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The Journal of Neuroscience, July 1, 2000, 20(13):4890-4903
Rectification and Rapid Activation at Low Ca2+ of
Ca2+-Activated, Voltage-Dependent BK Currents: Consequences
of Rapid Inactivation by a Novel  Subunit
Xiao-Ming
Xia,
Jiu-Ping
Ding,
Xu-Hui
Zeng,
Kai-Lai
Duan, and
Christopher J.
Lingle
Washington University School of Medicine, Departments of
Anesthesiology, and Anatomy and Neurobiology, St. Louis, Missouri 63110
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ABSTRACT |
A family of accessory  subunits significantly contributes to the
functional diversity of large-conductance, Ca2+- and
voltage-dependent potassium (BK) channels in native cells. Here we
describe the functional properties of one variant of the subunit
family, which confers properties on BK channels totally unlike any that
have as yet been observed. Coexpression of this subunit (termed 3)
with Slo  subunits results in rectifying outward
currents and, at more positive potentials, rapidly inactivating (~1
msec) currents. The underlying rapid inactivation process results in an
increase in the apparent activation rate of macroscopic currents, which
is coupled with a shift in the activation range of the currents at low
Ca2+. As a consequence, the currents exhibit more
rapid activation at low Ca2+ relative to any other
BK channel subunit combinations that have been examined. In part
because of the rapid inactivation process, single channel openings are
exceedingly brief. Although variance analysis suggests a conductance in
excess of 160 pS, fully resolved single channel openings are not
observed. The inactivation process results from a cytosolic N-terminal
domain of the 3 subunit, whereas an extended C-terminal domain does
not participate in the inactivation process. Thus, the 3 subunit
appears to use a rapid inactivation mechanism to produce a current with
a relatively rapid apparent activation time course at low
Ca2+. The 3 subunit is a compelling example of
how the subunit family can finely tune the gating properties of
Ca2+- and voltage-dependent BK channels.
Key words:
accessory subunits; K+ channels; BK
channels; Ca2+- and voltage-gated
K+ channels; mSlo channels; inactivation
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INTRODUCTION |
Ca2+-
and voltage-dependent K+ (BK-type)
channels, like the voltage-gated K+
channels, are composed of four homologous subunit peptides that
contribute to the ion channel pore (Atkinson et al., 1991 ; Adelman et
al., 1992; Butler et al., 1993; Shen et al., 1994), along with accessory subunits that regulate many important aspects of BK channel
function (McManus et al., 1995; Wallner et al., 1995, 1999; Dworetzky
et al., 1996; Tseng-Crank et al., 1996; Nimigean and Magleby, 1999; Xia
et al., 1999). Typically, BK channels exhibit a stereotypic, large
unitary conductance but exhibit considerable functional diversity in
regards to kinetic behavior, apparent Ca2+
dependence, and pharmacology (McManus, 1991). Thus, BK channels can
play a variety of physiological roles well suited to the demands of the
cells in which they are found. The functional diversity can, in part,
be accounted for by various splice variants of the subunit encoded
by the Slo loci (Adelman et al., 1992; Tseng-Crank et al.,
1994; Jones et al., 1998; Ramanathan et al., 1999). However, the family
of accessory subunits may, in fact, play a more important role in
defining the phenotypic properties of BK channels, including the
effective gating range and inactivation behavior (McManus et al., 1995;
Jones et al., 1998; Wallner et al., 1999; Xia et al., 1999), because
the functional variation arising from Slo splice variants
appears rather modest (Adelman et al., 1992; Tseng-Crank et al., 1994;
Saito et al., 1997; Jones et al., 1998; Ramanathan et al., 1999). To
date, including the subunit studied in this paper, four distinct
members of a mammalian subunit family have been identified [KCNMB1
(1) (Knaus et al., 1994); KCNMB2 (2) (Wallner et al., 1999; Xia
et al., 1999); KCNMB3 (3) (Riazi et al., 1999; Brenner et al., 2000;
Uebele et al., 2000); KCNMB4 (4) (Wickenden et al., 1999; Brenner et
al., 2000; Meera et al., 2000; Wallner et al., 2000)], each of which
appears to confer unique functional properties on the resulting BK
channels. Definition of the functional properties of the subunits
is an essential step in understanding the diversity of phenotypic
properties of BK channels in native cells.
Here, we describe the functional properties of new subunit (termed
3) that confers onto BK channels properties totally unlike any that
have as yet been described. Genomic sequence encoding a portion of this
subunit has been described recently (Riazi et al., 1999), although the
subunit we have identified contains differences in the N terminus from
that proposed. Coexpression of this new subunit with Slo subunits results in an extremely rapidly inactivating, Ca2+- and voltage-dependent current with
pharmacological properties characteristic of previously described BK
channels. However, the currents have several properties unlike other BK
currents, including rectification of outward current and extremely
flickery, unresolvable single channel openings. The effects of this
particular subunit family member illustrate an extreme example of
the diverse ways ion channel function can be altered by an accessory subunit.
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MATERIALS AND METHODS |
Identification of the full-length cDNA clone of
3. Using homology searching of human expressed sequenced
tagged (EST) databases based on BK 1 (Knaus et al., 1994), an
avian subunit (Oberst et al., 1997), and 2 (Wallner et al.,
1999; Xia et al., 1999) sequences, several partial cDNA sequences
(GenBank accession numbers AA761761, AA195511, AA823768, and
AA236968) suggestive of a new subunit family member were
identified. These cDNA clones were obtained from Genome Systems (St.
Louis, MO) and resequenced with BigDye Terminator Kit (Applied
Biosystems, Foster City, CA). The four EST cDNA clones exhibited
sufficient overlap to define most of the 3 sequence, including the
3' terminus, but terminated at a position aligning within TM1 defined
by the other subunits. Thus, the EST cDNA clones failed to identify
a full-length clone and lacked the start codon at the 5' end. To search
for the 5' coding sequence, a nested PCR was performed on human heart
cDNA library (Stratagene, La Jolla, CA), from which several of the EST
clones had been identified. The two rounds of PCR were performed with a
vector-specific primer and a 3-specific primer (first PCR, Lacmer
and 5'-CATCATGGCAAACCCCAGCATCAC-3'; second PCR, T3 primer and
5'-GAAGATCTCCCAGCATCACGGCTCGGTCCTCTCC-3', with pfu polymerase,
30-36 cycles, 96°C for 30 sec, 50°C for 45 sec, and 72°C for 2 min). The PCR products were then purified and digested with
EcoRI and BglII overnight and then gel purified
and subcloned in pSK plasmid (Stratagene). Sequencing revealed that one
pSK clone contained an extra 500 base pairs upstream of the 3
sequence. The continuity of this sequence to the rest of 3 was
justified by the fact that 46 base pairs at the 3' end of the
PCR-generated fragment were identical to sequence obtained from the
partial 3 sequence identified by the EST sequences. This new
sequence identified by PCR define 19 additional amino acids at the 3
N terminus that precede an upstream stop codon in the same reading frame.
Tissue distribution of the 3 message. Northern
blots were performed on membranes purchased from Clontech (Palo Alto,
CA) containing ~2 µg of poly(A+) RNA
per lane from various human tissues. The PCR fragment of full-length
3 was 32P-labeled as the hybridization
probe. The vendor adjusted the RNA loading of each lane based on
previous blots using human -actin cDNA as probe. The experimental
conditions used were described previously (Xia et al., 1999).
