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The Journal of Neuroscience, December 15, 1999, 19(24):10706-10715
Xenopus Embryonic Spinal Neurons Express Potassium
Channel Kv Subunits
Meredith A.
Lazaroff,
Alison D.
Hofmann, and
Angeles B.
Ribera
Department of Physiology and Biophysics, University of Colorado
Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
Developmental regulation of voltage-dependent delayed rectifier
potassium current (IKv) of
Xenopus primary spinal neurons regulates the waveform of
the action potential. IKv undergoes a
tripling in density and acceleration of it activation kinetics during
the initial day of its appearance. Another voltage-dependent potassium
current, the A current, is acquired during the subsequent day and
contributes to further shortening of the impulse duration. To decipher
the molecular mechanisms underlying this functional differentiation, we
are identifying potassium channel genes expressed in the embryonic
amphibian nervous system. Potassium channels consist of pore-forming
( ) as well as auxiliary ( ) subunits. Here, we report the primary
sequence, developmental localization, and functional properties of two
Xenopus Kv genes. On the basis of primary sequence,
one of these (xKv2) is highly conserved with Kv2 genes identified
in other species, whereas the other (xKv4) appears to identify a new
member of the Kv family. Both are expressed in developing spinal
neurons during the period of impulse maturation but in different
neuronal populations. In a heterologous system, coexpression of xKv
subunits modulates properties of potassium current that are
developmentally regulated in the endogenous
IKv. Consistent with xKv4's unique
primary sequence, the repertoire of functional effects it has on
coexpressed Kv1 subunits is novel. Taken together, the results
implicate auxiliary subunits in regulation of potassium current
function and action potential waveforms in subpopulations of embryonic
primary spinal neurons.
Key words:
potassium channels; Kv1 subunits; auxiliary Kv
subunits; Xenopus embryos; electrical excitability; spinal
cord neurons
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INTRODUCTION |
In several systems,
voltage-dependent potassium current (IKv)
undergoes developmental regulation and dynamically regulates emerging
patterns of neuronal excitability (for review, see Spitzer and Ribera,
1998 ). In embryonic amphibian spinal neurons, maturation of
IKv converts long-duration action
potentials to brief sodium-dependent spikes (Barish, 1986; O'Dowd et
al., 1988; Lockery and Spitzer, 1992). Maturation of
IKv involves a tripling in its density
and acceleration of activation and inactivation kinetics (Barish, 1986;
O'Dowd et al., 1988; Ribera and Spitzer, 1990). Definition of the
molecular mechanisms that underlie development of excitability requires
identification of the potassium channels expressed in embryonic
Xenopus spinal neurons.
Voltage-dependent potassium channels are oligomeric proteins composed
of pore-forming (Kv) and auxiliary subunits. Kv subunit genes
belong to one of several related subfamilies (Kv1-Kv9). Kv1
subunits are expressed and contribute to potassium current maturation
in the embryonic Xenopus spinal cord (Ribera and Nguyen, 1993; Jones and Ribera, 1994; Ribera, 1996). Auxiliary cytoplasmic Kv subunits tightly associate with Kv1 subunits (Rehm and
Lazdunski, 1988; Parcej et al., 1992; Rettig et al., 1994; Scott et
al., 1994; Rhodes et al., 1995, 1996; Nakahira et al., 1996; Sewing et
al., 1996). The majority of studies of vertebrate Kv gene function
have examined channels expressed in heterologous systems, and
consequently their in vivo function is not yet defined. In heterologous systems, the effects of Kv subunits are diverse and
range from increasing surface expression/current density (Shi et al.,
1996; Accili et al., 1997a) to accelerating activation and
inactivation kinetics (Rettig et al., 1994; England et al., 1995a,b;
Majumder et al., 1995; Heinemann et al., 1996; Morales et al.,
1996). In addition, Kv subunits share sequence and structural similarity with aldo-keto reductases, raising the possibility that they
function as enzymes with as yet unspecified substrates (McCormack and
McCormack, 1994; Gulbis et al., 1999).
Although little is known regarding the in vivo function of
Kv subunits, less is known about their contribution to emerging properties of neuronal excitability [but see, Butler et al. (1998)]. We sought to determine whether embryonic spinal neurons express Kv
subunits, and if so, whether their mRNAs are detectable in spinal
neurons that express xKv1 subunits (Ribera, 1990; Ribera and Nguyen,
1993). Here we report that Xenopus embryonic spinal neurons
express Kv subunit genes during the period of maturation of
IKv. One of these (xKv2) appears to
be the Xenopus ortholog of mammalian Kv2 on the basis of
high amino acid identity (97%). The other gene shares less identity
(<73%) with previously identified Kv genes. We propose that it is
a new member of the Kv family, xKv4. Heterologous coexpression of
xKvs with xKv1 subunits leads to modulation of properties of
potassium current that are developmentally regulated in the endogenous
IKv: current density, activation
kinetics, and extent of inactivation. In situ hybridization indicates that xKv2 and xKv4 are expressed contemporaneously with
previously studied Kv1 genes in subpopulations of spinal neurons.
Taken together, the results implicate xKv subunits in regulation of
IKv and emerging excitability of
embryonic spinal neurons.
