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Volume 17, Number 1,
Issue of January 1, 1997
pp. 32-44
Copyright ©1997 Society for Neuroscience
A Novel Subunit for Shal K+ Channels Radically Alters
Activation and Inactivation
Timothy Jegla and
Lawrence Salkoff
Department of Anatomy and Neurobiology, Washington University
School of Medicine, St. Louis, Missouri 63110
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Shal (Kv4) potassium channel genes encode classical subthreshold
A-currents, and their regulation may be a key factor in determining neuronal firing frequency. The inactivation rate of Shal channels is
increased by a presently unidentified class of proteins in both
Drosophila and mammals. We have cloned a novel Shal
channel subunit (jShal
1) from the jellyfish Polyorchis
penicillatus that alters Shal currents from both invertebrates
and vertebrates. When co-expressed with the conserved jellyfish Shal
homolog jShal1, jShal1 dramatically changes both the rate of
inactivation and voltage range of activation and steady-state
inactivation. jShal1 provides fast inactivation by a classic N-type
mechanism, which is independent of its effects on voltage dependence.
jShal1 forms functional channels only as a heteromultimer, and
jShal1 + jShal1 heteromultimers are functional only in a 2:2 subunit
stoichiometry.
Key words:
K+ channel;
Shal;
inactivation;
Polyorchis;
a-current;
heteromultimer;
jellyfish;
diploblast;
-subunit
INTRODUCTION
Transient K+ currents that are active
at subthreshold potentials (A-currents) (Connor and Stevens, 1971a
) are
a dominant K+ conductance during the interspike interval
and have a major influence on the firing frequency of neurons (Connor
and Stevens, 1971b). Of the four subfamilies of genes that encode the
channel-forming 
-subunits of voltage-gated K+ channels
(Shaker, Shab, Shal, and Shaw), the Shal (Kv4) subfamily most closely
matches the description of the classical subthreshold A-current. The
importance of subthreshold A-currents is underscored by the fact that
Shal is the most highly conserved voltage-gated K+ channel
subfamily in higher triploblastic metazoans (Salkoff et al., 1992).
Recent genetic and molecular evidence supports the idea that Shal
channels underlie classical subthreshold A-currents in neurons of both
vertebrates and invertebrates. Serodio et al. (1994) have shown that
the major subthreshold A-current expressed in the rat brain is likely
to be encoded by Shal genes. Shal channels in the mammalian brain are
located primarily on the dendrites and cell bodies of neurons (Sheng et
al., 1992), where their placement might influence spiking behavior.
Using mutant analysis, Tsunoda and Salkoff (1995a,b) also found Shal
currents in cell bodies of neurons from the fruit fly
Drosophila, suggesting that the intracellular location and
function of these channels is conserved across species.
Shal inactivation rate is regulated by an unknown mechanism in both
vertebrates and Drosophila. Shal currents vary almost 50-fold in inactivation rate in Drosophila embryonic
neurons. Most in vivo Shal currents inactivate far more
rapidly than currents expressed when Shal cRNA is injected into
Xenopus oocytes (Pak et al., 1991). In mammals, the
inactivation rates of Shal currents can be increased by co-expression
in Xenopus oocytes with low molecular weight fractions of
mRNA from rodent brain (Chabala et al., 1993; Serodio et al., 1994).
Both results suggest a mechanism for increasing the rate of
inactivation in Shal channels that is not intrinsic to Shal
-subunits. Because Shal currents influence the duration of the
interspike interval (Connor and Stevens, 1971b), their inactivation
rates may determine neuronal firing patterns. Thus, understanding the
mechanism controlling Shal inactivation rates may be important for
understanding how neuronal firing patterns are generated.
We have explored the evolutionary origins of Shal channels in a
primitive metazoan and have discovered a molecular mechanism for
regulating the inactivation rate of Shal currents. We show here that
Shal is highly conserved in the jellyfish Polyorchis penicillatus, a diploblastic coelenterate. Because diploblasts are
the most primitive metazoans to have nervous systems, our results show
that Shal currents were present in the first neurons that evolved in
the last common ancestors of diploblasts and triploblasts, ~700
million to 1 billion years ago (Morris, 1993). One
Polyorchis Shal homolog, jShal1, appears to function only
as a heteromultimer in concert with a Shal -subunit. Compared with
homomultimeric Shal channels consisting of Shal -subunits alone,
Shal + Shal heteromultimers produced currents that inactivate far
more rapidly. In contrast to the cytosolic 
-subunits that provide
rapid inactivation in mammalian Shaker K+ channels (Rettig
et al., 1994), jShal1 is homologous to the -subunits of
voltage-gated K+ channels.
MATERIALS AND METHODS
Cloning. Amplification and isolation of fragments of
jShal1 and jShal1 from Polyorchis
penicillatus genomic DNA was performed as described in Jegla and
Salkoff (1995) and Jegla et al. (1995). Briefly, the degenerate primers
5
-TCGGAATTCTATGACTACTGTTGGNTAYGGNGA-3 and
5-ACCTCTAGAGGTAGTGCTATTRYNAGNACNCC-3, which are derived from the
consensus amino acid sequences of the P-domain (MTTVGYGD) and the S6
domain (GVL(T/V)TIAL) of voltage-gated K+ channels, were
used to amplify initial fragments. These were size-selected,
reamplified using overlapping nondegenerate primers, and then subcloned
to allow for isolation of individual fragments.
