(Received for publication, October 29, 1996, and in revised form, February 18, 1997)
From the Washington University School of Medicine, Departments of
Anesthesiology and Neurobiology and Anatomy,
St. Louis, Missouri 63110
cDNA libraries from rat chromaffin cells and PC12
cells were screened for homologs to the mouse mSlo gene
that encodes a large conductance, calcium (Ca2+)- and
voltage-activated potassium channel (BK channel). One Slo variant contained sequence encoding a cysteine-rich, 59-amino acid
insert for a previously described site of alternative splicing. This
insert is reminiscent of zinc-finger domains. The exon was found in RNA
from pancreas, anterior pituitary, cerebellum, and hippocampus.
Expression in Xenopus oocytes of a Slo
construct containing this exon conferred a 30 to
20 mV shift of the
conductance-voltage curve. A previously uncharacterized alternative
splice junction near the C-terminal end of Slo was also
identified. In contrast to BK channels in rat chromaffin cells, none of
the Slo variants exhibited inactivation when expressed in
Xenopus oocytes. PCR screening of chromaffin cell RNA
failed to reveal a homolog of an accessory
subunit known to
influence Slo channel function. Furthermore, a
-subunit-dependent Slo channel activator,
dehydrosoyasaponin I, was without effect on chromaffin cell BK current.
The results argue that an accessory subunit may not be a required
component of the native chromaffin cell BK channel.
In contrast to the extended gene families of the voltage-gated K+ channel families, molecular diversity in large conductance, voltage- and calcium (Ca2+)-activated channels (termed BK1 channels) appears to arise largely as a function of alternative splicing of a single gene product encoded by the Slo locus both in mammals (1-3) and Drosophila (4, 5). However, considerable functional diversity in BK channels exists among different tissues (6), the origins of which remain to be fully explained. Rat chromaffin cells express either of two phenotypically distinct forms of BK current (7, 8). One, termed BKi current, exhibits rapid inactivation following current activation by depolarization and elevated intracellular calcium ([Ca2+]i), while the second current, termed BKs, exhibits the more usual sustained activation as a function of [Ca2+]i and voltage.
The primary purpose of the present work was to identify and
functionally characterize alternatively spliced forms of Slo
found in chromaffin cells and PC12 cells, a rat pheochromacytoma line. One Slo alternative splice variant was found in both
chromaffin cells and PC12 cells and encodes a pattern of cysteine
residues with similarity to cysteine-rich regions in a number of other proteins. Slo constructs containing this insert exhibit a 20-mV leftward shift in the apparent voltage dependence of gating at a given
[Ca2+] relative to constructs lacking this exon. Another
variant also found in chromaffin cells was alternatively spliced at the
C terminus, but was functionally indistinguishable from mSlo. However,
no variants so far identified account for the inactivating phenotype of
some chromaffin cell BK channels. Thus, inactivation behavior of
BKi channels may not be governed by the properties of a
particular Slo subunit itself. We then investigated whether a
homolog of a known accessory
subunit of Slo channels (9, 10)
might be found in these cells. Using both functional criteria and
PCR-based screens for a
subunit, our results indicate that such an
accessory subunit is not a component of BK channels in chromaffin
cells. These results do not eliminate the possibility that other
accessory subunits, different from the previously characterized
-subunit (9), are present in these cells.
PC12 cDNA libraries (courtesy of Drs. Jim Boulter (Salk
Institute) and N. Gautam (Washington University School of Medicine)) and a commercial mouse chromaffin cell cDNA library
(CLONTECH) were screened at high stringency by
using the entire mSlo coding sequence as a probe. The probe
was labeled with [32P]dCTP. Eight independent cDNA
clones were isolated from the PC12 library. Two cDNAs contained
virtually complete coding sequences with the exception of some
N-terminal sequence and initiation methionines. Variation in N-terminal
sequences was investigated by PCR screening of the cDNA libraries
with primers containing vector-specific and gene-specific sequence. One
presumptive initiation methionine was obtained by this method, and this
N terminus was verified in rat chromaffin cells using the RACE (rapid
amplification of 5 ends) method (11).
