ALA Instruments). Inside-out patches were
recorded with an EPC-7 amplifier (List Electronics) for single channel
and Axo200B (Axon Instruments) for multiple channel analysis. Single or
multiple channel current recordings were filtered at 0.5-2 kHz,
sampled at 5 kHz, and then stored digitally direct via the PCLAMP (Axon
Instruments) and "Aquire" (Bruxton Corp.) software packages for
subsequent offline data analysis.
In patches containing a single active channel, channel open activity
was assessed by an open probability (Po) as follows.
A 50% threshold criterion was used to detect an event, and all events
were visually confirmed. The dwell time at each level was determined
using FETCHAN software (Axon Inst.). From this, the
Po was calculated using the equation derived by
Spruce et al. (34). Patches containing multiple channels (
For single channel kinetic analysis, idealized current reconstructions
for each recording were generated using "TAC/TACfit" program
(Bruxton Corp.) based on a half-maximal amplitude threshold criterion.
Open and closed time histogram analysis was performed on recordings
demonstrating only a single level of channel activity. Burst durations
were resolved at a critical time cutoff of 3.5 ms to distinguish a gap
between two bursts from a closure within a burst (31, 32) (Equation 1).
sur2 Exon 17 Exhibits Tissue-restricted Alternative
Splicing--
Our initial characterization of sur2 included
the description of an alternatively spliced coding region variant,
SUR2(
Solution hybridization and RNA protection with a labeled antisense RNA
probe spanning exons 13-18 (bp 1879-2254 of the coding region) was
employed both to confirm the RT-PCR splicing data and to quantitate the
relative mRNA tissue expression level for each of the three
identified splice variants in the mouse (Fig. 1B). Digestion
temperatures and RNase concentrations were varied to determine the
optimal protection/digestion conditions, and relevant controls were
included with each gel to ensure proper probe digestion (Fig.
2B, lanes 3 and 4).
Individual variants were indicated by their predicted protected
fragment sizes. A 375-nt protected fragment was present ubiquitously
and represented an mRNA species containing both exons 14 and 17 which is predicted to be the full-length sur2 transcript.
The exon 17 deleted variant ( Tissue-restricted Expression of CTD with Exon 39 and Exon
40--
Alternate sur2 CTD usage was recently demonstrated
by Isomoto et al. (22). We have adopted a nomenclature based
on exon usage, thus isoforms SUR2A and SUR2B correspond to exons 39 and 40, respectively. Exon 40 was demonstrated to be present in all tissues
and is highly homologous to the sur1 CTD. S1 nuclease protection analysis of the carboxyl-terminal alternate exon usage was
employed to corroborate the results reported by Isomoto et al. (22), as well as quantitatively determining which major carboxyl-terminal isoform was expressed in any single tissue. A
single-stranded 32P-labeled antisense cDNA probe
spanning exons 35-39 was generated and hybridized to total RNA in
solution. Exon 39 variants resolved as a 378-nt fragment, whereas exon
40 species, which could not protect the last 131 nt of the probe,
resulted in a 247-nt protected fragment (Fig. 1C). A second
probe corresponding to a 125-bp stretch of Exon 14 Splicing Only Occurs with Carboxyl Tail Exon
39--
Demonstrating three mid-coding region sur2 splice
variants (full-length, Exon 17 Splicing Occurs Independently with Respect to Splicing of
the CTD--
A similar PCR/restriction digestion approach was not
available to determine the relationship between exon 17 splicing and the alternate carboxyl terminus, as no unique restriction sites existed
in exon 17. Instead, primers were designed to reside entirely within
exons 17 and 39 or to straddle these exons and thus exclude them (Fig.
2B). The straddling primers annealed to the splice junction
asymmetrically; a majority of the 5' end of each primer annealed to one
exon leaving 3-4 bp of the primer's 3' end to anneal to the exon
following the splice junction. By this design, these primers could only
anneal to those sur2 variants lacking either exon 17 or 39 to provide a productively primed template for polymerase extension
(schematic, Fig. 2B). The four designed primers
were used in combination to test each of the four possible splice forms
for exons 17/
In summary, five sur2 isoforms are generated by the
alternative splicing of the coding region: SUR2(39), SUR2( Single Channel Currents Resulting from the Coexpression of sur2
Splice Variants with mKir6.2--
Coexpression of four of the five
sur2 variants and mouse Kir6.2 in COS-1 or COS-7 elicited
currents with identical single channel conductance (Fig.
4, A and B), and
kinetic properties similar to cardiac and skeletal muscle
KATP (7, 32, 34). One variant, SUR2( Exon 17 Splicing Alters ATP Sensitivity--
The four exon
14-containing sur2 variants were inhibited by ATP in a
dose-dependent manner, with a 2-fold difference in ATP sensitivity based on the presence or absence of exon 17. ATP in the
presence of 0.2 mM MgCl2 was rapidly applied to
the cytoplasmic side of the patch membrane at concentrations varying
from 1 to 1000 µM. To avoid bias from any channel
run-down, the concentration of ATP was stepped in both increasing and
decreasing concentrations. Fig.