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MATERIALS AND METHODS |
Animals. Xenopus embryos were produced by
in vitro fertilization and staged according to Nieuwkoop and
Faber (1967).
Isolation of Xenopus Kv cDNAs, DNA sequencing, and
analysis. 32P-labeled probes directed
against the entire coding regions of rat brain Kv1 and Kv2 genes
(kindly provided by Drs. Ken Nakahira and James Trimmer, Department of
Biochemistry, SUNY Stony Brook, Stony Brook, NY) were used to screen a
Xenopus tadpole brain cDNA library (gift of Dr. Nicholas C. Spitzer, Department of Biology, University of California San Diego, La
Jolla, CA) at reduced stringency. One full-length clone (xKv-A) and
two partial-length clones (xKv-B1p, xKv-B2p) were obtained. The
missing 5' ends of the partial clones were obtained by further
screening of cDNA libraries using either conventional methods or
PCR. All isolated clones were sequenced over both strands. DNA
sequences were read and entered into a computer using a GEL READER
interface and software (CBS Scientific, Del Mar, CA) and analyzed using
DNASTAR software (Madison, WI).
Whole-mount in situ hybridization. The
nonradioactive whole-mount detection method (Harland, 1991) was used
with minor modifications as described previously (Ferreiro et al.,
1993; Burger and Ribera, 1996). cRNA sense and antisense probes
corresponding to the 3' untranslated region (UTR) and 3' coding region
of xKv-A, xKv-B1, and xKv-B2 were used. For xKv-A, the
probe contained 700 bp of coding region and 150 bp of 3' UTR; for both
xKv-B1 and xKv-B2, the probes contained 500 bp of coding region
and 700 bp of 3' UTR. cRNA probes were synthesized in the presence of
digoxigenin-labeled UTP (Boehringer Mannheim, Indianapolis, IN) and
hybridized to whole-mount albino embryos. After removal of probe,
embryos were incubated with alkaline phosphatase-conjugated
anti-digoxigenin antibody (Boehringer Mannheim). The alkaline
phosphatase reaction product was developed in the presence of
chromogenic substrate (Boehringer Mannheim). Whole-mount embryos were
either cleared in Murray's solution (2:1 benzyl benzoate/benzyl
alcohol) for photography or embedded in plastic (JB-4 embedding kit;
Polysciences, Warrington, PA). Photography of whole mounts and sections
was performed with Kodak Ektachrome 160T film using appropriate color filters. Photographic images were digitized (Nikon Cool Scanner), and
composite figures were constructed using Adobe Photoshop Software.
Oocyte recording. The entire coding regions of xKv1.1 and
xKv1.2 potassium channel genes were previously cloned into the pSP64T expression vector, and cRNA was synthesized as described (Ribera
and Nguyen, 1993). An EcoRI-XhoI fragment
containing the entire coding region of xKv4 along with 671 bp of 3'
UTR was cloned into the pCS2+ expression vector (Rupp et al., 1994;
Turner and Weintraub, 1994); the xKv2-pCS2+ expression construct was constructed by insertion of a PCR-generated cDNA encoding xKv2 into
pCS2+. Synthesis of capped cRNA was achieved by linearization of a
xKv-pCS2+ expression vector with NotI followed by
in vitro transcription with SP6 RNA polymerase (Promega,
Madison, WI) in the presence of ribonucleotide triphosphates (Pharmacia
Biotech, Piscataway, NJ) and cap analog (Boehringer Mannheim). RNA
concentrations were determined spectrophotometrically and confirmed by
electrophoresis in agarose-formaldehyde gels. Stage VI oocytes were
removed and defolliculated as described previously (Ribera, 1990;
Ribera and Nguyen, 1993; Burger and Ribera, 1996). xKv and xKv1
cRNAs were either injected alone or coinjected at ratios of 1-30:1
(xKv/ xKv1). For two-electrode voltage-clamp (TEVC) experiments,
oocytes were injected with 50 nl of RNA solution (Kv1 RNA, 2.5-5
µg/ml; Kv RNA, 75-150 µg/ml). Higher expression was required
for macropatch experiments, and oocytes were injected with 100 nl of a
more concentrated RNA solution (Kv1 RNA, 5-21 µg/ml; Kv RNA,
150-630 µg/ml). Oocytes were incubated at 18°C in Barth's
solution containing the following (in mM): 88 NaCl, 1 KCl, 0.41 CaCl2, 0.33 Ca(NO3)2, 2.4 NaHCO3, 0.82 MgSO4 and 5 Na
HEPES, pH 7.4.
TEVC methods were used [Axoclamp 2A amplifier (Axon Instruments,
Foster City, CA) or Oocyte Clamp OC-725C (Warner Instruments, Hamden,
CT)] to record voltage-activated outward currents induced in oocytes
24-48 hr after injection of RNA. For detailed analysis of activation
kinetics, data were obtained 48-72 hr after injection using the
cell-attached macropatch configuration and a patch-clamp amplifier
(Axopatch 200B amplifier, Axon Instruments) to avoid the constraints
imposed by the large capacitative transients of the whole oocyte. Data
acquisition and analysis were accomplished with the pCLAMP suite of
programs and AxoGraph software (Axon Instruments). For TEVC recordings,
currents were sampled at 100-200 µsec and filtered at 5-10 kHz. For
macropatch recordings, currents were sampled at 30-100 µsec and
filtered at 5 kHz, and data from 10 runs were averaged to improve the
signal-to-noise ratio. The leak and capacitative transient currents
were subtracted using the P/4 protocol of the Clampex program (pCLAMP).