Total RNA was extracted
from freshly isolated tissues (50-200 mg) by using a modified
Chomczynski and Sacchi method (TriZol reagent, Life Technologies,
Inc.). The first strand cDNAs were synthesized from the total RNA
by reverse transcriptase (SuperScript II, Life Technologies, Inc.) by
use of oligo(dT) primers. cDNAs were then used either for
evaluation of the presence of different alternatively spliced variants
or for isolation of unknown 5 end variations using the RACE
method.
By using nested primer sets encoding the leading and trailing sequences adjacent to splice junction 2 (1, 2), alternatively spliced exons for this site were amplified from various tissues. The first round, outside primer sequences were: TTTAGGATTTTTCATCGCAAGTGA (sense) and GTGAAACATTCCAGTGGAGTCGTA (antisense). The second round, inside primer sequences were: TACTGCAAGGCCTGTCATGATGAC (sense) and GTGAAACATTCCAGTGGAGTCGTACTT (antisense).
The RACE reactions utilized a gene-specific antisense primer close to
the initiation methionine in the mSlo coding sequence. Rat
chromaffin cell total RNA was reverse-transcribed with a gene-specific primer: CCGCCAGAGCAAGATGATGAAGA. Excessive primer was removed by
GeneClean II (BIO 101 Inc.), and the poly(A) sequence was added at the
5 end of the cDNAs by terminal transferase (Life Technologies, Inc.) for 15 min at 37 °C. The primer sequences were as follows. Primer set I: TAACCCTCACTAAAGGGAACATCGATTTTTTTTTTTTTTTTT
(sense) and gene-specific primer I TGAGCGCATCCATC (antisense); primer set II: GCGCAATTAACCCTCACTAAAGGGAACA (sense) and gene-specific primer II CCGCCAGAGCAAGATGATGAAGA (antisense).
Since none of the PC12
clones were full-length, the functional role of the unique splice site
2 exon was investigated by subcloning a 557-base pair
HindIII-NheI piece encoding the PC12 Slo
59-amino acid insert into an mSlo expression vector. For
convenience, we used segments of a mouse cDNA (mbr5 (Ref. 1)),
which were known to encode amino acid sequence identical to the rat
Slo channel. To this we added 5, 3
, and alternative exon
sequence as mentioned in the text. To make these constructs, we
utilized two unique restriction sites, HindIII and
NheI, which are conserved in both mouse (mSlo)
and rat (PC12) sequences.
For expression studies, capped cRNA was prepared by run-off
transcription with T3 RNA polymerase (mMessage mMachine, Ambion). Approximately 50 nl (1 µg/) of cRNAs were injected in stage IV Xenopus oocytes. The oocytes were incubated for 1-6 days in
ND96 and maintained at 17 °C.
Rat chromaffin cell cultures followed standard procedures used in this laboratory (12-14). Recordings were made from cells maintained in culture for 2-10 days.
Electrophysiological MethodsThe vitelline membrane from injected oocytes was removed with fine forceps in hypertonic saline. All recordings were made at room temperature. The oocytes were bathed in a standard extracellular solution (ND-96: 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 2.5 mM sodium pyruvate) during patch formation. For inside-out membrane patches, the pipette solution contained 140 mM potassium methanesulfonate, 20 mM KOH, 2 mM MgCl2, 10 mM HEPES, adjusted to pH 7.00 with 5% methanesulfonic acid. Just prior to excision of inside-out patches, the solution bathing the patch was changed to a 0 Ca2+ solution. Cytosolic solutions of defined [Ca2+]i were prepared as described (15) with all [Ca2+] verified with a Ca2+-sensitive electrode (catalog no. 93-20, Orion). The predominant anion in these solutions was methanesulfonic acid. For outside-out recordings, the patch pipette solution was the 100 µM CaCl2 solution.