5A shows representative
current records for the four functional sur2 variants. The
open probability, or the averaged current at each ATP concentration,
was normalized to that in the absence of ATP. The normalized current
values, or relative current, represent the ATP block and are summarized
in Fig. 5B. The concentration-dependent block of
ATP was quantified by fitting the mean data points with a sigmoidal
Hill equation (Fig. 5B). Half-maximal inhibitory
concentrations, K1/2, and the Hill coefficients, H, for ATP block of channel activity were determined and are
listed in Table I. The data show
significant differences between variants with exon 17 and variants
without exon 17. Deletion of exon 17 increased the
K1/2 for ATP block approximately 2-fold, regardless
of CTD usage. The Hill coefficients for all variants are approximately
1 and the differences among them are insignificant.
Next, we explored whether the differences in single channel gating
could account for the reduced ATP sensitivity. Given the observation
that CTD usage did not affect ATP sensitivity, we restricted the study
of single channel kinetics to variants with exon 39. The single channel
behavior of SUR2(39) was compared with SUR2(
The distributions of gaps between bursts for both variants required at
least two exponents to fit (Fig. 6B), in close
correspondence to previously reported data from cardiac and skeletal
KATP which has been described by a four-state linear
sequential model (32, 35). The suitability of such a model, consistent
with the model we derived from our duration histogram analysis (Fig.
7A), was tested independently
by subjecting our raw single channel recordings to a maximum likelihood
computational method for the modeling of an aggregated Markov process,
as described by Qin et al. (36). The resulting four-state
linear sequential model generated by this method was identical to the
model based on our histogram analysis and to the previously described
models (32, 35). The rate constants for this kinetic scheme, summarized
in Fig. 7A, were calculated from the parameters determined
by the maximal likelihood fits to the dwell time histograms (Fig.
6B). At 1 µM ATP, SUR2(39) differed
significantly from SUR2( Contribution of Alternative Splicing of sur2 to KATP
Channel Diversity--
KATP channel properties vary widely
in different cell types, particularly with regard to nucleotide block
and drug sensitivity (3, 4, 37, 38). This diversity may meet the
various physiological and pathophysiological roles of KATP
in the wide range of tissues. Many factors such as pH, cell membrane
composition, and other nucleotides affect ATP and pharmacological
sensitivity of KATP and contribute to the observed
diversity in tissue response (3, 4, 9, 39-41). In addition, channel
subunit isoform composition may also intrinsically regulate
KATP response. The cloning of cDNAs for
KATP subunits, sur1 (14), sur2 (15,
16), Kir6.1 (19) and Kir6.2 (20), and the demonstration of distinct nucleotide and pharmacological sensitivities between recombinant KATP (16, 20) indicated that a degree of the observed
KATP diversity may result from the assembly of different
channel subunit isoforms. Specifically, the sur subunit has
been found to confer characteristic features for KATP
including drug sensitivity and ADP activation. The distinct tissue
expression patterns reported for sur1 and sur2
isoforms likely contribute to observed tissue-specific KATP
channel diversity. Furthermore, Isomoto et al. (22)
demonstrated that the alternative CTD usage of SUR2(40) results in
recombinant KATP with altered response to the
antihypertensive drug diazoxide. They argued that the properties of the
reconstructed channel correspond to the native smooth muscle
KATP. Here, we identify alternative splicing of exon 17, which alters nucleotide sensitivity by 2-fold. The alternate usage of
exon 17 in sur2 follows a consistent pattern with previously
observed tissue-specific differences in ATP sensitivities for
KATP and as such may be a contributing factor to the
overall tissue differences in ATP sensitivities. For example, cardiac myocytes, which express sur1, full-length sur2,
and SUR2(
KATP molecular heterogeneity is further extended with the
identification of five different sur2 variants (Fig. 2). The
exon selection and relative transcriptional levels occur in a
tissue-specific pattern with
Previously, we described the identification of an sur2
variant lacking exon 14, SUR2(
The approach of chimeric SUR/Kir fusion proteins has provided strong
evidence that the KATP complex consists of a
hetero-octamer, with the sur and Kir subunits in equal
stoichiometry (17, 18). Of potential consequence, sur1 and
sur2 demonstrate overlapping expression in heart and
possibly skeletal muscle (16, 46), and sur2 and SUR2(
As both full-length and SUR2( ATP Block and the Underlying Kinetic Mechanism of Reconstructed
KATP--
A novel finding in theses studies is the
alternation in ATP block in response to exon 17 usage. We found that
the K1/2 for ATP block were around 40 µM for full-length sur2 and 90 µM for SUR2(
The ATP block of striated muscle KATP has been described
with a four-state linear sequential model (32, 35), similar to the
model presented in Fig. 7. In this model the transition O
To date, including this report, three isoforms of recombinant
KATP have been described at the single channel level as
follows: SUR2(39), SUR2( Structural Basis of Exon 17 Affecting KATP
Function--
The peptide segment encoded by exon 17 is a part of the
NBF1 structure and is in close proximity to the Walker A motif. Its location suggests an intimate association between this segment and the
putative ATP binding pocket of NBF1. The highly conserved Walker A
motif is believed to be a critical part of a site for ATP binding and
hydrolysis (52, 53). A consensus structure model has been proposed for
the nucleotide binding pocket of NBF-containing proteins which consists
of a series of six
Our finding of the reduction in ATP sensitivity with the removal of
exon 17 suggests that the exon 17 segment contributes to nucleotide
responsiveness of the Kir6.2-sur2 complex. However, our
electrophysiological data are insufficient to distinguish between the
direct involvement of exon 17 structure in ATP binding causing channel
block or an indirect effect on the mechanism of ATP block. Shyng
et al. (29) proposed a model for sur as an ATP-supersensitizing switch. In this model, the sur subunit
enhances the intrinsic ATP block of Kir. ATP hydrolysis at one or both of sur's NBFs uncouples the ATP hypersensitivity
sur imposes on Kir. Our finding suggests that the capability
of SUR2(
In conclusion, we have described five distinct sur2
transcripts resulting from the alternative splicing of the single
sur2 gene, and we demonstrated that the alternative splicing
of exon 17 affects the ATP block sensitivity of recombinant
KATP. Together with the tissue-specific expression,
alternative splicing of sur2 may contribute a meaningful
role in determining KATP physiological functions.