A complete jShal1 genomic clone was obtained using the
initial jShal1 fragment to probe a Polyorchis
genomic library (provided by Dr. Warren Gallin, University of
Alberta) under high-stringency conditions (Butler et al., 1989) and
sequenced. The coding region of jShal1 consists of three
exons, as indicated in Figure 1. Two exons encoding the
N-terminal, S1-S6, and neighboring C-terminal cytoplasmic regions were
predicted based on their high homology to dShal and mShal. The third
exon encoding the poorly conserved distal C-terminal region was
identified by amplification from an oligo dT-primed
Polyorchis cDNA library using a jShal1-specific sense primer in the S6 region
(5-CCTGGTAAACTA-GTTGGTAGTATTTGCTCA-3) and an antisense primer
corresponding to the library vector sequence (5-TCCGGTCGACGTAGAGGG-GAATAAATCGCCATA-3). Construction of the Polyorchis genomic library and of cDNA libraries from
neuronally enriched Polyorchis penicillatus tissue samples
have been described previously (Gallin, 1991).
Fig. 1.
jShal1 and jShal1 amino acid sequences.
Jellyfish Shal homologs jShal1 and jShal1 are compared with Shal
channels from Drosophila (dShal; Wei et al., 1990) and
mouse (mShal, Kv4.1; Pak et al., 1991). Identical residues are shown in
reversed lettering (white on black). Predicted
transmembrane domains (S1-S6) and a K+ channel pore motif
(P-domain; Hartmann et al., 1991; Yool and Schwarz, 1991) are
underlined. Also underlined is the
cytoplasmic N-terminal domain (T1), which is believed to mediate
subfamily-specific assembly of voltage-gated K+ channel
subunits (Li et al., 1992; Shen et al., 1993; Shen and Pfaffinger,
1995). Large italic letters and plus
symbols mark five evenly spaced positively charged residues
found in S4 voltage sensor (Papazian et al., 1991) and seven positively
charged residues near the N terminal of jShal1 that are part of a
motif similar to N-terminal inactivation ball motifs (Murrell-Lagnado
and Aldrich, 1993). The positions of two introns conserved in jShal1,
dShal, and mShal are marked with arrows. Of these two,
only the intron position in the P-domain motif is also found in
jShal1. A third jShal1-specific intron near S6 is labeled with an
arrow bracketed by asterisks. Residue
numbers are shown on the right. Asterisks by the residue numbers at the end of the dShal and mShal sequences indicate that they are incomplete. The GenBank accession numbers for
jShal1 and jShal1 are U78642[GenBank] and
U78641[GenBank], respectively.
[View Larger Version of this Image (95K GIF file)]
The jShal1 genomic sequence was obtained in two
sequential rounds of inverse PCR (Ochman et al., 1988). The genomic DNA
used for inverse PCR was prepared by first cutting with a desired
restriction enzyme and then ligating at concentrations of 2 ng/µl or
less to promote self-circularization. In the first round of inverse PCR, a 1.4 kb fragment of jShal1 was amplified from DNA
prepared with BglII using sense
(5-CAAGTCTAGATGATAGGCTCTATGTGTTGCTTGAT-3) and antisense
(5-AACTAAGCTTGGATGGTAACAGGAACAACATC-3) primers derived from the
original P-domain-S6 jShal1 fragment. Sequencing revealed that the fragment included a BglII site at the
beginning of S2 and extended through the 3 end of the open reading
frame. Inverse PCR was also performed on genomic DNA prepared with
HindII using jShal1-specific primers
(5-CAGTTGTTTAGTTATAACCCTCTCC-3) and (5-GCCAAAGAAAAGGTGGGGTCTTCAC-3)
located 5 of a HindII site in S3. A 900 bp fragment
obtained in this screen was found to extend through the 5 end of
jShal1 coding sequence. The 3 coding region of jShal1
was confirmed by PCR amplification from a Polyorchis cDNA
bank by the same method as for jShal1, but with the
jShal1-specific sense primer used in the first round of
inverse PCR. A stop codon was found just 3 of S6 in two cDNAs and is
also present in the genomic sequence. The 5 coding region was not
found in any cDNA clones but was instead determined from genomic
sequence. Our analysis, based on several observations, indicated that
no introns interrupt coding in this region. First, no consensus
sequences for acceptor or donor splice junctions were found in this
region. Second, the codons used in this predicted 5 region of
jShal1 match the coding bias we have observed for six
Polyorchis K+ channel genes (data not shown).
Finally, this region has an A/T content of 64%, which falls within the
range we have observed for Polyorchis coding sequence
(60-65%), but well below the range we have observed for
Polyorchis introns (72-76%).