For recordings from inside-out patches from chromaffin cells, the cells were bathed with an extracellular saline (in mM: 140 NaCl; 5.4 KCl, 10 HEPES, 1.8 CaCl2, and 2.0 MgCl2, pH 7.4) during seal formation and bathed with a 0 Ca2+ saline (in mM: 140 KCl, 20 KOH, 10 HEPES, 5 EGTA, pH 7.0) during excision of the patch. The pipette saline contained the following: (in mM) 140 KCl, 20 KOH, 2 MgCl2, 10 HEPES, pH 7.0, adjusted with 1 N HCl, with 200 nM apamin to block small conductance, voltage-independent, Ca2+-activated SK-type currents. For cytosolic solutions of defined Ca2+, the composition was identical to that used for the 0 Ca2+ solution, except that HEDTA was used to buffer 4 and 10 µM Ca2+ solutions. Estimates of free [Ca2+] were determined as described previously (7, 8).
For patches from either oocytes or from chromaffin cells, solution exchange and drug applications were accomplished with a multibarrel solution delivery system (7, 13). Charybdotoxin (CTX) and dehydrosoyasaponin I (DHS-1) were gifts from colleagues at the Merck Research Laboratories (Rahway, NJ). CTX obtained from Peninsula Laboratories was used in some experiments. All other chemicals were from Aldrich or Sigma. Osmolarity was measured by dew point (Wescor Osmometer) and adjusted within 3% (internal saline, 290; external saline: 305). In experiments with CTX or apamin, each agent was added directly to the extracellular saline without additional adjustments. Removal of inactivation from chromaffin cell BK channels was accomplished by brief exposure to 0.3-0.5 mg/ml trypsin as described (7).
Currents were recorded with an Axopatch 1C amplifier (Axon Instruments,
Foster City, CA). Voltage protocols and data acquisition were done
using programs from pClamp (Axon Instruments). Fitting of current
waveforms or extracted data was done using a Levenberg-Marquardt search
algorithm to obtain non-linear least-squares estimates of function
parameters. Conductance-voltage curves were fit with a single Boltzmann
function, G(V) = Gmax/(1 + exp(V50 V)/k), where
Gmax reflects the maximal conductance,
V50 is the voltage of half-maximal activation,
V is the activation potential, and k is the slope
factor reflecting the voltage dependence of conductance.
Estimates of drug dissociation constants (Kd) were
made by fitting simultaneously the entire time course of blockade and
recovery at one or more drug concentrations to a first order blocking
reaction (open blocked), with forward blocking rate given by
f[D], where [D] is the concentration of blocking drug, and reverse blocking rate given by b. Data points were
evaluated over two time regimes where t0 = 0 is
the time of drug application and t1, the time of
washout of drug (Equations 1 and 2).
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(Eq. 1) |
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(Eq. 2) |
Slo Homologs in Rat Chromaffin Cells and PC12 Cells
cDNA libraries from PC12 cells were screened at high stringency
for mSlo homologs. Eight unique clones were identified
(PC1-PC8). Two cDNAs encompassed most of the Slo coding
region, but lacked an initiator methionine and the carboxyl end. The
amino acid sequence was identical to the mSlo sequence, mbr5
(1), differing only at an alternative splice site termed splice
junction A by Butler et al. (1) or splice junction 2 by Tseng-Crank et al., (2). Since this site
corresponds to the location of the second splice site in the mammalian
Slo sequence, we will refer to this site as splice site 2. The extents of the eight cDNAs are diagrammed onto the
mSlo sequence in Fig. 1A.