For TEVC recordings, the bath consisted of Barth's solution and the
electrode solution consisted of 3 M KCl and 10 mM HEPES, pH 7.4. Electrode resistances ranged between 0.1 and 0.7 M . Oocytes were not used if the holding current exceeded
 200 nA at a potential of 80 mV or if the resting membrane potential
was positive to 40 mV. For macropatch recordings, the bath consisted
of 115 mM KCl, 1.8 mM EGTA, and 10 mM HEPES, pH 7.4, and electrodes were filled with Barth's
solution. Electrode resistances ranged between 1.5 and 2.5 M.
Current amplitudes were measured 50-55 msec into a 60 msec
pulse (steady-state level), except for data obtained on coexpression of
xKv4. In the latter case, peak values were measured to avoid the
effects produced by inactivation; induction of inactivation was
analyzed separately (see Fig. 7). Conductance-voltage
(G-V) relationships were determined for
recordings from oocytes that did not saturate the amplifier at the most
depolarized voltage step (+100 mV). Conductance was determined by
dividing current amplitudes by the driving force assuming an internal
K+ concentration of 100 mM and EK = 116 mV; recordings that did not achieve a plateau value of
conductance (Gmax; determined from Boltzmann fits) were excluded. Normalized G-V
plots were obtained by dividing values of G by maximum
G, Gmax, as determined from a fit to the Boltzmann equation. The Boltzmann parameters,
V1/2 and k, were obtained
from fits of the Boltzmann equation to G-V plots. Time to half-maximum (t1/2) was
determined as the time required to achieve half-maximum amplitude
during a 60 msec voltage step. Current densities were determined by
dividing current amplitudes by the oocyte surface area, assuming a 1 mm
diameter (~106
µm2). Levels of statistical significance
were assessed using the nonparametric Mann-Whitney test; p
values <0.05 were indicative of statistical significance.
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RESULTS |
Identification of Xenopus Kv Potassium
Channel Subunits
Given the high identity among previously identified Kv
subunits, we began our search for Xenopus Kv clones by
screening cDNA libraries with probes derived from rat (kindly provided
by Drs. K. Nakahira and J. S. Trimmer, Department of Biochemistry, SUNY, Stony Brook, NY). One full-length (xKv-A) and two
partial-length cDNA (xKv-B1p and xKv-B2p) clones were isolated.
The missing 5' ends of the partial clones were obtained through a
combination of further library screening and PCR-based approaches (see
Materials and Methods) to yield cDNA clones containing complete open
reading frames (xKv-B1 and xKv-B2, respectively).
The xKv-A coding region contains 1101 base pairs (bp) and predicts a
protein of 367 amino acids and a molecular weight (MW) of 40 kDa (Fig.
1). Alignment of the predicted amino acid
sequence of Xenopus Kv-A with mammalian Kv sequences
reveals >97% identity with Kv2 protein (Table
1). In contrast, only 76-77 and 62-67% exists with Kv1 and Kv3 subfamily members. On the basis of these comparisons, we consider Kv-A to be the Xenopus ortholog
of Kv2 and refer to it as xKv2.
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Figure 1.
Xenopus Kv subunits. The
predicted amino acid sequences of Xenopus xKv2 and
xKv4 coding regions are aligned against mammalian Kv1, Kv2,
and Kv3 sequences [hKv1.1 and hKv2.1 (McCormack et al.,
1995); hKv3.1 (Leicher et al., 1998)]. Gaps (dots)
have been introduced to improve the alignment. Residues that are
identical to those of xKv2 are indicated by a hyphen.
Overall, xKv2 and xKv4 share 71% identity at the amino acid
level (Table 1). -Helices and -sheets are shaded
light and dark gray, respectively;
residues that contribute to the putative active site are indicated by
an overlying asterisk (Gulbis et al., 1999).
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xKv-B1 and xKv-B2 both contain open reading frames that are 1203 bp in length. They share 93 and 94% overall identity at both the
nucleotide and predicted amino acid levels over the coding region.
Kv subunits belong to the superfamily of aldo-keto reductases; for
this superfamily, Jez et al. (1997) proposed that  97% amino acid
identity is indicative of allelic forms. Given their 94% amino acid
identity and 94 and 89% nucleotide identity of coding regions and 3'
UTR, respectively, xKv-B1 and xKv-B2 sequences are likely to be
allelic variants. Consistent with this, both genes have similar
mRNA expression patterns (data not shown). Only results for the
xKv-B1 allelic form are presented below.
The coding sequence of xKv-B1 predicts a protein containing 401 amino acids (44 kDa MW) (Fig. 1). Alignment of the predicted amino acid
sequence of xKv-B1 with mammalian Kv subfamily members reveals
69-74, 70-73, and 70-74% identity with mammalian Kv1, Kv2,
and Kv3 subfamily members (Table 1). In contrast, xKv2 is 97%
identical to Kv2 genes previously cloned in other vertebrate species. Within a species (e.g., human), the different Kv genes (i.e., Kv1, Kv2, Kv3) share ~70-75% identity (Table 1),
which is the degree of identity found between xKv-B1 and previously identified Kv genes. On this basis, xKv-B1 appears to
identify a new Kv subfamily, and we refer to xKv-B1 as
xKv4.