17) predominates in skeletal
muscle and gut and is also expressed in bladder, fat, heart, lung,
liver, and kidney. Polymerase chain reaction and restriction digest
analysis of sur2 cDNA demonstrate the existence of at
least five sur2 splice variants as follows: SUR2(39),
SUR2(40), SUR2(
17/39), SUR2(
17/40), and SUR2(
14/39).
Electrophysiological recordings of excised, inside-out patches from COS
cells cotransfected with Kir6.2 and the sur2 variants
demonstrated that exon 17 splicing alters KATP sensitivity to ATP block by 2-fold from
40 to
90 µM for exon 17 and
17, respectively. Single channel kinetic analysis of SUR2(39)
and SUR2(
17/39) demonstrated that both exhibited characteristic
KATP kinetics but that SUR2(
17/39) exhibited longer mean
burst durations and shorter mean interburst dwell times. In sum,
alternative splicing of sur2 enhances the observed
diversity of KATP and may contribute to tissue-specific
modulation of ATP sensitivity.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell of the pancreas (5, 6). In these cells, KATP
couple metabolic stresses to membrane excitability (5) and therefore play a key role in regulating physiological events such as muscle contraction, control of vascular tone, and insulin secretion (7-9). KATP are also generally characterized by their sensitivity
to inhibition by a class of antihyperglycemic drugs, sulfonylureas, and
to activation by a number of K+ opening drugs such as the
antihypertensive diazoxide and the vasodilative pinacidil (10, 11). The
properties of KATP with respect to nucleotide block,
activation, and drug responsiveness vary widely in the literature,
especially with respect to the tissue specificity (4, 6, 9, 12, 13).
The recent cloning of a number of KATP channel subunits
(14-16) has established a molecular framework underlying the
structural basis for functional diversity of KATP channels
in various cells.
-cell inward
rectifier), and a sulfonylurea receptor regulatory subunit,
sur1 or sur2, which are members of the ATP
binding cassette superfamily (ABC proteins) (14-16, 19-21). The
coexpression of both types of subunits yielded K+ currents
inhibitable by cytoplasmic ATP and sulfonylurea compounds (16, 20).
When the same pore protein, Kir6.2, is expressed either with
sur1 or sur2, the resulting channels exhibit
identical conductive properties but differ in their ATP block profile
and drug responses. Channels from sur1 were inhibited
half-maximally with 10 µM ATP and were responsive to the
potassium channel opener, diazoxide (20), whereas channels from
sur2 demonstrated a half-maximal block at 100 µM ATP and were insensitive to diazoxide (16).
-cell KATP
properties. sur2 identified by Inagaki et al.
(16) resembles the cardiac type KATP. A variant of the
sur2 carboxyl-terminal domain (CTD) arises from the excision
of the final 129 coding bp of sur2 (along with 48 bp of the
sur2 3'-untranslated sequence) and results in the expression
of a unique CTD from the use of 129 bp of the 3'-untranslated sequence
as coding sequence. This splice variant is described as SUR2B. SUR2B
yields recombinant KATP channels with restored diazoxide
sensitivity (22) and resembles a smooth muscle type KATP
(16, 20, 22). The expression pattern of these sur species
corresponds to their tissue-predictive functional properties. A third
sur2 variant lacking exon 14, named SUR2A (by coincidence,
as Isomoto et al. (22) denoted the original sur2
as SUR2A), has been identified only in heart (15). Each of the
identified sur2 variants results from a single exon
alternative splicing event. Examination of the mouse sur2
genomic structure reveals that the sur2 intron/exon
boundaries2 are comparable to
the published genomic structure of human SUR1, with 40 exons
available for the mRNA coding
region.3 Here, we adopt a
system of sur2 nomenclature based on the exons involved in
alternative splicing to clarify the identified sur2 variants, as well as future yet to be described variants resulting from
alternative splicing. The alternate carboxyl terminus usage of exon 40 replacing exon 39, SUR2B, is denoted as SUR2(40). Likewise, we identify
the sur2 isoform described by Inagaki et al. (16, 19, 20) that uses exon 39 as SUR2(39), and our previously identified
cardiac-restricted variant, SUR2A, is thus designated SUR2(
14/39).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Total RNA was isolated by TRIzol extraction (Life
Technologies, Inc.) from the indicated whole organ mouse tissues. The
RNA was primed with oligo(dT) and reverse-transcribed with Superscript II (Life Technologies, Inc.).