Expression constructs. The Xenopus oocyte
expression vector pBF was used to subclone all of the DNA constructs
(Xia et al., 1998). The mSlo subunit [GenBank
accession number L16912 (Butler et al., 1993)] was digested with
ClaI and filled in to generate blunt ends at the 5' end of
mSlo; a 3' SalI site was used to obtain a
blunt-end SalI fragment. The resultant mSlo was then subcloned into a HpaI-SalI-digested pBF
vector. The full-length 3 expression clone was generated by
overlapping PCR and DNA manipulation. The 3' part was obtained by
subcloning 3 EcoRI fragment from EST clone AA761761 to
the EcoRI-digested vector portion of AA195511, which
contains partial 3 3' end sequence. This intermediate construct,
which is a partial 3 sequence lacking the 5' end, was screened by
sequencing to define the correct orientation. The complete full-length
3 clone was then acquired by overlapping PCR (Xia et
al., 1999) (first round PCR primer, reaction A,
5'-AGGGATCCACTGCCAATGACAGCCTTTCCTG-3' and
5'-GGCAGCCTCTTGTGCACATCTAGTGGGTCTCCATC-3'; reaction B,
5'-GATGGAGACCCACTAGATGTGCACAAGAGGCTGAA-3' and
5'-TACTAGTCGACTTAAGATTTCTCTGCTCTTCCTT-3'; second round PCR primer,
5'-AGGGATCCACTGCCAATGACAGCCTTTCCTG-3' and
5'-TACTAGTCGACTTAAGATTTCTCTGCTCTTCCTT-3'; pfu polymerase, 30-36
cycles, 96°C for 30 sec, 52°C for 45 sec, and 72°C for 2 min).
The final overlapping PCR product was digested with BamHI
and SalI and subcloned in the
BamHI-SalI pBF oocyte expression vector (Xia et
al., 1998). Both strands of DNA were sequenced for verification.
3 N-terminal or C-terminal deletion constructs D3, D4, and D5 were
generated by pfu PCR with specific 3 primers (D3 primer, D4 primer,
and D5 primer). The 2 N terminus 3 chimera (construct D1) and
3 N terminus 2 chimera (construct D20) were generated by
overlapping PCR (Xia et al., 1999). To study the other
potential 5' alternative h3 variant (Riazi et al., 1999), we
amplified the 5' end from HEK293 genomic DNA using pfu PCR and then
used overlapping PCR to link the 5' sequence with the rest of the 3' h3 sequence. In the first round PCR, two PCR reactions were
performed using either human genomic DNA (A) or h3 DNA (B) as
templates (reaction A primers,
5'-ATATATCTAGACA CAGGTAGGCAGCAAATGAGATTATCC-3' and
5'-GGCAGCCTAT TGTGCACATCTAGTGGGTCTCCATC-3'; reaction B primers, 5'-GATGGAGACCCACTAGATGTGCACAAGAGGCTGCC-3' and
5'-CTACGTCGACTTAAGATTTCTCTGCTCTTCCTT-3'; pfu DNA polymerase, 96°C for
30 sec, 50°C for 30 sec, and 72°C for 1 min, 36 cycles). The second
round PCR was performed using both reaction A and reaction B as
templates (primer, 5'-ATATATCTAGACACAGGTAGGCAGCAAATGAGATTATCC-3' and
5'-CTACGTCGACTTAAGATTTCTCTGCTCTTCCTT-3'; pfu DNA polymerase, 96°C for
30 sec, 50°C for 30 sec, and 72°C for 2 min, 36 cycles). The reaction product was then purified, digested with
XbaI and SalI overnight, gel-purified, and
subcloned into the XbaI-SalI pBF vector. Both
strands of DNA were sequenced for verification. This molecule is
referred to as Gmh3.
Expression in Xenopus oocytes. Methods of
expression in Xenopus oocytes were as described previously
(Xia et al., 1999). SP6 RNA polymerase was used to synthesize cRNA for
oocyte injection after DNA was linearized with MluI (Xia et
al., 1998). Fifty nanoliters of cRNA (10-20 ng/µl) was injected into
stage IV Xenopus oocytes harvested 1 d before. To
ensure a molar excess of subunits to Slo subunits,
we regularly injected : at 1:1 or 1:2 ratios by weight.
During electrophysiological recordings, oocytes were maintained in ND96
[(96 mM NaCl, 2.0 mM KCl, 1.8 mM
CaCl2, 1.0 mM
MgCl2, and 5.0 mM HEPES, pH 7.5)
supplemented with sodium pyruvate (2.5 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin 50 µg/ml]. Oocytes
were used 1-7 d after injection of cRNA.
Electrophysiology and analysis. Currents were recorded in
either inside-out or outside-out patches (Hamill et al., 1981). Patches
used for determination of conductance-voltage
(G-V) relationships contained large numbers
of channels and were typically from oocytes maintained for 3-7 d.
Generation of ensemble averages of channel openings was done with
patches with fewer channels from oocytes maintained for 1-3 d before
recording. Currents were typically digitized at 20-100 kHz.
Macroscopic records were filtered at 5-20 kHz (Bessel low-pass filter;
 3 dB) during digitization. Single channel records were filtered at 10 kHz (Bessel low-pass filter; 3 dB).
During seal formation, oocytes were bathed in ND96. After excision,
patches were quickly moved into a flowing 0 Ca2+ solution. For inside-out recordings,
the pipette extracellular solution was 140 mM potassium
methanesulfonate, 20 mM KOH, 10 mM HEPES, and 2 mM MgCl2, pH 7.0. Test
solutions bathing the cytoplasmic face of the patch membrane contained
140 mM potassium methanesulfonate, 20 mM KOH,
10 mM HEPES, pH 7.0, and one of the following: 5 mM EGTA (for nominally 0 Ca2+,
0.5 µM, and 1 µM
Ca2+ solutions), 5 mM HEDTA
(for 4 and 10 µM Ca2+
solutions), or no added Ca2+ buffer (for
60 µM, 100 µM, 300 µM, and 5 mM Ca2+ solutions). The
methanesulfonate solutions were calibrated to be identical to a set of
chloride-containing solutions with free Ca2+ determined from a computer program
(EGTAETC; E. McCleskey, Vollum Institute, Portland, OR). Calibration
was also performed against a commercial set of
Ca2+ standards [World Precision
Instruments (WPI), Sarasota, FL], which yielded values essentially
identical to our Cl -based standards. In
all cases, the desired free Ca2+ was
obtained by titrating the solution with calcium methanesulfonate until
the electrode measurement of the methanesulfonate-based solution
matched that of the chloride-based solution and the WPI calibration
solution. Local perfusion of membrane patches was as described
previously (Solaro and Lingle, 1992; Solaro et al., 1997).
Voltage commands and acquisition of currents was accomplished with
pClamp 7.0 or 8.0 for Windows (Axon Instruments, Foster City, CA).
Current values were measured using ClampFit (Axon Instruments), converted to conductances, and then fit with a nonlinear least squares
fitting program. As described in Results, conductances were determined
in three ways: from tail currents, from the peak current at a given
activation potential, and from steady-state current at a given
activation potential. G-V curves for activation were fit
with a Boltzmann equation with the form:
![G(V)=G<SUB><UP>max</UP></SUB>/(1+<UP>exp</UP>[(<UP>−</UP>V+V<SUB>0.5</SUB>)/k])](/content/vol20/issue13/fulltext/4890/img001.gif)
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(1)
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where V0.5 is the voltage of
half-maximal activation of conductance, and k is the voltage
dependence of the activation process (units of millivolts). At
room temperature, the net charge (z) moved between closed
and open conditions is given by 25.26/k. To evaluate
steady-state conductance-voltage relationships, a double Boltzmann
that included terms for both activation and block was used:
![G(V)=G<SUB><UP>max</UP></SUB>/(1+<UP>exp</UP>[(<UP>−</UP>V+V<SUB>0.5</SUB>)/k]+K<SUB><UP>b</UP></SUB>(0)*<UP>exp</UP>(<UP>−</UP>zFV/RT))](/content/vol20/issue13/fulltext/4890/img002.gif)
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(2)
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where V0.5 and k
correspond to the voltage of half-activation and voltage dependence of
activation, respectively, and Kb(0) is
the 0 voltage equilibrium constant for the blocking reaction, with
z, the fractional charge moved during the blocking reaction, and F, R, and T with their usual
meanings. This form of a Boltzmann equation can be related to specific
molecular steps for simple activation and blocking schemes in which
blockade proceeds strictly from open states, i.e., C   O B.
Ensemble averages were generated using our own software. Traces
obtained in 0 Ca2+ were used to generate
an idealized trace that was then subtracted from each record subsequent
to averaging of the leak-subtracted records.