Alternative Splice Variants
Two distinct alternative splice
forms were identified for splice junction 2. PC8, which has the longest
5 end, does not have any insert at splice junction 2. A second form
encodes a peptide of 59 amino acids and was found in three clones, PC1,
PC3, and PC5. These 59 amino acids share no sequence homology with any portion of other known proteins. However, a pattern of repeated cysteines beginning just upstream of the splice junction and including the 59 amino acids shares similarity both to variants of phospholipase A2 (PLA2) and to zinc-finger domains of protein
kinase C (PKC) and diacylglycerol kinase (DGK) (Table
I). In PLA2, the cysteines form disulfide
linkages, whereas, in PKC and DGK, the cysteines participate in
defining a zinc-binding site. By PCR methods, both splice junction 2 variants were found in both rat and mouse adrenal chromaffin cells. In
contrast to variation observed at splice junction 2, none of the clones
had an alternative exon at the previously described splice junction B
(1). The sequence encoding the 59-amino acid insert was confirmed three
times on both nucleotide strands.
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Previously published mSlo and hSlo homologs have been shown to differ at the C terminus. Specifically, following Q1187 there is sequence divergence between mSlo and hSlo (1, 3). Similarly, one cDNA clone from PC12 cells (PC7) contained sequence which was identical to mSlo and hSlo through Q1187, but which diverged downstream (Table II). This appears to represent a previously unrecognized splice site in both rodents and humans. Since four other splice sites were previously described in mammalian Slo forms (2), this new splice junction should be considered splice site 5.
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Since an initiator methionine was not
found in any of the PC12 cDNA clones, PCR methods were used to
isolate the 5 ends from RNA. Two sets of nesting primers were
designed. Sense primers were specific to the cloning vector
(pBlueScript, Stratagene); antisense primers were specific to PC12
cDNA sequence. From PCR screening, one potential 5
end was
isolated with the following sequence.
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Although N-terminal variation has also been obtained from mouse cDNA libraries (1), genomic Slo nucleic acid sequences do not reveal a mechanism by which such N-terminal diversity might be generated, because intron consensus splice junctions are not present in this region (3).
Tissue Distribution of the 59-Amino Acid InsertThe tissue distribution of the alternative splice variant encoding the 59-amino acid peptide was examined by splice-site specific RT-PCR (Table III). RT-PCR was used to amplify mRNAs from various tissues using sense and antisense primers to regions flanking this splice junction. Resulting PCR products were separated on 2% agarose gels. An approximately 442-base pair fragment, which encoded the 59-amino acid exon, was abundant in PC12, chromaffin cells, pancreas, cerebellum, and pituitary. It was present but less abundant in hippocampus. Spinal cord, smooth muscle, and skeletal muscle failed to reveal an exon of comparable size. The smaller and larger splice fragments were detected in relatively equal abundance in chromaffin cells. Partial sequencing of the 442-base pair band obtained from hippocampus revealed complete identity with the PC12/chromaffin cell exon. The exon is similar, but not identical, to an exon also present in human pancreatic islets (24). PCR fragments of other sizes were not detected in these tissues, indicating that only two alternative forms may be expressed in these tissues.
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Functional Properties of Slo Variants Found in PC12 and Chromaffin Cells
Functional Properties Conferred by Site 2 and Site 5 Alternative ExonscDNAs containing sequence encoding the 59-amino acid
insert were studied in a rat Slo background (mSlo and rat
Slo peptides are identical; see "Materials and Methods") and
also in association with the alternative N-terminal domains from
PC12/chromaffin cell cDNA. Currents resulting from expression of
various Slo constructs were studied in excised inside-out patches
from Xenopus oocytes. Patch currents resulting from
Slo channels containing the unique insert (Slo-zf; Fig.