-[32P]CTP (Amersham Pharmacia
Biotech) and T3 RNA polymerase (Promega). A
-actin loading control
probe was generated in a similar fashion using Tri-actin mouse 125 as a
template (Ambion).
-[32P]dCTP (Amersham
Pharmacia Biotech) with Klenow polymerase (Ambion). The labeled probes
were purified from the template by denaturing polyacrylamide
electrophoresis, and the band containing the probe was excised and
eluted from the gel overnight in TE at 37 °C. A
-actin loading
control probe was generated in a similar fashion using Tri-actin mouse
125 as a template (Ambion).
80 °C or was exposed to a phosphorimaging cassette (Molecular
Dynamics) for 3 h at room temperature for quantitation.
-cell inward rectifier-pCMV6b:GFP-greenlantern (Life Technologies, Inc.), using the
Superfect transfection system (Qiagen). The cells were used for
recording 72 h post-transfection.
5 channels) were calculated in a similar fashion. In macro-patch recordings (>5 channels), an apparent open probability (N
Po) was employed. The N
Po was calculated as the average current in a 10-s
time window divided by the known single channel current amplitude under
the same recording conditions.
where
(Eq. 1)
1 and
2 are the time constants
for closed intervals within a burst and between bursts;
a1 and a2 are the areas
of exponential fits corresponding to
1 and
2, and td, the "dead time" of
underestimated events, corresponds to double the sampling rate (400 µs). Distributions of open and closed time events were plotted
against a logarithmic time scale with event durations log-binned at
50-µs resolution. Exponential fits to the histograms were performed
by a maximum likelihood fitting strategy. Values for kinetic rate
constants were determined based on the relationships described by
Sakmann and Trube (33), Gillis et al. (31), and Alekseev
et al. (32).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
14/39), restricted to heart expression (15). Further
examination of the PCR products spanning this alternatively spliced
region suggested a novel coding region variant. Primers designed to
amplify from the coding region 2126-2415 bp (exons 16-21) resolved a
novel product from the full-length sur2 PCR product (Fig.
1A). This smaller variant
demonstrated wide tissue expression distribution in mouse and was
detected in every tissue examined. It was prominent in bladder, gut,
kidney, and skeletal muscle and was weakly detected in all other
tissues. The sequence of this product indicated it lacked 39 bp (13 amino acids), from bp 2190 to 2229 of the sur2 coding
region, corresponding precisely to a single exon, exon 17, based on the
human SUR1 genomic structure. Subsequent mapping of the
mouse sur2 genomic intron/exon boundaries demonstrated that
the splice junction for this species indeed corresponded to exon 17, with appropriate flanking splice donor/acceptor sequences (data not
shown). Previous work indicated that the beginning of exon 17 "wobbled" by ±1 amino acid, which was attributed to an alternate
recognition of the splice acceptor site at the intron/exon junction.3 This is a distinct event from the alternative
splicing of exon 17 we present here.
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Fig. 1.
Expression of alternatively spliced
sur2 exons in various mouse tissues.
A, RT-PCR spanning exons (Ex) 16-21. Exon 17 containing transcripts (Ex 17+) are represented by a 289-bp
product, whereas exon 17 lacking variants (Ex 17)
resolves as a 250-bp product. Both species are present in every tissue
examined. B, in solution hybridization RNase protection. An
antisense probe spanned exons 13-18, and the protected fragments are
indicated. Controls for probe digestion are represented by the
lanes probe + RNase (no target mRNA) and probe + sense (in vitro transcribed probe in the sense
direction). The
-actin species represents a 3-h exposure, and the
protected sur2 species represents an 18-h exposure.
C, in solution hybridization S1 nuclease protection. A
single-stranded, labeled cDNA probe spanned exons 35-39, and the
protected carboxyl-terminal fragments are indicated. The heart and
skeletal muscle lanes, as well as the
-actin species, represent a
3-h exposure, whereas the other protected sur2 species
represent an 18-h exposure.