Experiments were done at room temperature (21-24°C). All salts and
chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Simulation of currents. Simulation of currents was done with
custom software developed in the lab, based on manipulation of Q-matrices (Colquhoun and Hawkes, 1981) for up to 30 state models. For
the simulations in Figure 11, the following arbitrary model was
used:
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(Scheme I)
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with k1 = 1000 sec1,
k1 = 0.5 sec1,
kf = 5000 sec1, and
kr = 2000 sec1. The block and unblock transitions
were voltage-independent while the net charge movement associated with
each step along the activation pathway was 2.23e, divided between
activation and deactivation steps.
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RESULTS |
Identification of a BK subunit variant conferring very rapid
inactivation on BK channels
From searches of human EST databases, several cDNA clones of the
same gene were identified that show high homology with known BK channel
subunits, KCNMB1 (1) (Knaus et al., 1994), an avian subunit
(Oberst et al., 1997), and KCNMB2 (2) (Wallner et al., 1999; Xia et
al., 1999). The identification and isolation of the full-length cDNA
clone of the gene h3 was completed through nesting PCR and DNA
recombination as described in Materials and Methods. h3 shares
extensive homology with other subunits (Fig.
1). The coding region of 3 is
comprised of 771 base pairs and 257 amino acids. The h3 protein
shares 24% (62 of 257) identities and 37% (96 of 257) similarities
with h1 (191 amino acids). For comparison, Figure 1 also
includes sequence for an additional human neuronal subunit, KCNMB4
(4) (Wickenden et al., 1999; Brenner et al., 2000; Meera et al.,
2000; Wallner et al., 2000).
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Figure 1.
A family of subunits for the
Ca2+-dependent, voltage-gated K+
channel. A, Amino acid alignment of human KCNMB1 1
(h1), quail (c), human KCNMB2 (h3), human KCNMB3 (h4),
and human KCNMB4 (h4) subunits. Highlighted residues
are those with identity to aligned residues in at least one other subunit. The two predicted transmembrane segments (Knaus et al., 1994)
are marked with lines both above and
below. Residues known to be involved in CTX binding of
h1 [amino acids 90-94 of the 1 subunit (Hanner et al., 1997)]
are marked with a row of asterisks. B, 5'
genomic sequence of KCNMB3 starting with proposed start codon ATG
(Riazi et al., 1999). The exon sequence is shaded with
the splicing elements in bold. The splicing site is
marked by an arrow. C, Standard example
of an exon-intron junction and the elements involved in mRNA splicing.
The intron sequence is italicized. The 5' and 3'
splicing sites are marked by arrows. The genomic
sequence for 3 contains several features characteristic of a
consensus splice site, including 12 pyrimidines just upstream of the AG
junction (bold CTCCCTTTCCCC in B),
separated by 2-24 nucleotides from an upstream branch site of seven
nucleotides. The subscripts indicate the percent
occurrence of the specified base (or type of base) at each consensus
position. Py, Pyrimidines; Pu, purines;
N, any bases. For mRNA splicing, the intron is defined
by a GT-AG rule, which corresponds to the 5' donor and 3' acceptor.
Besides the GT-AG, short consensus sequences are required for the
completion of splicing, such as a branch site (underlined
TCTTTAT in B) located ~18-40 nucleotides
upstream of the 3' site (Lewin, 1997).
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The nomenclature given in Figure 1 follows current general usage as now
defined through GenBank. In a previous publication from this lab (Xia
et al., 1999), we used the designation 3 for the then
unnamed KCNMB2 subunit, because identification of the avian subunit
(Oberst et al., 1997) preceded identification of KCNMB2 (2).
Although the relationship of the avian subunit to other mammalian
subunits remains to be clarified, the terminology used here follows
the GenBank designations for the mammalian subunit family.
With no signal peptide, the topology analysis suggests that 3
probably shares common structural features with the other subunits,
having two transmembrane domains with both N and C terminals residing
intracellularly. The 3 subunit is somewhat unusual in having
extended C-terminal sequence. There are two potential N-glycosylation sites (N-X-S/T) in the extracellular loop and one consensus PKC phosphorylation site
(Basic1-3-X0-2- (S*/T*)-X0-2-Basic1-3) located at the penultimate C-terminal residue. 3 shares with the
other subunits four conserved cysteine residues in the
extracellular segment.
In the midst of this work, Riazi et al. (1999) published a partial
genomic sequence, which, when combined with information from EST data
base searches, allowed them to propose a putative gene for a new BK subunit (Riazi et al., 1999). This putative gene, termed KCNMB3, is
identical with our sequence for the 768 base pairs preceding the 3'
stop codon (256 amino acids) but differs at the 5' end. The N-terminal
amino acid sequence of their putative KCNMB3 is 19 amino acids longer
than our 3 beginning from the initial Met. Comparison of their
genomic sequence with our cDNA sequence identifies a consensus 3'
splicing site in the genomic sequence just at the point of divergence
between the two sequences (Fig. 1A). The splicing
site is apparently used to generate an initiation ATG site in our cDNA
clone and suggests that this 3' splicing site may be used as an
alternative splicing site to generate multiple splicing variants (Fig.
1B,C) (Uebele et al.,
2000).
It should also be mentioned that, during completion of this work,
another group has reported recently on the functional properties of the
identical KCNBM3 variant described here (Brenner et al., 2000).
However, in this other study, the properties of currents when + 3 were presumably coexpressed exhibited little difference from
currents resulting from the subunit alone, in marked contrast to
the major differences described below.
Tissue distribution of h3 message
Northern blot analysis was used to assess the distribution of 3
mRNA in various human tissues. 3 message was detected in heart,
liver, pancreas, adrenal medulla, adrenal cortex, and stomach. Low-level expression of 3 was also detected in skeletal muscle, kidney, thyroid, testis, and small intestine (Fig.
2). The original blot also suggested
faint expression in the brain. To determine whether 3 expression
might be stronger in particular brain regions, we therefore tested a
panel of 14 different brain samples, but only low-level expression of
3 was observed (results not shown). The detected mRNA products are
apparently not homogeneous, either within the same tissue or among
different tissues. In human heart, as many as four products were
detected, 1, 1.8, 2.5, and 6 kb, whereas in other tissues, such as
liver, adrenal medulla, and adrenal cortex, only two or three products
were visible. However, in human pancreas, four products differing from
those of heart were detected, 0.5, 0.9, 1.5, and 2.5. Some of these
smaller products (0.5 and 0.9 kb) may be the degradation products of
the larger ones. In stomach, the major product was 1.5 kb. This
diversity in apparent message size, although possibly indicative of
degradation, is also consistent with the possible expression of
multiple splice variants of the 3 subunit. Based on our sequence, we
would expect transcript sizes on the order of at least 1.0 kb, although
with no information about noncoding regions, this is only a minimal estimate. Because of the possibility that multiple splice variants may
be present in different tissues, these results do not allow us to
identify which tissues express the particular N terminal we describe
here.
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Figure 2.
Distribution of 3 message in various tissues.
A, Northern blots show membranes containing human
message probed with radiolabeled human 3 sequence. Tissues are as
follows: 1, heart; 2, brain;
3, placenta; 4, lung; 5,
liver; 6, skeletal muscle; 7, kidney;
8, pancreas; 9, adrenal medulla;
10, thyroid; 11, adrenal cortex;
12, testis; 13, thymus;
14, small intestine; 15, stomach. The
minimal predicted transcript size for the 3 variant described here
would be expected to be ~1 kb.
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Ca2+ and voltage dependence of currents arising
from the 3 subunit
The h3 message was coexpressed with Slo subunits
in Xenopus oocytes. Currents obtained from inside-out
patches from oocytes expressing h3 and subunits were both
voltage- and Ca2+-dependent. Increases in
cytosolic Ca2+ resulted in shifts in
current activation at more negative potentials. In addition, at strong
depolarizations and higher Ca2+, + 3 currents exhibited a very rapid, although incomplete, inactivation (Fig. 3). The time constant
of the inactivating portion of current was ~0.7-1.5 msec (Fig.