2A) or from mSlo/rSlo (Fig. 2B)
were elicited by voltage-steps in the presence of 1-100
µM Ca2+. In comparison to mSlo
currents, channels containing the 59-amino acid domain were
activated by more negative voltages at comparable [Ca2+]i. Specifically, the presence of the exon
produces a 20 to
30 mV shift in the conductance-voltage relation,
suggesting an increase in apparent Ca2+ sensitivity when
compared with wild-type mSlo at a given membrane potential (Fig. 2,
B and C). V50 values for
activation of currents resulting from the expression vector containing
both the 59-amino acid insert and the 25-amino acid N-terminal sequence
(MWISH-slo-zf) were indistinguishable from those without the N-terminal
sequence (Fig. 2C). Slo channels containing the 59 amino acid insert at splice junction 2 were also comparable to mSlo
channels in terms of the ability of Mg2+ to shift
activation to more negative voltages (data not shown). Although
Mg2+ itself is unable to activate Slo channels, the
addition of 10 mM Mg2+ to a solution containing
100 µM Ca2+ results in an approximately
60-mV shift in the V50 for activation, which
is comparable to the shift caused by Mg2+ on mSlo channels
(25).
The functional consequences of the unique C-terminal exon found in
chromaffin cell Slo homologs were also examined both with (Slo-zf-3) and without (Slo-3
) the 59-amino acid insert. Currents were activated with 10 µM submembrane Ca2+
over a range of voltages. Neither Slo-zf-3
(16 of 16 patches) nor
Slo-3
(5 of 5 patches) constructs result in inactivating currents
(e.g. Fig. 3). The V50
for activation of Slo-zf-3
at 10 µM Ca2+ was
7.7 + 1.3 mV (2 patches; mean ± 90% confidence limit of fit),
while for the V50 for Slo-3
was 42.8 ± 2.1 (4 patches). These values are similar to those for constructs
containing the mSlo C terminus (e.g. Fig. 2C).
Thus, the chromaffin cell 3
terminal does not appear to influence the
activation properties of mSlo. Furthermore, for constructs containing
the chromaffin cell C terminus, currents activated by 400-ms
voltage-steps to +60 mV exhibited no inactivation, indicative that the
3
addition does not influence inactivation properties of Slo.
To examine the basis for the shift in apparent Ca2+
sensitivity of the Slo-zf constructs, the time courses of current
activation and deactivation were examined at 4, 10, 30, and 100 µM Ca2+ over a range of voltages. These were
reasonably well fit by single exponential functions, although, in some
cases, currents could be better described by two exponential components
(26). In Fig. 4A, the time constants derived
from single exponential fits to the current activation time course are
plotted for mSlo and the Slo-zf construct for voltages positive to 0 mV. Over this range, Slo-zf currents activate somewhat faster than mSlo
currents at identical [Ca2+]. Differences in time
constants of current deactivation between Slo-zf and mSlo are somewhat
more pronounced. In Fig. 4B, deactivation and activation
time constants for a set of patches at either 4 or 100 µM
Ca2+ are plotted for both mSlo and Slo-zf. Relative to mSlo
currents, Slo-zf currents activate somewhat more rapidly and deactivate clearly more slowly at a given Ca2+ and voltage. The
V50 for activation corresponds approximately to
that voltage where the activation time constant and deactivation time
constant at a given [Ca2+] are identical. As a
consequence, differences in both activation time and deactivation time
between mSlo and Slo-zf contribute to the negative shift in the
V50 for activation of conductance, although
effects on deactivation may be somewhat more important.
Sensitivity to CTX
The inactivating variant of BK current (BKi) found in native rat chromaffin cells is approximately an order of magnitude less sensitive to blockade by CTX than the noninactivating variant (Kd~ 1-4 nM).2 We therefore tested whether the presence of the 59-amino acid insert might alter the sensitivity of Slo channels to CTX. Currents were recorded in outside-out patches activated by voltage-steps to +60 mV with 100 µM Ca2+ in the recording pipette. The time course of onset and recovery from CTX blockade was used to define the blocking efficacy. As shown in Fig. 4C, application of 20 nM CTX leads to an almost complete blockade of Slo-zf currents. Both the onset and recovery of blockade proceed with an approximately single exponential time course; the time course of the reduction and recovery in current amplitudes resulting from steps to +60 mV can be reasonably fit by the first-order blocking model described under "Materials and Methods." The estimated Kd for blockade by CTX was 2.2 nM at +60 mV, comparable to blockade of BKs currents by CTX in native rat chromaffin cells.2 Thus, the 59-amino acid insert is unlikely to account for the reduced CTX sensitivity of inactivating BK channels in native chromaffin cells.