17), 312 nt, was alternatively spliced
in a number of tissues but was predominant in bladder, heart, and
skeletal muscle, and the exon 14 deleted SUR2(
14), a 243-nt
fragment, was present in heart alone. No evidence was seen that an
sur2 variant exists that lacks both exons 14 and 17. This
would be presented as a 179-nt fragment by RNase protection or as a
460-bp product in the previously reported RT-PCR (15). Quantitation of
expression levels by phosphorimaging indicate that SUR2(
17) was
expressed in highest levels in heart, skeletal muscle, and bladder when normalized to the
-actin loading control (n = 3 for
each tissue, quantitation data not shown). In heart, full-length
sur2 and SUR2(
14) were expressed at nearly similar
levels. SUR2(
17) was the predominant species observed in skeletal
muscle and in gut. In bladder, full-length sur2 was
expressed at nearly equal levels as SUR2(
17). Modest levels of
sur2 were present in all tissues and were expressed at least
5-10-fold greater than SUR2(
17).
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Fig. 2.
Relationship between midcoding region exon
usage and carboxyl-terminal usage. A, PCR restriction
digestion to evaluate exon 14 and CTD usage. Primers defined PCR
products that spanned bp 1639-5240 of the full-length sur2
transcript, amplified from oligo(dT)-primed mouse heart cDNA. The
products were digested and resolved by agarose gel electrophoresis in
two steps as indicated in the schematic. Exon 39 and exon 40 species
resolved as 760- and 581-bp fragments, respectively. B, exon
17 and carboxyl-terminal usage. Primer combinations and their
corresponding sur2 targets are depicted in the
schematic. Each lane of the resolved PCR products indicates
the primer set employed and the sur2 species amplified
according to the diagram. The presence of a 2-2.2-kilobase pair
amplified product indicates the existence of the particular
sur2 variant; the lack of a product indicates its absence.
The 9th to 14th lanes represent a relevant
positive and negative control for each primer using 10 ng of the
indicated cDNA template.
-actin served as a
loading control. Quantitation of protected fragments by phosphorimaging
(n = 2 for heart and skeletal muscle) indicated that
the heart contained both exon 39 and 40 sur2 variants in
nearly equal amounts, while skeletal muscle expressed approximately 2-fold greater levels of exon 39 compared with exon 40 (Fig.
1C). Weak expression of exon 39 was also noted in adipose
tissue and in bladder but at much lower levels than the corresponding
expression of the exon 40 variants. The exon 40 variant was present in
all tissues.
14, and
17) and two carboxyl-terminal
tails (exons 39 and 40) suggests that six possible sur2
variations could be generated if the mid-coding region splice events
occur independently of alternate carboxyl-tail usage. To determine if
exon 14 splice variants occurred with both exon 39 and exon 40, long
PCR was employed spanning exons 9-40 (Fig. 2A). Exon 14 and
exon
14 amplified products could be identified and separated by a
unique HaeII restriction site present in exon 14, 500 bp 3'
to the sense primer. From these amplified products, HaeII
digestion yielded PCR products differing by 500 bp. The digested
products were separated by gel electrophoresis, and the exon 14 and
exon
14 fragments were excised from the agarose gel. The presence or
absence of exon 17 (± 35 bp), present only within exon 14+ variants,
resolved as a nearly indiscernible doublet indicated as 14+
in Fig. 2A. These two exon 17 species, as both possessed
exon 14, were copurified for the subsequent digestion step. The
gel-purified fragments were digested a second time with a unique
XbaI site 5' to exon 39. Exon 39 and 40 variants resolved as
760- and 581-bp fragments, respectively. The results demonstrated that
exon 14 containing sur2 variants were present with both exon 39 and 40 CTD tails, but the SUR2(
14) variant, which was expressed in heart alone, only existed with the exon 39 CTD.
17 and 39/40, resulting in a 2-2.2-kilobase pair
amplified product (depending on the primer pair) if the variant was
expressed in the tissue or no amplified product if the tissue lacked
the variant. Heart and skeletal muscle were tested, as most of the
other tissues were known to contain only the exon 40 CTD (Fig. 1 and
Ref. 22). As such, these tissues must contain either 17/40 or
17/40
as determined by the earlier RT-PCR and the S1 nuclease (Fig. 1). Fat
and bladder, which also express minor levels of exon 39, were not
examined at this time. As seen in Fig. 2B, both skeletal
muscle and heart expressed all four of the possible exon 17 variants.
14/39),
SUR2(
17/39), SUR2(40), and SUR2(
17/40). Their tissue expression
pattern is summarized in Fig. 3.
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Fig. 3.
Summary of sur2 alternative
splicing. A, a summary of the expression patterns for
each sur2 variant in mouse tissues. * indicates these
tissues have not yet been tested for mid-coding region/CTD
combinations, but the individual exon splicing events have been
determined. B, representative schematic of the
sur2 topology and the locations of the identified
alternatively spliced exons. C, the amino acid sequence of
NBF1. The alternatively spliced exons are highlighted in
gray, and the Walker A and B motifs are
boxed.