4A). The current
inactivation time constant exhibits a moderate dependence on membrane
voltage (Fig. 4B) and reaches a
Ca2+-independent rate at least at the more
positive activation potentials (Fig. 4C). Despite the rapid
time course of inactivation, inactivation of current is incomplete up
to a voltage of +190 mV, and current inactivates to a steady-state
level that is dependent on voltage but independent of
[Ca2+]. The substantial level of
sustained current after inactivation at the most positive activation
potentials indicates that both the onset of block and recovery from
block that underlie the blocking reaction must be rapid. Furthermore,
as will be seen in more detail below, there is a range of voltages and
[Ca2+] over which the rapidity of the
inactivation is such that current is reduced, despite the fact that no
time-dependent inactivation is discernible in the traces. In such
cases, the current does not appear to be inactivating but does exhibit
strong rectification. Another interesting aspect of the currents
is that, even at moderate Ca2+, e.g.,
4 µM, at a potential of +60 and more positive,
currents rapidly reach their peak value in ~0.5-2 msec.
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Figure 3.
Coexpression of the 3 subunit with the
Slo subunit produces rapidly inactivating,
Ca2+- and voltage-dependent current.
A, Traces show currents obtained from an
inside-out patch from a Xenopus oocyte injected with
cRNA encoding both human 3 and mouse subunits. Channels were
activated by voltage steps from 140 to +180 mV with 0, 1, 4, 10, and
300 µM Ca2+. The voltage protocol is
shown at the top. B, Faster time base
records of the currents in A are shown for both
activation and inactivation and for deactivation. Note that the largest
tail current amplitude at 120 mV greatly exceeds the steady-state
current level during even the most positive activation steps,
indicating that there must be extensive channel unblocking before the
peak of the tail current. Also, note that, at 1 and 4 µM
Ca2+, the maximal tail current at 120 mV is larger
than the maximal peak current activated during the voltage step to +180
mV. This suggests that, at lower Ca2+, many channels
become blocked during the rising phase of outward current.
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Figure 4.
Voltage and Ca2+ dependence of
the inactivation time constant of 3 currents. A,
Traces on the left show normalized currents (from same
patch as Fig. 3) activated with 300 µM
Ca2+ for +60, +100, +140, and +180 mV. In each case,
the best fit of a single exponential function to the current
inactivation time course is plotted over the trace, yielding the time
constant of inactivation ( i). i
was 1.26, 0.77, 0.62, and 0.57 msec for +60, +100, +140, and +180 mV,
respectively. Traces on the right show normalized
currents (also from Fig. 3) activated at +140 mV for 1, 4, 10, and 300 µM Ca2+. Fitted single exponential
functions are also plotted. i was 1.49, 0.74, 0.65, and
0.62 msec for 1, 4, 10, and 300 µM, respectively.
B, i is plotted as a function of command
potential for four different [Ca2+]. For each
Ca2+, there is a small increase in apparent
inactivation rate with more depolarized command potential. Each point
is the mean ± SEM with four patches for 1 µM
Ca2+, 10 patches at 10 µM, nine
patches at 300 µM Ca2+, and five
patches for 1 mM Ca2+.
C, i is replotted as a function of
[Ca2+]. At any voltage, there is little indication
of any Ca2+ dependence to i at
Ca2+ of 10 µM and higher.
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To illustrate the unusual features of the 3 currents, Figure
5 compares the properties of currents
through channels resulting from the subunit alone, + 3, and
+ 1, when studied with identical salines and stimulation
conditions. Examination of these sets of currents suggests that the
3 subunit, in addition to producing inactivation, shifts the
activation range of the resulting channels to more negative potentials
at a given Ca2+, similar to the 1
subunit. Additionally, the 3 subunit produces a distinct slowing of
the deactivation time course at a given Ca2+ and voltage, relative to the subunit alone. However, at Ca2+ of 10 µM and higher, this slowing of deactivation is less than observed for the 1 subunit. The inactivation mediated by this 3
subunit differs from inactivation for the previously described 2
subunit in several ways (Wallner et al., 1999; Xia et al., 1999).
First, 3 inactivation is much more rapid (~1 vs ~20 msec). Second, there is significant steady-state current during 3
inactivation compared with 2 inactivation. Third, at potentials
negative to 0 mV, no time-dependent inactivation is observed for the
3 currents.
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Figure 5.
Comparison of currents resulting from alone,
+ 3, and + 1. A, Traces
show currents resulting from expression of alone activated by the
indicated voltage protocol with 10, 60, and 300 µM
Ca2+. B, Traces show
+ 3 currents under the same conditions as in A.
C, Traces show currents resulting from + 1 expression under the same conditions as in A and
B, except that the potential both before and after the
activation step was 180 mV. The + 3 combination
results in currents that, at a given Ca2+, are
activated at more negative potentials than for subunits alone but
somewhat more positive than for + 1 subunits. This can be most
clearly seen in the tail currents. At 60 µM, the step to
120 mV results in only slight current activation for the 3
construct but produces almost 50% activation for the 1
construct.
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In the remainder of this paper, we will focus on qualitative aspects of
the currents arising from the 3 subunit, including the macroscopic
conductance-voltage behavior, pharmacology, and the appearance of
single channel openings. Furthermore, we will present results
suggesting that an important functional role of the inactivation
process may be to produce Ca2+-dependent
K+ current with a relatively fast apparent
rate of macroscopic current activation at lower
Ca2+.
Properties of conductance-voltage curves arising from coexpression
of + 3 subunits
G-V curves were determined in three separate ways
(Fig. 6A). First,
G-V curves were determined from the tail current amplitude after repolarization from various command potentials. Second, conductances were determined from the peak currents measured during each test step. Third, conductances were determined from the
steady-state current remaining at the end of each test step.
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Figure 6.
The voltage dependence of activation and
steady-state properties of inactivation revealed in
conductance-voltage curves. A, Examples of
G-V curves obtained from tail current amplitude
(open triangles), from the peak current (inverted
filled triangles) measured during the command step, and from
the steady-state current at the end of the voltage step
(filled triangles) are shown for one patch bathed
with 300 µM Ca2+. For tail currents,
each point corresponds to the conductance measured 110 µsec after the nominal imposition of the repolarizing voltage step to
120 mV from a given command potential. Reduction of conductance at
the most positive potentials probably reflects some slow cumulative
block from contaminating ions. B, A repolarizing step to
160 mV was used to compare tail current amplitude and time course
either from just after the peak of outward current during a step to
+160 mV or after development of the steady-state current level at +160
mV. Despite the fact that the current near the peak is over twofold
larger than the steady-state current, the tail current amplitude in
each case is essentially identical. d after
repolarization near the peak outward current was 0.70 msec, whereas
after repolarization from the steady-state current level it as 0.97 msec. C, Tail current conductances (from the same patch
as in A) were determined for 0 (filled
circles), 0.5 (open circles), 1 (filled diamonds), 4 (open
diamonds), 10 (filled triangles), and 300 (open triangles) µM
Ca2+ . The solid lines are single
Boltzmann fits (Eq. 1) to the G-V curves. Values were
as follows: for 0 µM,
V0.5 = 113.3 mV, k = 15.3 mV; for 0.5 µM,
V0.5 = 67.1 mV, k = 15.97; for 1 µM, V0.5 = 40.4 mV, k = 15.4 mV; for 4 µM,
V0.5 = 16.2 mV, k = 15.9 mV; for 10 µM, V0.5 = 32.2 mV, k = 16.08; for 300 µM,
V0.5 = 70.5, k = 17.47 mV. D, Steady-state conductances were determined
from the average current level at the end of voltage steps to a given
activation potential and plotted as a function of command voltage.
Solid lines are fits of the double Boltzmann function
given by Equation 2. Values for V0.5 for
activation and k for activation were constrained to
those obtained in C. E, Peak conductances
were calculated from peak currents at each activation potential using a
0 mV reversal potential and plotted as a function of command potential.
The lines simply connect the values. F,
Voltages at which conductance is half-activated are plotted as a
function of [Ca2+] for alone, + 1,
+ h3, and + 2( 33) in which the 2 inactivation has
been deleted. Error bars show SD. For + 3, points at 0, 10, and
300 µM correspond to 17 patches, points at 1, 4, 1000, and 5000 µM correspond to 6-10 patches, and points at
0.5 µM corresponds to three patches. For alone, each
point corresponds to four patches, for + 1, six patches, and for
+ 2(33), at least five patches for each point.