Kv genes share sequence and structural similarities with the
NAD(P)H-dependent oxidoreductase superfamily (McCormack and McCormack,
1994; Gulbis et al., 1999). The xKv proteins show the same extent of
sequence identity (13-49%) with these enzymes as do mammalian Kv
subunits. Although aspects of the xKv4 sequence are novel, motifs
that define characteristic -- barrel structures and critical
active site residues are conserved (D119, Y124, and K154 of xKv4)
(Fig. 1), indicating that xKv4 belongs to the subunit cluster of
aldo-keto reductases (Jez et al., 1997; Gulbis et al., 1999).
xKv2 and xKv4 transcripts are present in excitable tissues of
the embryo
Whole-mount in situ hybridization was performed to
determine the expression patterns of xKv2 and xKv4 genes in
Xenopus embryos. Embryos ranging in age between 1 and 2 d [Stage (St) 8-36] were examined, because maturation of
IKv in spinal neurons occurs during this period. Action potentials are first detected in sensory spinal neurons at 20 hr (St 18) (Baccaglini and Spitzer, 1977). At this time,
potassium currents are of small density (Barish, 1986; O'Dowd et al.,
1988). During the subsequent day, there is a progressive increase in
IKv density and consequent shortening
of the action-potential duration. Appearance of
IKv in myocytes (somitic tissue) is
delayed with respect to that in neurons but also undergoes functional upregulation once it is present (25 hr, St 23) (Ribera and Spitzer, 1990).
In the developing embryo, excitable tissues express xKv2 in a
spatially and temporally dynamic pattern (Fig.
2). xKv2 transcripts are first
detected in somites (26 hr, St 24). Within 9 hr, (St 29, 35 hr), Kv2
is detected in the spinal cord as well. One hour later (St 30, 36 hr),
xKv2 mRNA expression is downregulated in somites and upregulated in
the spinal cord. By St 35-36 (50 hr), xKv2 expression is limited to
the spinal cord and no longer present in the somites. In transverse
sections of 2 d embryos (St 35), xKv2 mRNA expression is
detected only in dorsal spinal neurons, including the mechanosensory
Rohon-Beard cells.
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Figure 2.
xKv2 and xKv4 transcripts are present in
excitable tissues of developing Xenopus embryos. Embryos
were hybridized as whole mounts to antisense or sense cRNA probes
corresponding to either xKv2 (A-F) or xKv4
(G-I). The in situ hybridization
signal is recognizable as a deep blue-purple precipitate. The
whole-mount embryos (A-C, G-I) are oriented
with their dorsal sides up and anterior ends to the
left. Embryos that were hybridized to sense control
probes are identified by s; these demonstrate the
background signal (caused by trapping of probe in internal cavities),
which is higher in older embryos. The transverse sections
(D-F) are oriented dorsal side
up. A, At 26 hr (St 24), xKv2 mRNA is
prominent in developing somites. B, Between 1 and 2 d (top, middle, and bottom
embryos are St 28, 29, and 30, respectively), the pattern of
expression of xKv2 mRNA undergoes a dramatic change. Initially the
signal predominates in the somites but with time diminishes in this
tissue and begins to appear in the spinal cord
(arrowheads). At St 35-36 (50 hr; C),
the signal is no longer observed in the somites but is present in the
spinal cord and head. D, In a transverse section through
a 1 d embryo hybridized to xKv2 antisense probe, the signal is
present in the developing somites. E, In a transverse
section of a 2 d embryo hybridized to xKv2 antisense probe, the
signal is detectable in the dorsal spinal cord, where Rohon-Beard cells
reside. F, In a transverse section of a 2 d embryo
hybridized to a sense control probe, no signal is present.
G-I, Whole-mount embryos hybridized to
xKv4 antisense probe. At all stages, the signal predominates in the
nervous system. In the older embryos (e.g., I),
the signal is stronger. Comparison of H and
I (xKv4) with B and C
(xKv2) reveals that xKv4 mRNA localizes to a more ventral
position in the spinal cord than does xKv2. Scale bar:
A, G, 1 mm; B,
C, 1.5 mm; H, 1.2 mm;
I, 1.1 mm; D-F, 700 µm.
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xKv4 expression is limited to the nervous system in a spatially and
temporally dynamic pattern. Kv4 transcripts are first detected in
the brain and only the most anterior regions of the spinal cord (St 24, 26 hr). Nine hours later (St 29), Kv4 mRNA is detected in posterior
regions of the spinal cord as well. During the subsequent day (Fig.