14/39), failed to
generate current. The current-voltage relationship for each variant
demonstrated weak inward rectification and an estimated reversal
potential of approximately
58 mV (Fig. 4B), a value close
to the K+ equilibrium potential of
65 mV. The single
channel conductance was approximately 30 pS for all variants at a
membrane potential of 0 mV. In addition, each coexpressed variant
exhibited characteristic KATP channel inhibition at 1 mM cytoplasmic ATP. Application of 100 µM
tolbutamide or 1 µM glyburide to exon SUR2(39) or
SUR2(
17/39) expressing variants showed roughly equal channel block
(data not shown). These results demonstrate that the four
sur2 variants possessing exon 14 yield typical
KATP.
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Fig. 4.
Electrophysiological recordings or COS-7
cells expressing sur2 splice variants cotransfected
with Kir6.2. A, current traces of single channels
recorded from KATP generated by coexpressing mouse Kir6.2
and the indicated mouse sur2 variants. C and
O represent closed and open channel
levels, respectively. The holding potential was 0 mV for all traces.
[K+]i/[K+]o = 140/10
mM/mM. Outward current is plotted as an upward
deflection. B, single channel current I-V relationships for
reconstituted KATP. The current amplitudes were measured
from all points amplitude histograms. Each data point represents 1-6
patches.
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Fig. 5.
Dose-dependent responses of ATP
for sur2 splice variants expressed in COS-6
cells. A, representative current recordings of ATP
block for reconstituted KATP from the coexpression of
Kir6.2 and the indicated sur2 variants. Currents were
filtered at 200 Hz. The holding potential was set at 0 mV.
[K+]i/[K+]o = 140/10
mM/mM. Outward current is plotted as an upward
deflection. i = 0 indicates the level where all
channels are closed, and individual channel levels are
marked to indicate the number of channels per patch. ATP was applied to
the internal face of the membrane at the micormolar concentrations
indicated. The fast downward deflections in the data traces correspond
to solution change artifacts. 1 µM ATP was included in
the internal solutions whenever the ATP concentration is not provided.
B, dose-response relationships of ATP block for each
sur2 variant coexpressed with Kir6.2. The curves
represent fits of the experimental data to the Hill equation:
Irel = 1/{1
+([ATP]/K1/2(ATP))H}, where
Irel is the current amplitude relative to the
peak current at 1 µM ATP,
K1/2(ATP) is the concentration for
half-maximal ATP block, and H is the Hill coefficient. The
data points are the mean ± S.D. from the number of experiments
listed in Table I. * indicates a significant difference of
p < 0.05 between the relative current levels for the
exon 17+ variants versus the exon 17 variants, as
determined by the paired Student's t test.
ATP block of KATP expressed from SUR2 splice variants and
Kir6.2
17/39) at low (1 µM) and high (50 µM) internal ATP, in
symmetrical K+ (140 mM) at a membrane potential
of
60 mV. Both variants exhibited kinetics characteristic of striated
muscle KATP, resolving into long bursts of channel openings
that contained brief intraburst closures separated by interburst gaps
(Fig. 6A) (7, 34, 35). Open
and closed time constants were obtained using a maximal likelihood fit
to the dwell time histograms. The distribution of open and closed times
within a burst for each variant were accurately described with a single
exponential function. The averaged time constants,
O and
C1, for SUR2(39)-
O = 3.19 ± 0.09 ms
and
C1 = 0.37 ± 0.02 ms (n = 7)
and SUR2(
17/39)-
O = 3.30 ± 0.10 ms and
C1 = 0.36 ± 0.04 ms (n = 14) were
not significantly different from each other nor did they differ from
the corresponding averaged
O and
C1
values measured in the presence of 50 µM internal ATP, which were SUR2(39)-
O = 3.12 ± 0.15 ms and
C1 = 0.35 ± 0.01 ms (n = 8) and
SUR2(
17/39)-
O = 3.04 ± 0.19 ms and
C1 = 0.37 ± 0.01 ms (n = 8).
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Fig. 6.
Kinetic properties for SUR2(39) and
SUR2( 17/39). A, representative
single channel current records for excised inside-out patches of the
indicated sur2 exon 17 variant coexpressed with Kir6.2 in
COS-7 at
60 mV. [K+] were equal in the pipette and bath
(140/140 mM/mM). Patches were either excised
into 1 µM ATP or first excised into 1 µM
ATP and rapidly switched to 50 µM ATP. B,
representative open time, closed time, and burst distributions from a
single recording for each sur2 variant at the indicated ATP
concentrations. Closed time histograms of the gaps between burst
represent distributions exceeding the critical
tcutoff of 3.5 ms and are fit to two exponents.
Inset, closed time histograms representing distributions
before imposition of the critical tcutoff. Burst
distributions represent open state durations defined by gaps exceeding
the critical tcutoff of 3.5 ms and fit to two
exponents.