For graphical purposes, points in nominally 0 Ca2+
were plotted at 0.05 µM.
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Visual inspection of the G-V curves determined either from
the tail currents or the peak currents (Fig. 6A)
indicates that a similar maximal conductance can be obtained in both
cases. This would seem surprising given the extensive blockade observed
in the 3 currents before the repolarizing voltage step. This
similarity of the maximal conductance determined from the tail current
G-V curves to the maximal conductance in the peak
current G-V curves suggests that most channels that are
blocked at the end of the activation step must recover essentially
instantaneously during the deactivation step. If this is true, a
deactivation step at the peak of outward current should elicit the same
tail current amplitude as a deactivation step after a steady-state
level of inactivation is achieved. This is, in fact, observed (Fig.
6B). Because of this rapid recovery from block, the
tail current G-V curves therefore appear to provide a
direct indication of the fraction of channels activated by a voltage
step to different test potentials, despite the extensive inactivation
during the test step. It must be kept in mind, however, that the tail
current G-V curves, in fact, represent not only channels
that are open before the deactivation step but also channels that were blocked.
Conductances from tail currents were calculated from a repolarizing
step to 120 mV after steps to different activation potentials as
shown for one patch in Figure 6C. Over all
Ca2+, a similar absolute maximal
conductance is observed from the tail currents, even with 0 Ca2+. With elevations in
Ca2+, the G-V curves show the
leftward shift characteristic of BK-type channels. In contrast to the
tail current G-V curve, the steady-state conductance (Fig.
6D) exhibits a marked voltage-dependent reduction at
more positive potentials, and the absolute amount of steady-state conductance (and current) at all Ca2+ from
10 µM and higher is relatively similar at each
voltage above 0 mV. Because over these
[Ca2+] the tail current G-V
curves indicate that all channels are essentially maximally activated
at any potential above 0 mV (Fig. 6C), this indicates that
the blocking reaction is essentially
Ca2+-independent at 10 µM Ca2+ and
higher. Even at 1 and 4 µM
[Ca2+], at sufficiently high voltages,
the steady-state conductance also appears to asymptote to values
comparable with those at higher Ca2+.
Thus, qualitatively the blocking process appears to be
voltage-dependent but primarily independent of
Ca2+.
The steady-state conductance was fit in each case with the double
Boltzmann given by Equation 2 containing a term for current activation
and a second term for a voltage-dependent blocking reaction
(Fig. 6D). Both the
V0.5 and k for the
activation of conductance were constrained to values obtained from fits
to the tail current G-V curves shown in Figure
6C. The resulting double Boltzmann functions then provided
an estimate of the voltage dependence of the blocking reaction with
values given in the figure legend. The reduction of conductance at each
potential positive to 0 mV was essentially identical for all
Ca2+ of 10 µM and
higher. This is the result expected for a
Ca2+-independent, voltage-dependent
blocking process in which the channel activation process is maximally
activated over this range of conditions. Results from 17 patches
studied with [Ca2+] from 10 µM through 5 mM yielded
similar conclusions (Table 1).
In the simplest case, Equation 2 arises from the predicted equilibrium
condition for a simple three-state model (C O B) in which
a voltage-dependent blocking process arises only from open channels.
The fitted values for z then provide an indication of the
amount of charge movement that occurs during the blocking process. The
equivalence of the charge movement during the blocking process over the
range of 10 µM to 5 mM
Ca2+ suggests that, irrespective of the
blocking mechanism, the equivalent of a total of ~0.33 elementary
charges are moved across the membrane in association with the
transition(s) from the open to the blocked condition.
When the identical fitting procedure was used to compare tail current
and steady-state G-V curves at 0, 0.5, 1, and 4 µM Ca2+, Equation 2 was less successful at accounting for the steady-state currents.
Specifically, if the values for the blocking reaction at low
[Ca2+] were constrained to those
obtained from fits to the steady-state G-V curves at higher
Ca2+, this resulted in large
underestimates of the value for maximal conductance at a given
Ca2+. If, on the other hand, the maximal
value of conductance is constrained to be in agreement with the value
obtained from fitting the tail current G-V curves, the
resulting values for Kb(0) for block
and the resulting voltage dependence deviate substantially from those obtained at higher Ca2+. Qualitatively,
the discrepancy between estimates of the tail current peak conductance
and steady-state current peak conductance after accounting for block
suggests that, at lower Ca2+, more
channels are in blocked states than expected based on the expectations
of the simple blocking scheme considered here. A more extensive
analysis of the blocking mechanism will be presented elsewhere.
In summary, the voltage-dependent reduction of steady-state current
observed with the 3 subunit is consistent with the movement of
~0.33 charges moving across the membrane during the transition from
fully open states to blocked states. At high
Ca2+, the steady-state features of block
are generally consistent with a simple equilibrium between open and
blocked states. At low Ca2+, there appears
to be more block than might be expected based solely on block of open
states alone.
The plot of peak conductance as a function of voltage reveals two other
interesting aspects of this current (Fig. 6E). First, at 10 µM Ca2+ and
above, the peak conductance activated at positive potentials, in
general, is similar in most patches to the maximal conductance determined from tail currents, although in some cases the peak conductance never reaches that defined by the tails (Fig. 3). However,
for Ca2+ well below 10 µM, the maximal conductance activated at
positive potentials grossly underestimates the conductance determined
from the tail currents. An explanation for this difference is that, at
more modest Ca2+, the rapid rate of
current inactivation relative to a slower rate of current activation
reduces the fraction of channels that are open at any time. Thus, when
the underlying activation rates of the current are slow, the rapidity
of the inactivation process will simply result in a rectifying current.
This will be examined more closely below. Second, the relationship
between peak conductance and activation voltage exhibits a novel double
hump appearance, which is consistently observed in all patches. Such a
behavior can, in fact, be predicted from blocking models in which rates of inactivation are rapid relative to rates of activation over a
particular range but for which activation exceeds inactivation rates at
more positive potentials (C. J. Lingle, unpublished observations).
Because the inactivated channels do, in fact, recover from inactivation
extremely rapidly, the tail G-V curves provide a reasonable indication of the Ca2+ and voltage
dependence of entry of channels into activated states. For comparison
with the 3 subunit, we have also examined patches from oocytes
expressing alone, + 1, or + 233 [2 with the
N-terminal inactivation domain removed (Xia et al.,
1999,)]. Figure 6F plots the voltage of
half-activation as a function of [Ca2+]
for all four channel types. The 1 and 2 subunits show the typical
marked shift in V0.5 relative to alone with less difference at lower Ca2+.
In contrast, + 3 produces a marked increase in sensitivity at
Ca2+ concentrations below 10 µM but produces less of a shift than the 1
subunit at higher Ca2+. These results
suggest that the 3 subunit may increase the sensitivity of BK
channels to submicromolar or low micromolar
Ca2+ compared with BK channels modulated
by other subunits. Unlike the 1 and 2 subunits, the 3
subunit appears to shift BK gating at low
Ca2+ as well as higher
Ca2+, causing a shift of the
V0.5 versus
Ca2+ curves, which is somewhat parallel to
that obtained with alone.
In summary, the macroscopic + 3 currents exhibit a number of
features that may be important in defining their physiological roles.
First, the currents appear more sensitive to lower
Ca2+ than BK channels formed from other
known subunits. Second, the channels exhibit rapid but incomplete
inactivation, such that the macroscopic current exhibits a marked
steady-state rectification at positive potentials. Third, outward
current can be reduced by the inactivation process even when visible,
macroscopic inactivation is not observed (for more details, see Fig.
11). A cautionary remark regarding physiological interpretation of
these results is that these measurements were all done in symmetrical
K+ solutions and at room temperature.