Is an Accessory Subunit an Obligatory Part of Chromaffin Cell BK Channels?
Recently, a gene encoding an accessory subunit that is
associated with the BK channel of bovine arterial smooth muscle (10) has been cloned (9). This subunit enhances the apparent
Ca2+ sensitivity of cloned mSlo channels (27) and hSlo
channels (3, 28). Similar to its interaction with other Slo variants, the bovine
subunit, when coexpressed with Slo
subunits
containing the 59-amino acid insert, shifted the apparent
Ca2+ dependence of current activation (six of six patches).
For these six patches, the V50 for activation of
conductance with 10 µM Ca2+ exhibited
considerable variability, ranging from
48.6 to
109 mV. This
compares to a V50 for activation at 10 µM of about
10 mV for Slo-zf constructs in the absence
of a
subunit (Figs. 2C and 3B). This negative
shift shows that the 59-amino acid insert does not reduce the ability
of the Slo
subunit to interact with the bovine
subunit. Similar
-subunit-induced shifts were also observed when the unique
chromaffin cell N-terminal domain was appended to the Slo-zf
sequence.
A unique feature of the subunit is that it permits the triterpenoid
glycoside BK channel agonist (29), DHS-I, to shift the apparent
Ca2+ dependence of activation (27). Similarly, the bovine
subunit confers sensitivity to DHS-I on Slo-zf channels (Fig.
5, A and B). These results would
argue that, if BK channels in chromaffin cells are normally associated
with an homologous
subunit, those channels should be sensitive to
DHS-I. However, when chromaffin cells are exposed to 500 nM
DHS-I, there is no indication that the sensitivity of BK channels to
Ca2+ is altered (Fig. 5, C and D).
This lack of effect of DHS-I was observed in seven patches containing
BKi channels and one patch containing exclusively
BKs channels (result not shown). We did note that in some
patches there was a tendency for a slight enhancement of BK current in
the presence of DHS-I at more negative activation voltages. This
appeared to be associated with a longer apparent burst duration once a
BK channel opened. However, this effect was insignificant in comparison
to effects of DHS-I on Slo channels coexpressed with the
subunit.
Coupled with the observation that the Ca2+ dependence of
the Slo-zf homolog alone is similar to that of BK channels in normal rat chromaffin cells (14), the lack of effect of DHS-I suggests that
association of endogenous BK subunits in chromaffin cells with the
known type of subunit does not occur. Alternatively, if chromaffin
cell BK channels contain a homologous
subunit as an integral
portion of the channel protein, this subunit does not influence the
apparent Ca2+ sensitivity of the channel and this subunit
must be insensitive to DHS-I.
Finally, rat chromaffin cell RNA was screened with degenerate PCR
methods by using primers based on the cloned bovine subunit to test
for the presence of a
subunit. Two positive controls were used in
these experiments, the bovine BK
cDNA clone (9) and first
strand cDNA from rat smooth muscle (stomach). Amino acid stretches
with low sequence degeneracy were chosen as primer sites. The first
round of PCR was carried out with a primer set that has degenerate
sequences at the 3
ends. Degeneracy was 8-fold for the sense primer,
and 16-fold for antisense primer. The PCR reaction was diluted 50 times. One µl was used as a template for the second round
non-degenerate PCR reaction. Using the above protocol, the cDNA
from rat smooth muscle and cDNA from the cloned
subunit
(control) generated a 245-base pair fragment. However, the cDNA
from adrenal chromaffin cells and a negative control reaction without
cDNA template did not generate any PCR products.