17/39) for rate constants directed away from
the open state: k23 = 5.8 s
1
versus 3.4 s
1 (p < 0.05) and
kO2 = 6.9 s
1 versus 5.4 s
1 (p < 0.05). At 50 µM
ATP, both variants exhibited significant increases in the rate
constants directed away from the open state (kO2
and k23), but the magnitude of the change was
similar: kO2 for SUR2(39) increased 2.9-fold
versus a 2.3-fold increase in kO2 for
SUR2(
17/39), and k23 for SUR2(39) increased
4.6-fold versus a 3.8-fold increase in
k23 for SUR2(
17/39). An appreciable difference between two variants was reflected in the rate constant directed toward the open state, k2O, at 50 µM ATP. Whereas k2O at 1 µM ATP showed no difference between the variants, a
2-fold decrease in k2O was observed for SUR2(39)
at 50 µM ATP. The k2O for
SUR2(
17/39) remained unchanged with increases in the ATP concentration. The difference in k2O was
reflected both in the calculated mean burst duration and in the mean
interburst gap duration for each variant (Fig. 7B). The mean
burst duration of SUR2(
17/39) in 1 µM ATP, 1.5-fold
longer than that of SUR2(39) (220 ± 14 ms versus
166 ± 13 ms), did not differ from its duration at 50 µM ATP. In contrast, the mean burst duration for SUR2(39) decreased 2-fold between 1 and 50 µM ATP. The mean
interburst gap durations did not differ significantly between the two
variants at 1 µM ATP. The mean interburst gap duration
for SUR2(39) increased 7.2-fold at 50 µM ATP (from
45 ± 15 to 325 ± 58 ms), whereas SUR2(
17/39) increased
only 1.9-fold (32 ± 5 to 63 ± 26 ms).
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Fig. 7.
Summary of single channel kinetics.
A, rate constants corresponding to the intraburst and
interburst transitions were calculated using the set of parameters that
define the open and closed distributions (as
represented in Fig. 6B) based on the relationships defined
by Gillis et al. (31) and adapted by Alekseev et
al. (32). B, average burst and interburst gap durations
were calculated based on the reciprocal values of the rate constants
leading away from the given states, as determined by Sakmann and Trube
(33): burst = (k1O + kO1)/(k1O
·kO1) and
interburst (C3 and
C2) = (k23 + k32)/(k32·k2O).
Error bars represent ± S.E., and the
brackets between each column indicate a statistically
significant difference as determined by the paired Student's
t test of p < 0.05 (n = 6-14 patches).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
17), ATP block over the range 20-100 µM has
been reported (4, 42); vascular smooth muscle, likely only expressing
full-length sur2,
30 µM (43); kidney,
expressing full-length sur2 and SUR2(
17),
100
µM (44); skeletal muscle, which predominantly expresses SUR2(
17),
130 µM (7); and the
-cell, which
expresses sur1,
10-20 µM (3). It is
important to note, however, that Kir6.2 is expressed primarily in the
pancreas and in heart (20), suggesting that sur2 must
associate with another Kir, possibly Kir6.1, in other tissues and thus
may possess significantly different ATP inhibition properties. Indeed,
when SUR2(40) has been coexpressed with Kir6.1 the resulting
sulfonylurea-sensitive channels were unlike traditional
KATP in that they did not spontaneously open when excised
into an ATP/free bath solution (45).
14 usage restricted to heart and
17
predominant in skeletal muscle and gut and high in bladder.
Interestingly, as exon 39/40 usage has already proven to discriminate
diazoxide sensitivity, it is conceivable that exon 17 and
17 may
prove to be targets for unique drug interactions, thus providing
KATP drug/tissue specificity for some tissues, such as
bladder or gut.
14/39) (15). In this study we tested the functional expression of this variant with Kir6.2 and found that it
yielded undetectable currents. This information is important in the
following two respects: first, it reveals that the peptide segment for
exon 14 may be critical to form functional channels; second, this
variant has the potential to serve as a negative suppressor of the
sur2 and so KATP expression. However, we did not
find an obvious suppressing effect in the test of the coexpression of
this variant with SUR2(39) using transient
transfection.4 Direct methods
to assay the levels and subcellular expression patterns of
SUR2(
14/39) will be needed to confirm this result.
17)
variants show overlapping expression in most tissues (Fig. 1,
A and B). If the subunit isoforms and variants are coexpressed in the same cell, it is possible that individual KATP subunits could coassemble into heterogeneous
KATP octameric complexes. This would significantly expand
the KATP subunit combinations possible. Considering that
our tissue distribution data were collected from the whole organs that
contained different types of cells, it still remains to be determined
whether the variants are indeed coexpressed in a single cell.
17) mRNA species are present in
most tissues where sur2 is expressed, the alternative
splicing of exon 17 may provide an intriguing mechanism to regulate
KATP sensitivity with respect to the cellular metabolic
environment. Recently, Akao et al. (47) demonstrated that
the Kir6.2 subunit of the cardiac KATP is regulated at the
transcriptional level in response to induced cardiac ischemia, and we
have observed altered sur2 transcript levels in cardiac and
skeletal muscle tissues of rats subjected to fasting.2
Additionally, Xie and McCobb (48) observed an example of
environmentally controlled splice regulation by demonstrating that
stress hormones could regulate the alternative splicing of a
K+ channel transcript. This suggests transcriptional or
post-transcriptional regulation (including exon usage) may have a role
for altering, or resetting, overall ATP sensitivity of KATP
current in cells experiencing periods of metabolic stress. Thus, by
expressing different sur2 variants in different cells within
a tissue, or within a single cell to form chimeric channels which may
also involve a dynamic regulation of subunit expression levels, the alternative splicing of sur2 reported here is a significant
mechanism employed in vivo to broaden the repertoire of
KATP.