+ 3 channels exhibit unusual single
channel openings
A hallmark of BK channels is a characteristic large single channel
conductance. To examine the properties of channels arising from + 3 coexpression, we attempted to identify patches with small numbers
of channels. An example of a patch from an oocyte expressing + 3
subunits is shown in Figure 7. We have
been unable to observe single channel openings that are fully
resolvable for any such patch studied over a range of
Ca2+ and activation conditions. Rather,
such patches typically exhibit exceedingly brief openings of primarily
undefined amplitude, even at a bandwidth of 10 kHz. That such openings
do, in fact, represent the behavior of coexpressed + 3 subunits
is supported by the fact that ensemble averages of such openings reveal
the characteristic Ca2+ and voltage
dependence of activation and the rapid but incomplete inactivation of
the macroscopic currents (Fig. 7). Furthermore, ensemble variance
analysis suggests a single channel conductance in excess of 150 pS at
positive activation voltages (data not shown). Because of the rapid
flickering behavior of these openings, this value most certainly
underestimates the true channel current. This rapid flickering behavior
is, in part, consistent with a rapid inactivation process of
~0.5-1.0 msec. However, the failure to resolve any channel openings
that approach the expected single channel current level for subunits alone suggests that other kinetic factors besides the rapid
inactivation may contribute to the unusual flickery behavior of the
single channels. To date, we have not been successful in identifying a
patch with only a single + 3 channel.
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Figure 7.
Channel openings resulting from coexpression of
+ 3 and exhibiting unusual gating behavior.
Traces in each column show channel
openings in an inside-out patch bathed with 0, 10, 60, or 300 µM Ca2+, respectively, from
left to right. Channel openings were
activated by steps to +100 mV (with the voltage protocol shown on the
top of the first column). Four
consecutive sweeps are shown below each condition. The
dotted line indicates a 25 pA current level (250 pS at
this voltage). The records were filtered at 10 kHz, and currents were
sampled at 100 kHz. Traces on the bottom
of each column show ensemble averages for each condition
shown above. In each case, at least 60 sweeps were
included in each average. Averages exhibit the rapid but incompletely
inactivating current seen for the macroscopic currents, despite the
fact that no fully resolved channel openings are seen. Single
exponential fits to the inactivation time course of the averaged
currents yielded values of 0.99, 1.37, and 1.18 msec for 10, 60, and
300 µM Ca2+, respectively, comparable
with the inactivation time constants of macroscopic currents.
Calibration: 30 pA applies to the individual current sweeps, whereas 15 pA applies to the averaged currents.
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Pharmacological properties of the + 3 channels
In addition to the characteristic shifts in gating with increases
in Ca2+, BK-type channels exhibit specific
pharmacological properties. Because of the unusual properties of the
currents resulting from + 3 coexpression, we examined the
pharmacological sensitivity of these currents to various compounds.
Extracellular 5 mM 4-aminopyridine was without effect on
+ 3 currents. BK currents are typically blocked by extracellular
application of tetraethylammonium (TEA) at concentrations less
than those affecting other voltage-dependent K+ channels. The effect of extracellular
TEA on either alone (Fig. 8A) or + 3
currents (Fig. 8B) was therefore examined. TEA
blocked + 3 currents with an IC50 of
0.59 ± 0.30 mM at +100 mV (Fig. 8C) compared with an IC50 of 0.43 ± 0.05 mM for block of alone. Over the range
of +20 to +100 mV, the voltage dependence and fitted value for
dissociation constant at 0 mV (for values, see legend to Fig.
8D) were similar between the two currents (Fig.
8D).
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Figure 8.
3 currents exhibit sensitivity to TEA
characteristic of other BK currents. A,
Traces show currents activated by the indicated
voltage-protocol (activation steps from 100 to +100 mV) from an
outside-out patch from an oocyte expressing the Slo subunit alone with 10 µM Ca2+ in the
recording pipette. Left panel shows currents in control
saline, middle panels show the effects of 1 and 30 mM TEA applied to the extracellular face of the membrane,
and the right panel shows currents after removal of TEA.
B, Traces show currents activated with
the same protocol used in A from an outside-out patch
expressing + 3 subunits. C, The fractional block
of current elicited at +100 mV is plotted as a function of
extracellular TEA concentration for both alone (open
circles) and + 3 (filled
circles). Fractional block was normalized to the amount of
block produced by 30 mM TEA. The IC50 values
for block were 0.43 ± 0.05 mM (n = 3) for with a Hill coefficient of 0.82, and 0.59 ± 0.30 mM (n = 4) for block of + h3 with a Hill coefficient of 0.94. D, Estimates of the IC50 for block by TEA
are plotted as a function of voltage. Fractional block in each case was
measured from the steady-state currents at +20 through +100 mV.
Solid lines are fits of the function
K(V) = K(0) *
exp(zFV/RT), where
K(0) indicates the IC50 for block by TEA at
0 mV, F, R, and T have
their usual meanings, and z is the fitted value for
fractional charge moved during the blocking reaction. For ,
K(0) = 0.18 ± 0.01 mM with
z = 0.28 ± 0.025, whereas for + 3,
K(0) = 0.22 ± 0.04 mM, with
z = 0.30 ± 0.076.
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Extracellular charybdotoxin (CTX) inhibited + 3 currents with an
IC50 of 80.0 ± 10 nM
(n = 4) as estimated from the time course of the onset
and recovery from CTX blockade at 100 nM (Saito et al., 1997; Xia et al., 1999), assuming that 100% block is defined by 30 mM TEA. This sensitivity of the 3
subunit appears similar to that of the 2 subunit, which is less than
for either + 1 or alone (Wallner et al., 1999; Xia et al.,
1999).
Thus, currents arising from coexpression of + 3 exhibit
pharmacological features characteristic of BK-type channels.
Inactivation results from the 3 N terminus but not the
C terminus
Inactivation mediated by the 2 subunit requires an N-terminal
structure. Compared with the 2 subunit, the 3 subunit contains a
somewhat shorter N terminus but also an extended C terminus that is
predicted to be cytosolic. We therefore wished to determine whether
either the N- or C-terminal sequences might play a role in inactivation
mediated by the 3 subunits. Removal of most of the C terminus of the
3 subunit (construct D3; n > 10 patches) had no
discernible affect on the ability of 3 subunits to produce inactivating currents when coexpressed with (Fig.
9A). In contrast, removal of
the N terminus (construct D4; n = 4 patches) resulted in complete removal of inactivation (Fig. 9B).
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Figure 9.
The 3 N terminus, but not the C terminus, is
responsible for inactivation of the + 3 currents.
A, Thirty-five amino acids from the C terminus of the
3 subunit (construct D3) were removed, and the resulting construct
was coexpressed with subunits. Currents retain the typical
inactivation behavior of the intact 3 subunit. Currents were
activated with the indicated voltage protocol with 10 µM
cytosolic Ca2+. B, Twenty-one amino
acids were removed from the N terminus of the 3 subunit (construct
D4), resulting in loss of the inactivation behavior (10 µM Ca2+). C,
Twenty-one amino acids from the 3 N terminus were used to replace 33 amino acids at the N terminus of the 2 subunit (construct D20). The
D20 construct exhibits the rapid, incomplete inactivation of the 3
subunit but the more negative activation range of the 2 subunit.
Currents were activated with 10 µM
Ca2+. D, The Gmh3 alternative
splice variant (Riazi et al., 1999) was expressed, and currents were
activated with 10 µM Ca2+. Note the
different time base for currents in D. The time constant
of inactivation for this construct at 10 µM and +100 mV
was ~50 msec, although the time course of decay is somewhat better
described with two exponential components.
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We also examined constructs in which the 2 N terminus and 3 N
terminus were swapped between the parent subunits. Replacing the
2 N terminus with a 3 N terminus (construct D20;
n > 10 patches) resulted in currents with the
characteristic rapid but incomplete inactivation of the full-length
3 subunit (Fig. 9C). In contrast, when the 3 N
terminus is replaced with the 2 N terminus (construct D1;
n = 4 patches; data not shown), relatively complete
inactivation with a time constant of 20-25 msec was observed, characteristic of the full-length 2 subunit.
Finally, we have also examined the functional properties of the
previously reported putatively genomic KCNMB3 alternative N-terminal
variant (Riazi et al., 1999). The alternative N-terminal variant
(construct Gmh3) also results in inactivating BK currents (n = 4 patches) (Fig. 9D), although
inactivation proceeds substantially more slowly
(i of ~50 msec).