2I), the spatial pattern of expression remains
constant, although the intensity of staining obtained is stronger,
suggesting upregulation of gene expression during this period.
xKv2, but not xKv4, increases functional expression of
xKv1 channels
Coexpression of mammalian Kv with Kv1 subunits leads to
increases in current density and acceleration of activation and
inactivation kinetics (Rettig et al., 1994; Majumder et al., 1995;
McCormack et al., 1995; Heinemann et al., 1995, 1996; Morales et al.,
1996; Shi et al., 1996; Uebele et al., 1996; Accili et al., 1997a,b). These properties of potassium current are of particular interest, because they are developmentally regulated in the endogenous
IKv. We coexpressed xKv with
xKv1.1 and Kv1.2 subunits, because the developing spinal
cord expresses the mRNAs for these -subunits (Ribera, 1990; Ribera
and Nguyen, 1993).
xKv1 and xKv mRNAs were coinjected into Xenopus
oocytes at several different ratios ranging between 1 and 30:1 (xKv/
xKv1). For ratios of 1-4:1, no effects are observed. At ratios of
15-30:1, similar effects are observed, which are statistically
significant at a ratio of 30:1 (xKv/xKv1). Thus, only data
obtained using the 30:1 ratio are presented.
Coexpression of xKv2 with either xKv1.1 or xKv1.2 subunits
increases current amplitude 1.6- and 2.0-fold, respectively (+20 mV)
(Fig. 3A). In contrast,
coexpression of xKv4 with either xKv1 subunit has no effect on
current amplitude. One possible explanation for the lack of effect of
xKv4 coexpression is that this auxiliary subunit was not expressed
at a sufficiently high level. However, xKv4 coexpression does have
functional effects on kinetic properties (see below). On this basis,
lack of expression of xKv4 subunits at the 30:1 ratio
(xKv/xKv1) is unlikely to account for the absence of effect on
current density.
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Figure 3.
Coexpression of xKv2, but not xKv4, with
xKv1.1 or xKv1.2 subunits leads to an increase in current
amplitude. A, Representative whole-oocyte recordings of
currents induced after injection of Kv1.1 (top) and
Kv1.2 (bottom) RNAs in the absence
(left) or presence of xKv2 (middle) or
xKv4 RNA (right). Currents were elicited by
depolarizing the membrane in 10 mV increments to potentials ranging
between 60 and +130 mV from a holding potential of 80 mV; currents
elicited by depolarizations to 60, 30, 0, +30, +60, +90, and + 120 mV are shown. B, Normalized current-voltage
(I-V) relationships for currents recorded
from oocytes injected with xKv1.1 (left) and Kv1.2
(right) ±xKv2 or xKv4 RNAs. Current amplitudes
were normalized to the mean current amplitude obtained at +50 mV for
xKv1.1 or Kv1.2 alone; numbers in parentheses = n. Data obtained in the presence of Kv2 ( ) are
significantly different from those obtained in its absence ( );
p  0.0002.
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Current densities for channels expressed in oocytes ranged between 1 and 10 pA/µm2, which includes the range
found in developing spinal neurons (0.5-1.5
pA/µm2 at +30 mV) (O'Dowd et al., 1988;
Jones and Ribera, 1994). On coexpression of xKv2, current densities
increased two- to threefold, just as
IKv density does in developing spinal neurons.
xKv1 and xKv2 modify functional properties of
xKv1 channels
The increase in current densities observed on coexpression of
xKv2 subunits may reflect changes in the conductance-voltage relationship of the current, the number of functional channels present
in the plasma membrane, or both. To distinguish between these
possibilities, we examined conductance-voltage relationships of
currents carried by the various subunit combinations. We determined the
maximum value of the
conductance-(Gmax) and
voltage-dependent parameters derived from fits to the Boltzmann
equation (V1/2 and k).
For Kv1.1 and Kv1.2 subunit-containing channels, coexpression of
xKv2 subunits leads to 1.6- and 1.8-fold increases in Gmax (Fig.
4A,B;
Table 2). These data indicate that an
increase in the number of functional channels contributes to the
increased current amplitudes obtained on xKv coexpression (Fig. 3).
The increase in Gmax found on xKv2
coexpression with xKv1.1 subunits accounts for the increase in
current density. For Kv1.2,xKv2 coexpression has a slightly
smaller effect on Gmax than it did on
current amplitudes. If the single-channel conductance is unaltered, the
result suggests that there are about twice as many functional channels
in the plasma membrane on coexpression of xKv2 with either xKv1.1
or xKv1.2 subunits. In contrast, xKv4 coexpression does not alter
either Gmax values (Fig.
4A,B; Table 2) or current amplitudes (Fig. 3).
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Figure 4.
Coexpression of xKv2 versus xKv4 subunits
differentially modulates steady-state activation properties of xKv1
channels. A, B, Conductance-voltage
(G-V) relationships indicate that coexpression
of either xKv1 subunit (A, xKv1.1;
B, xKv1.2) with xKv2 subunits results in an
increase in Gmax. In contrast, coexpression
of xKv1 subunits with xKv4 has no significant effect on
Gmax. C, D, Normalized
G-V curves for xKv1.1
(C) or xKv1.2 (D)
currents obtained in the absence or presence of xKv subunits.
Coexpression of xKv1.2 with either xKv2 or xKv4 subunits leads
to a depolarizing shift in the G-V
relationship (Table 2). In C, data obtained in the
presence of xKv4 ( ) are statistically different from those
obtained in its absence () in the range of 20 to +40 mV;
p values range between 0.0001 and 0.006. In
D, data obtained in the presence of xKv2 () or
xKv4 () are statistically different from those obtained in its
absence () in the range of +10 to +50 mV (xKv2) and 0 and 50 mV
(xKv4); p values range between 0.0001 and 0.04.