17) variants. Interestingly, the CTD usage
did not significantly affect ATP sensitivity. The
K1/2 for exon 17 containing sur2 are
similar to those previously reported (16, 22, 46). The Hill
coefficient, which is determined by the nature of the ATP binding
process, reportedly varies between 1 and 2 in both native striated
muscle KATP and recombinant KATP (5, 17, 22).
Here, under uniform conditions, we found that the Hill coefficient for
all four variants was approximately 1, suggesting that the overall ATP
binding to block the channels follows an apparent single site binding process.
C1 is
ATP-independent, whereas the transitions C3
C2
O are ATP-dependent processes. In agreement with the observation
in native cardiac KATP (35), our data indicated that the
four rate constants, k32,
k23, k2O,
kO2, all varied with internal ATP concentration, with k32 the least variable. The ATP dependence
of the rate constants leading to the open state,
k32 and k2O for the
SUR2(39) variant, indicated possible cooperativity of several
ATP-binding sites or the regulation of ATP block by a second binding
event at a different site. Negative cooperation was suggested for ATP
binding to the sur1 regulating ATP block, possibly occurring
at the Kir6.2 subunit (49). The 2-fold reduction in ATP sensitivity
associated with the deletion of the exon 17 segment which we observed
can be attributed to an elimination of k2O
sensitivity to ATP. The absence ATP sensitivity of
k2O and the relative insensitivity of
k32 to ATP for SUR2(
17) channels suggests
that SUR2's ATP-dependent regulation of ATP block is
ablated by the removal of exon 17 segment.
17/39), and sur1 coexpressed with
Kir6.2 (20, 50). A fourth channel has been described, an engineered
truncation of Kir6.2 lacking its carboxyl-terminal 26 residues
(Kir6.2
c26), which expressed as an ATP-sensitive channel in the
absence of the sur subunit (51). From a casual inspection of
the four different recombinant KATP channel burst lengths,
the burst duration sequence is SUR2(
17/39) > SUR2(39) > sur1 > Kir6.2
c26, in the absence of ATP (Fig. 7
(20, 51)). This sequence indicates the importance of sur in
the determination of the burst kinetics of this channel.
-coil/
-strands (54). According to this model,
the segment of exon 17 begins with the end of
-strand 2 and
encompasses a critical residue predicted to participate in nucleotide
hydrolysis. Alternatively, sequence homology between sur2
and other ABC proteins suggests that the exon 17 segment may comprise a
portion of the putative switch I region identified in G-binding
proteins which is predicted to affect nucleotide binding and/or signal
transduction upon nucleotide hydrolysis (23, 27). The NBF of some ABC
proteins has been shown to possess ATPase activity (26, 28). Although
Ueda et al. (25) have demonstrated ATP binding to
sur1, intrinsic ATPase activity by an sur has yet
to be directly demonstrated.
17) of enhancing ATP sensitivity is significantly diminished
or lost. In the context of the supersensitizing model,
17 could
result in reduced ATP affinity at NBF1 thus preventing the key
substrate for the hypersensitizing switch from binding, or
17
enhances the rate of ATP hydrolysis and facilitates an uncoupling to
the desensitized state, or perhaps both. Alternatively, should exon 17 play a role in the signal transduction through which a hydrolytic signal induces an sur conformational change to the
desensitized state, SUR2(
17) might then resemble an sur
locked into a desensitized conformation. To test the validity of these
interpretations, it will be important to determine ATP binding and
hydrolysis at NBF1 for the two splice forms.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical support of D. L. McClelland and Shujie Yu.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK-K08-02170 (to C. F. B.), R29 HL58133-01 (to Z. F.), RO1-HL57414 (to J. C. M.), RO1-GM36823, and RO1-GM54266 (to D. J. N.), and the Oscar Rennebohm Foundation (to J. C. M.).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.
¶ To whom correspondence should be addressed: Parke-Davis Pharmaceutical Research, 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 734-622-1282; Fax: 734-622-5668; E-mail: charles.burant{at}wl.com.
Current address: Dept. of Physiology and Biophysics, the
University of Tennessee, Memphis, TN 38163.
2 W. A. Chutkow and C. F. Burant, unpublished observations.
3 G. Gonzalez, L. Aguilar-Bryan, and J. Bryan, GenBankTM accession number L78208.
4 W. A. Chutkow and Z. Fan, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: KATP, ATP-sensitive potassium channel; CTD, carboxyl-terminal domain; SUR, sulfonylurea receptor; Kir, inward rectifying potassium channel; NBF, nucleotide binding fold; bp, base pair; nt, nucleotide(s); RT-PCR, reverse transcriptase-polymerase chain reaction.
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REFERENCES |
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