BK channel blockers do not compete with the
inactivation process
Inactivation of BK channels mediated by the 2 subunit N
terminus contrasts with N-terminal-mediated inactivation of
voltage-dependent K+ channels (Choi et
al., 1991) in that cytosolic channel blockers do not slow the rate of
current inactivation (Solaro et al., 1997; Xia et al., 1999),
suggesting that the inactivation domain of the 2 subunit does not
bind directly to sites at the ion channel mouth occupied by the
blockers. We did a similar test on inactivation of BK channels mediated
by the 3 N terminus, using the rapid cytosolic blocker TEA (Fig.
10). For a simple competition between blocker and any inactivation domain, 50% reduction of peak current amplitude is expected to result in a twofold prolongation of the inactivation time constant (Choi et al., 1991). Over a
range of TEA concentrations producing up to a greater than 50% block
of peak current, no prolongation of inactivation time course was observed. Similar, to blockade mediated by the 2 N terminus, TEA
does not alter the rate of onset of the block process mediated by the
3 N terminus.
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Figure 10.
Cytosolic application of TEA does not compete
with the native 3 inactivation process. A,
Traces show 3 currents activated by the indicated
voltage step with 300 µM cytosolic
Ca2+ and 0, 30, 60, or 100 mM TEA. A
trace was also recorded in 0 Ca2+ with 100 mM added TEA. TEA results in a concentration-dependent
reduction of peak and steady-state outward current but no effect
on the time constant of inactivation. B, The fold
increase of i by TEA and fold decrease in peak current
amplitude is plotted as a function of TEA. For a simple competition
between TEA and the inactivation mechanism, both relationships should
increase with [TEA] in an identical manner. Larger
symbols are means with SDs for estimates from five patches. The
smaller symbols represent individual determinations from
these five patches.
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Physiological consequences of the rapid blocking process: an
increase in apparent current activation rate at low
Ca2+ and rectification
As indicated earlier, the time constant of 3-mediated
inactivation exhibits only a small dependence on voltage or
Ca2+; over a range of potentials and
Ca2+, inactivation occurs with a time
constant of ~0.7-1.5 msec. This rate of current inactivation is
rapid relative to the expected rates of BK current activation at
moderate [Ca2+] and depolarization.
Thus, the modest Ca2+ and voltage
dependence of the macroscopic i suggests that,
at potentials at which activation of current is slow, the kinetics of
rapid block may contribute to the time course of apparent current activation. Specifically, if rates of equilibration between open states
and inactivated states are rapid relative to movement of closed
channels to open states, the relaxation of current activation can be
strongly determined by the kinetics of the equilibration between open
and inactivated. The ability of a rapid blocking process to increase an
apparent activation rate may seem somewhat unexpected at first glance.
However, this situation is simulated for an arbitrary simple current
activation scheme (Scheme I) in Figure
11A in which,
although the underlying molecular rates of channel activation are
identical, the presence of a rapid inactivation decreases the apparent
activation time constant and decreases the time-to-peak.
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Figure 11.
The rapid blockade results in current
rectification and an increase in apparent current activation rate.
A, Currents were simulated for Scheme I given in
Materials and Methods (C1  C2 C3 O I).
Traces on the left correspond to absolute
currents in arbitrary units, with open symbols
indicating traces for the same model when inactivation
does not occur. For the top panels, a voltage of
+40 mV was used, and for the bottom panels, the voltage
was +20 mV. On the right, traces from the
left were normalized to the same maximum amplitude to
show the change in time-to-peak and apparent activation rate with rapid
inactivation intact. B, 3 currents were activated
with depolarizing steps to +80 with 10 µM
Ca2+. Repolarizing steps to 120 mV were applied at
various times into the activation step to +80 mV. Tail current
amplitudes exhibit a slower increase, even at a time when peak outward
current exhibits slight inactivation. Open circles are
the peak tail current amplitudes scaled to allow comparison of the
relative conductances observed either at +80 mV or during the tail
at 120 mV. The open circles emphasize the slow
activation time course of the underlying channels and also the
extensive rectification of conductance during the step to +80 mV.
The right panel compares the normalized time course of
outward current activation and the time course of the increase in tail
current, with time constants given in the figure. C,
Currents were activated by steps to +60 mV with 10 µM Ca2+. Again, currents at +60 mV
exhibit extensive rectification with no visible time-dependent
inactivation. As shown in the right panel, the
activation time course of outward current is faster than the time
course of tail current development.
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To demonstrate this for + 3 currents, currents were activated in
the presence of 10 µM Ca2+
at +80 (Fig. 11B) and +60 (Fig. 11C) mV
and tail currents determined at various times of the depolarizing
activation step. Qualitatively, in both cases the rate of increase in
tail current amplitude as a function of command step duration is slower
than the apparent rate of current activation during the activation step
itself. The increase in tail current amplitude reflects the rate of
channels appearing in open and blocked states and thus represents the
net movement of channels into the open state(s). On the other hand, the
outward current observed during the activation step represents only
channels currently in open states. The right panels of
Figure 11, B and C, compare the normalized onset
of outward current with the time course of activation defined by the
tail currents. The observed rate of outward current activation is 1.5- to 3-fold than that determined from the increase in tail current amplitude.
The time constants of activation defined by the increase in tail
current amplitude correspond closely with values for mSlo current activation as a function of
[Ca2+] defined in other work (Cui
et al., 1997). Specifically, the value of 1.77 msec for the
activation time constant given in Figure 11B
corresponds well with that expected for 10 µM
Ca2+ at +60 mV. An apparent activation
time constant of 0.66 msec as observed for the rise time of outward in
Figure 11B would require an effective increase
in [Ca2+] at +60 mV in excess of
100 µM (Cui et al., 1997, their Eq. 3).
It should also be noted that, although in some cases there is no
visible inactivation during the command step, the steady-state level of
current in each case exhibits marked rectification because of
rapid block that occurs during the rising phase of current activation.
Thus, rapid block confers on these channels two unique characteristics.
First, it increases the observed rate of macroscopic current activation
at moderate Ca2+ compared with channels
that lack rapid block. Second, it results in rectification of current.
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DISCUSSION |
We have described the functional properties of a new member of the
BK accessory subunit family, which confers a number of unique and
potentially physiologically important characteristics on the resulting
BK channels. First, compared with the 1 and 2 subunits (Wallner
et al., 1999; Xia et al., 1999), the
V0.5 for activation of this 3
variant is shifted to more negative voltages at lower concentrations of
Ca2+ (below 10 µM), whereas at higher concentrations of
Ca2+, the
V0.5 is less shifted. Second, the 3
subunit confers an extremely rapid but incomplete blockade of the
resulting BK channels. Third, the rapidity of the blocking process is
such that it plays a primary role in defining the apparent macroscopic
activation kinetics of current and its rectification properties over
some activation conditions. Fourth, because of rapid flickering
behavior, the properties of single channels containing 3 subunits
are unlike any known BK channel. The properties of these currents
suggest that, compared with other BK channel variants, they are likely to be more rapidly activated at lower
Ca2+. It will be important to determine
the properties of this channel under more physiological ionic conditions.
The properties of this 3 subunit variant add significantly to the
diversity of functional properties conferred on BK channels by the subunit family. In fact, subunits may play the critical role in
defining the tissue-specific phenotypic properties of BK channels in
various cells. Examination of Northern blots from the subunits so
far examined suggests that there is some tissue overlap among various
subunits. For example, from our work it is clear that both the 2
and 3 variants are expressed in the adrenal medulla (Xia et al.,
1999; this study). If different subunits are expressed in the same
cells and, as is likely, assemble into the same single channels, this
will add enormously to the variation in BK channel phenotypes,
providing cells with an even greater capacity to precisely tune the
gating range of the mature BK channels.
Where are channels containing the 3 subunit found?