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Next, we examined the possibility that coexpression of either xKv2
or xKv4 affects voltage-dependent properties of steady-state activation. In fact, coexpression of xK4, but not xKv2, with xKv1.1 shifts the activation curve to more positive voltages (Fig.
4C); V1/2 is increased by
~5 mV (Table 2). Such a shift in
V1/2 on Kv coexpression would lead
to a reduction in current amplitude in the range of 20 to +40 mV.
However, current amplitudes are unaffected by xKv4 coexpression
(Fig. 3).
Similar analyses were performed for Kv1.2-containing channels.
Coexpression of either xKv2 or xKv4 leads to small depolarizing shifts in the G-V relationship (Fig.
4D; Table 2). Such shifts would lead to a decrease in
current amplitude in the range of +10 to +50 mV. However, current
amplitude is unaffected by xKv4 coexpression and increased by
xKv2 coexpression. On the basis of these data, the increased current
amplitudes observed on xKv2 coexpression are most likely caused by
an increase in channel number rather than changes in steady-state
voltage-dependent properties of the expressed channels.
xKv subunits affect activation kinetics of Kv1 currents
The endogenous IKv undergoes a
developmentally regulated acceleration of activation kinetics (O'Dowd
et al., 1988). Mammalian Kv subunits accelerate activation kinetics
of Kv1 currents (Heinemann et al., 1995, 1996; Uebele et al., 1996). In
whole-oocyte recordings, xKv1 currents activate more rapidly on
coexpression of either xKv2 or xKv4 (Fig.
5). The time to half-maximum current
(t1/2) decreases, indicating that
activation of the current is accelerated. Larger effects are observed
when xKv subunits are coexpressed with xKv1.2 versus xKv1.1.
The effects of xKv coexpression are voltage dependent and less
pronounced with increasing depolarization. Overall, coexpression of an
xKv subunit can lead to a 25-50% decrease in
t1/2, which is reminiscent of the 50%
decrease in t1/2 observed for the
endogenous IKv during maturation of
the action potential.
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Figure 5.
Coexpression of xKv with xKv1 subunits
accelerates kinetics of activation. Time to half-maximum activation
(t1/2) as a function of voltage is
plotted for recordings obtained from whole oocytes injected with
Kv1.1 (left) or Kv1.2 (right) cRNAs
in the absence and presence of xKv2 or xKv4 cRNAs. Representative
currents are presented in Figure 3. Data obtained in the presence of
either xKv are significantly different from those obtained in its
absence; p values are all <0.0001. The number of
oocytes recorded from are as follows: Kv1.1 alone, 72; Kv1.1 + xKv2, 70; Kv1.1 + xKv4, 35; Kv1.2 alone, 83; Kv1.2 + xKv2, 82; Kv1.2 + xKv4, 49.
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To avoid the large, long-lasting capacitative transients obtained in
whole-oocyte recordings that compromise analysis of kinetics of current
activation, we also examined kinetics of activation in recordings from
oocyte macropatches (Fig. 6). The values
of t1/2 obtained from the macropatch
data are smaller than those obtained for whole-oocyte recordings (Fig.
5 vs Fig. 6), which most likely reflects the better temporal resolution
of the macropatch configuration. Nonetheless, as found for whole-oocyte
recordings, coexpression of xKv2 or xKv4 with xKv1.1 leads to
a decrease in the activation t1/2.
xKv coexpression effects are voltage-dependent and greater at less
depolarized potentials. At voltages positive to +60 and +70 mV (xKv2
and xKv4, respectively), the effects are no longer significant.
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Figure 6.
Activation of xKv1 currents recorded in oocyte
macropatches is accelerated when xKv subunits are coexpressed.
Representative macropatch recordings (A) and time
to half-activation (t1/2)-voltage
relations (B, C) are presented for currents recorded
from oocytes injected with xKv1.1 (A,
left; B) or xKv1.2 (A,
right; C) coexpressed with xKv
subunits. Currents were elicited by depolarizing the membrane in 10 mV
increments to potentials ranging between 60 and +130 mV from a
holding potential of 80 mV; the currents elicited by depolarizations
to 60, 30, 0, +30, +60, +90, and +120 mV are shown. In
B, data obtained in the presence of Kv2 are
significantly different from those obtained in its absence in the range
of +10 to + 60 mV, except at +40 mV; p values range
between 0.02 and 0.05. Data obtained in the presence of Kv4 are
significantly different from those obtained in its absence in the range
of 30 to +70 mV; p values range between 0.0005 and
0.04. In C, data obtained in the presence of Kv2 are
significantly different from those obtained in its absence in the range
of 30 to +60 mV; p values range between 0.0006 and
0.02. Data obtained in the presence of Kv4 are significantly
different from those obtained in its absence in the range of +10 to +60
mV; p values range between 0.02 and 0.04.