The major findings of interest in this study are as follows. First, a unique alternative splice variant that defines a cysteine-rich region is found in Slo variants isolated from rat chromaffin cells and some other tissues. Second, we have described a fifth potential alternative splice site in mammalian Slo. Third, several arguments suggest that a known accessory subunit of BK channels is probably not a component of BK channels in chromaffin cells.
Slo Alternative Splice Variants and Their Relationship to Native BK Currents in Chromaffin CellsWe have identified variants of Slo channels found in cDNA and RNA from rat chromaffin cells and PC12 cells. Two splice variants, one not previously noted in any other tissues, are present at a previously described splice junction (A in the terminology of Butler et al. (1); 2 according to Tseng-Crank et al. (2)). In addition, a fifth mammalian alternative splice junction near the C terminus was also found in chromaffin cell Slo variants, and a unique C-terminal sequence was identified.
The unique splice site 2 variant encodes a peptide with a pattern of repeated cysteine residues, which as discussed below, may serve to define a secondary structure critical for interaction of this region with some other molecule. The greater apparent Ca2+ sensitivity of Slo variants containing this 59-amino acid insert in comparison to mSlo channels increases the number of alternatively spliced Slo variants that have distinct functional properties (2, 30). As observed in other studies, alternative splice variation may be important in defining the ability of particular BK channels to open as a function of Ca2+ or voltage.
Normal adult rat chromaffin cells are known to express two phenotypic
variants of BK channels (8). One form exhibits rapid inactivation (7),
while the second form is relatively non-inactivating and similar to BK
channels described in many other cell types (8). None of the various
Slo constructs expressed in the present work exhibit inactivation, and
the molecular basis for inactivation of BK channels in chromaffin cells
remains undetermined. However, as a result of our work, a number of
factors can now be eliminated as possible elements of the inactivation
mechanism. First, a novel N-terminal sequence found in rat chromaffin
cells does not result in BK channel inactivation. Second, the 59-amino
acid insert found in chromaffin cell Slo variants does not confer
inactivation on Slo currents when expressed in Xenopus
oocytes. Third, an accessory subunit (Kv1; Ref. 31), which has been
shown to produce inactivation in Shaker homologs, but not other
mammalian voltage-dependent channels, is also unable to
produce inactivation of Slo channels.3
Fourth, the C-terminal splice variant found in chromaffin cell Slo
channels does not produce inactivation. The possibility that additional
Slo variants, which may affect inactivation, remain to be identified in
chromaffin cells cannot be completely excluded.
The sensitivity of inactivation to cytosolic trypsin argues that the inactivating domain is probably a cytosolic peptide (7). The slowing of inactivation during trypsin digestion argues that there are multiple inactivation domains per channel (32).4 Furthermore, inactivation is unaltered and stable following patch excision. The following explanations for BK inactivation remain to be explored. First, a currently unidentified N-terminal sequence in rat chromaffin cells may be competent to produce inactivation. Since there is no genomic basis for N-terminal variation in Slo, this possibility seems unlikely. Second, an unidentified homolog of the known accessory subunits may produce inactivation. Third, an unknown type of accessory subunit may produce inactivation. Fourth, unknown post-translational modifications specific to particular Slo variants or particular expression systems may confer inactivation properties. Work in progress hopes to address these possibilities.
It remains a possibility that the 59-amino acid insert, although not conferring inactivation itself, may be correlated with one of the two phenotypic variants of BK channel found in chromaffin cells, perhaps the inactivating variant. However, none of our present data allows us to make such a correlation. Nevertheless, the presence of this exon in chromaffin cells, PC12 cells, and pancreatic beta cells, all of which express inactivating BK channels (7, 32), is consistent with the proposal that Slo subunits containing the 59-amino acid insert are integral components of BKi channels. However, contrary to this hypothesis, this alternative exon is also found in GH3 cells in which inactivating BK channels have not, as yet, been observed. Therefore, the function of this splice variant may be unrelated to the inactivation properties of BK current, but critical to some other functional role characteristic of neurosecretory cell types.