The channel openings and macroscopic currents resulting from + 3 subunits exhibit the Ca2+ and voltage
dependence characteristic of BK channels. Yet, both the unusual single
channel properties and the rapid inactivation and rectification appear
unique among Ca2+- and voltage-dependent
K+ currents so far described. An
intriguing issue then is why has such a current not yet been observed
in any native cell. This could arise from a number of reasons. First,
in patches with few channels, the flickery behavior of the channels
might be difficult to recognize as valid channels. Second, the strongly
rectifying behavior of the macroscopic currents means that in a
whole-cell recording such a current might be only a minor contributor
to the overall outward current. Third, it is possible that this subunit is only expressed in cell types that have not as yet been extensively studied. Although message encoding this subunit family was found in
adrenal gland, heart, lung, and kidney, and, to some extent, in the
nervous system, the Northern blots provide no information about the
distribution of the particular variant we have studied electrophysiologically. A final possibility is that, although message
for this variant is found in mRNA, this particular variant is never
actually expressed. However, we consider it likely that the restricted
expression of some subunits for BK channels may result in a variety of
functionally novel BK-type currents that have yet to be identified in
native cells.
Comparison of inactivation mediated by the 2 and
3 subunits
Inactivation of the BK channels mediated by the 2 subunit,
although showing some features reminiscent of ball-and-chain
inactivation of voltage-dependent K+
channels (Hoshi et al., 1990), exhibits features suggesting that a
different type of inactivation may be involved. In particular, cytosolic blockers of native inactivating BK channels or cloned 2
channels do not compete with the native inactivation domain (Solaro et
al., 1997; Li et al., 1999; Xia et al., 1999), and furthermore, blocked
channels do not recover through the open state to return to closed
states (for native BKi channels, Solaro et al.,
1997; for + 2 channels, C. J. Lingle, unpublished
observations). It is possible that the 3 subunit might shed light on
the 2 inactivation mechanism if the two shared similar features.
Both the 2 and 3 subunits produce inactivation mediated by
trypsin-sensitive N-terminal domains of the subunits. 3 inactivation is much more rapid but incomplete, indicating that both the onset and
recovery from inactivation mediated by the 3 subunit are more rapid.
Like the 2 subunit, cytosolic blockers of the BK channel do not slow
the 3 inactivation process. Qualitatively, we can only say that, in
accordance with standard blocking models, neither the 2 nor 3
inactivation domain appears to bind to the mouth of the ion permeation
pathway as defined by cytosolic blockers. This leaves open the
possibility that both inactivation domains act at the same site.
Despite the kinetic differences, it also remains possible that both
subunits act in the same way, only differing perhaps in the relative
binding affinities of the inactivation domains.
Another unusual aspect of block produced by the 3 subunit was that,
although recovery from block during repolarization to negative
potentials was essentially instantaneous, we observed differences in
the tail current time course after repolarization at the peak of
outward current versus repolarization during steady-state block. We
note here that this result (Fig. 6B), by itself,
argues strictly against a simple block and unblocking of open channels. If all blocked channels recover very rapidly to the same open states,
the tail currents should decay similarly. This is not observed. Rather,
this phenomenon requires that during repolarization blocked channels
recover to an open state that is different from that occupied by
channels at the peak of outward current. Thus, inactivation mediated by
both the 2 and 3 subunits exhibits features that are inconsistent
with the behavior expected for simple, open channel block (Hoshi et
al., 1990).
Rapid activation of BK current at
moderate Ca2+
An important property of current resulting from the 3 subunit
is the relatively rapid current activation rate at low
Ca2+. How does this occur? By coupling a
very rapid but partial blocking reaction with slow activation kinetics,
the approach to steady-state after a step change in voltage reflects
both the underlying activation rate and the inactivation rate. Rapid
block, although producing marked current rectification, essentially
decreases the rise time of the outward current by twofold to threefold
over what it would be in the absence of the inactivation process.
Because of this, an important role of the 3 subunit may be to
increase effective current activation rates. Is a twofold to threefold
increase in a current activation rate significant? As pointed out in
Results, an increase in apparent rate of activation of this magnitude
corresponds to an over 10-fold increase in effective
[Ca2+] (Cui et al., 1997). The impact of
this effect in a native cell would also critically depend on the
density of channel expression.
Another unusual feature of the 3 variant is the more negative
V0.5 for activation than other
variants (1, 2) at [Ca2+] below 10 µM. In fact, the shift in
V0.5 produced by the 3 subunit relative to alone appears to be relatively similar at all
[Ca2+], whereas for 1 and 2 there
appears to be larger effects at higher
[Ca2+] (Wallner et al., 1996, 1999).
This raises the possibility that the mechanism by which the 3
subunit affects apparent Ca2+ sensitivity
may differ from the mechanism by which the 1 and 2 subunits may
shift gating. As we noted above, the slowing of deactivation tail
currents when channels close from the steady-state blocked condition
indicates that channels can reside in more open states during the
inactivated condition than during the peak of outward current. If the
additional open states are, in fact, associated with the inactivation
process itself, the occurrence of inactivation will increase the
fraction of channels that appear in open states as monitored by the
tail currents. This suggests that the blocking mechanism alone may
account for much of the 3-induced shift in V0.5 for observed at lower
Ca2+. Such a shift would be similar over
all Ca2+, because it would reflect
exclusively the Ca2+-independent
equilibrium between open and blocked states.
If this hypothesis is correct, this would indicate that the
inactivation mechanism of the 3 subunit has multiple physiologically important effects on the resulting BK current. Not only does the rapid
blockade produce rectification, but it also increases the apparent
current rise time and causes a shift in the
V0.5 of activation at low
Ca2+. It will be particularly interesting
to determine how the remarkable properties conferred by this particular
3 variant are translated into
Ca2+-mediated regulation of cellular
excitability in a real cell.
| |
FOOTNOTES |
Received Feb. 23, 2000; revised April 10, 2000; accepted April 24, 2000.
This work was supported by National Institutes of Health Grant DK46564
to C.L. We thank Anne Benz and the C. Zorumski laboratory for providing
us with oocytes and Jamie Thorp for technical assistance. We thank
Ying-Wei Wang for results obtained with 2-33 shown in Figure
6D.
Correspondence should be addressed to Chris Lingle, Washington
University School of Medicine, Department of Anesthesiology, Box 8054, St. Louis, MO 63110. E-mail: clingle{at}morpheus.wustl.edu.
Dr. Duan's present address: University of Science and Technology of
China, School of Life Science, Department of Neurobiology and
Biophysics, Hefei, Anhui 230027, China.
| |
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W. Marcotti, S. L. Johnson, and C. J. Kros
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X. Sun, X. Q. Gu, and G. G. Haddad
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X.-M. Xia, J.P. Ding, and C. J. Lingle
Inactivation of BK Channels by the NH2 Terminus of the {beta}2 Auxiliary Subunit: An Essential Role of a Terminal Peptide Segment of Three Hydrophobic Residues
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H Pawelzik, D I Hughes, and A M Thomson
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X. Qian, C. M. Nimigean, X. Niu, B. L. Moss, and K. L. Magleby
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Y. Imaizumi, K. Sakamoto, A. Yamada, A. Hotta, S. Ohya, K. Muraki, M. Uchiyama, and T. Ohwada
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P. Orio, P. Rojas, G. Ferreira, and R. Latorre
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P. Jin, T. M. Weiger, Y. Wu, and I. B. Levitan
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Y.-W. Wang, J. P. Ding, X.-M. Xia, and C. J. Lingle
Consequences of the Stoichiometry of Slo1alpha and Auxiliary beta Subunits on Functional Properties of Large-Conductance Ca2+-Activated K+ Channels
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E. S. L. Faber and P. Sah
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[Abstract]
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D. Bentrop, M. Beyermann, R. Wissmann, and B. Fakler
NMR Structure of the "Ball-and-chain" Domain of KCNMB2, the beta 2-Subunit of Large Conductance Ca2+- and Voltage-activated Potassium Channels
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C. E Armstrong and W. M Roberts
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F. Van Goor, Y.-X. Li, and S. S. Stojilkovic
Paradoxical Role of Large-Conductance Calcium-Activated K+ (BK) Channels in Controlling Action Potential-Driven Ca2+ Entry in Anterior Pituitary Cells
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P. V. Lovell and D. P. McCobb
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A. C. Gerlach, C. A. Syme, L. Giltinan, J. P. Adelman, and D. C. Devor
ATP-dependent Activation of the Intermediate Conductance, Ca2+-activated K+ Channel, hIK1, Is Conferred by a C-terminal Domain
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TOP
ABSTRACT
INTRODUCTION