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Coexpression of xKv subunits with xKv1.2 subunits also
accelerates current activation (Fig. 6A,C).
t1/2 values are reduced by as much as
a factor of 2. Effects are voltage dependent, being more pronounced at
less depolarized membrane potentials. In sum, Kv coexpression
accelerates the kinetics of activation, and the specific effects depend
on the particular combination of - and -subunits.
xKv4 subunits increase and accelerate inactivation of
xKv1 currents
The endogenous IKv undergoes a
developmentally regulated change in inactivation properties (Ribera and
Spitzer, 1990). Because mammalian Kv1 and Kv3 subunits enhance
inactivation of Kv1 currents (Rettig et al., 1994; Heinemann et al.,
1995, 1996; Majumder et al., 1995; McCormack et al., 1995; Morales et
al., 1996; Accili et al., 1997a; Leicher et al., 1998), we also
determined whether xKv subunits are capable of modulating
inactivation of channels containing either xKv1.1 or xKv1.2
subunits. Inactivation was evaluated by calculating the ratio of the
steady-state versus peak-current amplitudes achieved during a pulse
(Iss/Ipk). This ratio has a value close to 1 for sustained currents and decreases when
inactivation is apparent. Typically, xKv1.1 and xKv1.2 currents
have Iss/Ipk
values of 0.98 (n = 74 and 29, respectively; +100
mV), as expected for sustained currents.
Consistent with results found for Kv2 of other species, coexpression
of xKv2 has no effect on inactivation of either Kv1.1 or Kv1.2
currents (Fig. 7A). However,
coexpression of xKv4 with either xKv1.1 or xKv1.2 subunits
induces inactivation (i.e., Iss/Ipk 0.9) in
79 and 100%, respectively, of oocytes. For those oocytes having
Iss/Ipk values
0.90 (+100 mV), inactivation was examined over a more extended range
of voltages (Fig. 7B). xKv4 increased the extent of
inactivation of both xKv1.1 and xKv1.2 currents in a
voltage-dependent manner, with more inactivation observed at more
depolarized voltages. These effects of xKv4 coexpression resemble
those reported for mammalian Kv1 and Kv3 subunits (Rettig et al.,
1994; England et al., 1995a,b; Heinemann et al., 1995, 1996; Majumder
et al., 1995; Morales et al., 1996; Accili et al., 1997a).
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Figure 7.
Coexpression of xKv4 induces inactivation of
both xKv1.1 and xKv1.2 currents. A, Coexpression
of xKv4, but not xKv2, induces inactivation of xKv1 channels
(*p 0.0001 vs Kv1 alone). B, For
both xKv1.1 and xKv1.2 subunits, coexpression of xKv4 induces
a voltage-dependent inactivation; more inactivation is observed at
stronger depolarizations.
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DISCUSSION |
Our interest in Kv subunits derives from the fact that they
modulate properties of voltage-dependent potassium current that are
developmentally regulated in the IKv
of Xenopus spinal neurons. Although it is well known that
IKv undergoes changes in density and
kinetics of activation and inactivation, the underlying molecular mechanisms have yet to be defined. We find that xKv genes are expressed in spinal neurons of Xenopus embryos during the
period of action-potential maturation. Although one xKv gene
(xKv2) is highly conserved with mammalian forms, the other (xKv4)
is unique with respect to both its primary sequence and functional effects. Coexpression of xKv with xKv1 subunits modifies those properties of potassium current that are required for maturation of the
impulse and excitability.
xKv2 shares >97% identity with mammalian Kv2. In contrast,
xKv4 shares only 69-74% identity with mammalian Kv1, Kv2, and Kv3. The level of identity found between the different Kv families within a species (e.g., human Kv1 vs Kv3) is only
70-75%. On this basis, xKv4 appears to identify a new Kv
family. This conclusion is further supported by xKv4's novel
functional effects, as discussed below. Although we screened several
embryonic cDNA libraries and did extensive RT-PCR using embryonic mRNA,
we have isolated only xKv2 and xKv4. It is possible that at later
stages of development Xenopus expresses genes that are
orthologous to mammalian Kv1 or Kv3. Kv1 is not a
mammalian-specific gene, because Rajeevan et al. (1999) recently
isolated a chick Kv1.1 ortholog that is 95% identical to mammalian
Kv1.1. Previous Kv cloning efforts have focused on adult stages.
Thus, it is possible that xKv4 reveals a Kv subfamily that is
preferentially expressed during embryonic development.
Consistent with xKv2's high primary sequence identity to mammalian
forms, the functional effects of xKv2 coexpression are similar to
those reported for mammalian Kv2. Mammalian Kv2 coexpression increases the amplitude of Kv1.2 and Kv1.4 currents (McCormack et
al., 1995; Accili et al., 1997a) and surface expression of Kv1.1,
Kv1.2, and Kv1.6 subunits (Shi et al., 1996); mammalian Kv2
also accelerates Kv1.5 activation kinetics (Heinemann et al., 1996;
Uebele et al., 1996). Similarly, xKv2 increases current density and
Gmax and also accelerates activation
kinetics when coexpressed heterologously with xKv1.1 and xKv1.2 subunits.