A similar 60-amino acid peptide is encoded by an alternative splice variant found in human islet cells (24). The islet cell insert contains an additional upstream arginine and differs at two amino acids within the insert. This human pancreatic islet Slo variant has not as yet been functionally expressed.
Possible Role of the Cysteine-rich DomainConserved patterns of cysteine residues are a common feature of many proteins and are usually indicative of the formation of peptide loops that are critical in defining key structural features of the protein. In the case of zinc-finger domains, such cysteines along with key histidine residues form coordination foci for the zinc ion (20, 21, 33, 34). Formation of the zinc-finger loops thereby defines a secondary structure critical for interaction of the zinc-finger domain with other molecules. Such zinc-binding domains are known to mediate interactions of proteins with nucleic acids (35), with other proteins (34, 36), and with phospholipids (33). The role of the cysteines in PLA2 variants is somewhat different. In this case, disulfide linkages between particular cysteines helps to orient residues that are critical for Ca2+ binding and catalytic activity (17, 19). It has been proposed that the similarities in the cysteine-rich motif in PLA2 and PKC reflect similar functional roles (37, 38). The similarity of the cysteine pattern in the Slo-zf variant both to the known zinc-finger domains of PKC and DGK and to the cysteines defining disulfide linkages in PLA2s is provocative. Thus, irrespective of whether the cysteine residues in this Slo variant form a zinc-finger domain or participate in disulfide linkages, it seems likely that this region defines peptide loops anchored by the cysteines. A common feature of this region in PKC variants, DGK, and PLA2 is the ability to bind a metal ion and also a phospholipid. At present, we have no evidence that would allow us to infer whether a metal ion remains tightly associated with this region in the Slo-zf variant. However, as a guide to future investigation, we propose that this region defines a metal binding site, which may serve to mediate interactions with some other molecule, perhaps an unknown type of accessory subunit.
Accessory Subunits in Chromaffin Cell BK Channel FunctionPrevious work indicates that Slo peptides in
bovine smooth muscle are associated with an accessory subunit (10).
The ability of this subunit to increase the apparent Ca2+
sensitivity of cloned Slo variants (3, 27, 28) has led to the view
that the relatively greater sensitivity of some smooth muscle BK
channels to Ca2+ relative to cloned Slo channels may
result from the association of Slo peptides and
subunits.
Furthermore, the coexpression of the Slo peptide and
subunits
also confers a sensitivity to the diterpene, DHS-I (27).
In contrast to the situation in smooth muscle, the apparent
Ca2+ dependence of cloned chromaffin cell Slo channels
is similar to that previously described for native BK channels in
chromaffin cells (8, 14). Although this could be a fortuitous
consequence of the two different membrane environments, more simply it
would suggest that an accessory subunit is not a required component of chromaffin cell BK channels. Furthermore, although the cloned chromaffin cell Slo variants can interact with the bovine
subunit in a fashion similar to other Slo variants, native BK
channels in chromaffin cells are insensitive to DHS-I. These
experiments lead to either of two conclusions. Either a related
subunit is not an obligatory component of functional BK channels in
chromaffin cells or, alternatively, there is a different
subunit
variant in the chromaffin cells which does not confer sensitivity to
DHS-I and which does not significantly alter the apparent
Ca2+-sensitivity of native chromaffin cell BK channels
relative to the sensitivity of the cloned channels when expressed in
oocytes. Although we cannot exclude the possibility that unknown
accessory subunits may interact with chromaffin cell Slo peptides,
the results argue that a
subunit, similar to that found in smooth
muscle, is not a required component of BK channels in all cells.