The most obvious functional effect of xKv4 coexpression is induction
of inactivation of Kv1.1 and Kv1.2 channels (Fig. 7). In this
respect, xKv4 is similar to mammalian Kv1 and Kv3, which have
been shown to increase inactivation of coexpressed Kv1 subunits
(Rettig et al., 1994; Heinemann et al., 1995, 1996; Majumder et al.,
1995; McCormack et al., 1995; Morales et al., 1996; Accili et al.,
1997b; Leicher et al., 1998). Induction of inactivation by Kv1
subunits is thought to involve an N-terminal ball domain and critically
depends on residues that are sensitive to oxidation (Rettig et al.,
1994; Wang et al., 1996). In mammalian Kv3 as well as in the
newly reported xKv4, similar residues (cysteine and serine preceding
several positively charged amino acids) exist, indicating that
induction of inactivation by Kv1, Kv3, and xKv4 may share a
common mechanism of action. However, only Kv1.1, a Kv1 splice
variant, has been shown to induce inactivation of both Kv1.1 and
Kv1.2 channels as does xKv4 (Heinemann et al., 1996). However,
coexpression of xKv4 also accelerates activation kinetics, an effect
not observed on coexpression of either mammalian Kv1 or Kv3 with
Kv1.1 or Kv1.2 channels (Heinemann et al., 1995, 1996; Leicher et
al., 1998). Furthermore, xKv4 coexpression leads to depolarizing
shifts in the activation curve (Fig. 4). In contrast, coexpression of
Kv1 or Kv3 subunits typically leads to hyperpolarizing shifts, if
any (Pongs et al., 1999). Taken together with xKv4's unique primary
sequence, these results support the designation of xKv4 as a new
Kv family member.
The two xKv genes have different expression patterns, suggesting
that they operate in a nonoverlapping population of neurons. The
dorsally restricted expression of xKv2 within the spinal cord (e.g.,
Rohon-Beard cells) is reminiscent of the expression pattern found
previously for xKv1.1 (Ribera and Nguyen, 1993), suggesting that
xKv1.1 and xKv2 subunits form Kv complexes
in vivo. The earliest that xKv1.1 mRNA is detected in
Rohon-Beard cells is St 26, several hours after xKv2 mRNA is
detected. xKv2 is transiently detected in somitic tissue between St
24 and 30 (26-35 hr). The earliest that
IKv can be consistently recorded from
developing myocytes is St 24 (26 hr). Thus, just as in neurons, Kv2
is expressed as soon as would be required to contribute to formation of
the first functional potassium channels.
xKv4's expression pattern differs both spatially and temporally
from that of xKv2. Spatially, xKv4's expression domain is
limited to the nervous system but extends more ventrally within the
developing spinal cord. Temporally, xKv4 has a pronounced anterior-posterior gradient in its expression pattern, which is less
obvious for xKv2. The expression pattern of xKv4 is similar to
that of a recently identified xKv1 gene, xKv1.3 (A. Hoffman and
A. Ribera, unpublished data), suggesting that xKv1.3 and xKv4
form Kv complexes in vivo. However, coexpression of subunit mRNAs or proteins is not a reliable indicator of the final channel stoichiometry, because examination of mammalian brain Kv channel complexes indicates that only a subset of possible channel combinations actually exists (Rhodes et al., 1997; Shamotienko et al., 1997).
In Xenopus spinal neurons, developmental regulation of
IKv is synchronized, despite the
heterogeneity in neuronal identities as well as patterns of potassium
channel gene expression (Ribera, 1996). Accordingly, each neuronal
subpopulation must upregulate IKv via
molecular mechanisms that are specific to the potassium channel
isoforms expressed by that particular neuronal subtype. However, the
different neuronal populations must also temporally coordinate their
developmental increases in IKv
density. The results presented here indicate that in those neuronal
subpopulations using xKv1 channels, xKv mRNAs are present and
their coexpression is able to induce relevant developmental changes in
IKv. Because xKv genes are
expressed contemporaneously with Kv1 subunits, their potential
contribution to functional upregulation of
IKv would be regulated at a level
subsequent to transcription. In this regard, the sequence and
structural similarities between Kv subunits and aldo-keto reductase
enzymes raise the possibility that developmental changes in either
cellular redox potentials or concentration of substrate drive Kv
modulation of potassium channel function (McCormack and McCormack,
1994; Gulbis et al., 1999). It is possible to overexpress wild-type and
mutant forms of ion channel subunits in the Xenopus embryo
(Jones and Ribera, 1994; Ribera 1996) and thus test experimentally how
Kv subunits function in vivo.
| |
FOOTNOTES |
Received Aug. 3, 1999; revised Sept. 24, 1999; accepted Sept. 27, 1999.
This work was supported by National Institutes of Health (NIH) Grant
F32 NS10250 and American Heart Association Grant CWFW-07-98 (M.A.L.),
and NIH Grants T32 NS07083 and RO1-NS25217 (A.B.R.). We thank
Dr. Kurt Svoboda for generous assistance in preparation of Figure 2,
and Drs. Ken Nakahira and Jim Trimmer for providing the rat Kv1 and
Kv2 cDNAs.
The cDNA sequences reported have been assigned GenBank accession
numbers AF172144 (xKv2) and AF172145 (xKv4).
Correspondence should be addressed to Angeles B. Ribera, Department of
Physiology and Biophysics, C-240, University of Colorado Health
Sciences Center, Denver, CO 80262. E-mail:
angie.ribera{at}uchsc.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