Alignments and phylogenetic trees. The amino acid alignment
(see Fig. 1) was generated using Microgenie (Beckman, Palo Alto, CA) and optimized by eye. Phylogenetic trees were constructed from
these alignments by maximum parsimony, as implemented in the PAUP
computer program (Swofford, 1993). Only sections of the T1 region and
membrane spanning core (S1-S6) that have relatively conserved lengths
(and thus certain alignment) among the Shaker, Shal, Shab, and Shaw
subfamilies were used for tree building. Tree lengths were calculated
using a step matrix that weighted changes between amino acids according
to the minimum number of nucleotide changes that were necessary to
achieve the change. A heuristic search for optimal trees was performed
using random addition to generate initial trees. Tree bisection and
reconnection were then used to optimize the trees. The consensus
maximum parsimony tree (see Fig. 2) was constructed from the 18 shortest trees found in this search.
Fig. 2.
Phylogenetic analysis places jShal1 and jShal1
in the Shal subfamily. A consensus maximum parsimony tree of jShal1,
jShal1, and 15 voltage-gated K+ channel genes
representing the Shaker, Shab, Shal, and Shaw subfamilies is derived
from the 18 most parsimonious trees found in a heuristic search.
Numbers indicate the percentage of times a particular branch point was
observed in these trees. For the unresolved branchings seen in the
Shaker subfamily, no single pattern was observed in >50% of the trees
from which the consensus is derived. Branches defining each subfamily
are bracketed at the right edge of the figure. Shaker homologs are from rat (rKv1.1, X12589; Baumann et al.,
1988), Drosophila (dShak, M17211; Papazian et al.,
1987), Aplysia (aShak, M95914; Pfaffinger et al., 1991),
a platyhelminth (pShak, L26968; Kim et al., 1995), and the
jellyfish Polyorchis (jShak1 and jShak2, U32922
and U32923; Jegla et al., 1995). Shab subfamily members are from rat
[rKv2.1, X16476, Frech et al. (1989); IK8 and K13, M81783 and M81784,
Drewe et al. (1992)], Drosophila (dShab, M32659, Butler
et al., 1989), and Aplysia (aShab, S68356, Quattrocki et
al., 1994). The Shaw subfamily is represented by rKv3.1 [rat, M68880,
Rettig et al. (1992)] and dShaw [Drosophila, M32661,
Butler et al. (1989)]. The Shal subfamily includes sequences from
mouse [mKv4.1, M64226, Pak et al. (1991)] and
Drosophila [dShal, M32660, Wei et al. (1990)] as well
as jShal1 and jShal1.
[View Larger Version of this Image (24K GIF file)]
Expression vector construction. The complete open reading
frame of jShal1 was united by removing the two introns from
the genomic clone. The 3 intron was removed from jShal1 in
two subcloning steps. First, the C-terminal cDNA fragment (which
spanned from S6 to the C terminal) was subcloned into Bluescript II
SK+ (Stratagene, La Jolla, CA) using an SpeI
site in S6 and an EcoRI site from the cDNA library vector.
Second, a genomic fragment spanning from an XbaI site 5 of
the initiator methionine to the SpeI site in S6 was cloned
into the SpeI site of this cDNA subclone. The P-domain
intron was removed using overlap extension PCR off this template (Ho et
al., 1989). The final overlap extension product was produced using a
sense primer (5-TTACGAATTCG-
AAT GGTGACATAGGCGCTT-3) that adds a consensus translation initiation sequence (underlined) (Kozak, 1987) surrounding the jShal1
initiator methionine and an antisense primer from the Bluescript
vector. This product was cut with EcoRI and cloned into the
EcoRI site of the Xenopus oocyte expression
vector pBSMXT (Wei et al., 1994). The coding region was then sequenced
and compared with the jShal1 genomic clone to confirm that
no PCR-introduced mutations existed.
A similar overlap extension protocol was used to remove both introns
from the open reading frame of jShal1. The final product was made
with a sense primer
(5-ATATGGATTATTCGGTTACTTC-CACTGCAAC-3) that
introduced a consensus translation initiation sequence (underlined) to
the jShal1 initiator methionine and an antisense primer
(5-CTTATCTAGATCAATCTTCTTCGCTAGCCTTCA- TTTGAATTATTGGGACAGG-3) that
includes the jShal1 stop codon and spans coding sequence on both
sides of the 3 intron. It was cut at flanking BamHI and
XbaI sites introduced in the primers and subcloned into the
pOX expression vector. Four individually amplified clones were
sequenced and compared with each other as well as with the original
inverse PCR-generated jShal1 clones, allowing PCR-introduced
mutations to be identified. A jShal1 expression vector clone
containing two silent mutations was used in all physiological experiments. The pOX vector was constructed by inserting
Xenopus -globin 5 and 3 untranslated sequences
contained in pBSMXT (Wei et al., 1994) into new restriction sites in
pBluescript II KS+ (Stratagene). Briefly, the Xenopus
-globin 5 untranslated and an NheI site was inserted
between the KpnI and SalI sites of the vector,
while an XhoI site and the Xenopus -globin 3 untranslated were inserted between the XbaI and
NotI sites.
Expression and electrophysiology. Capped cRNAs were prepared
by run-off transcription with T3 RNA polymerase using the mMessage mMachine kit (Ambion, Austin, TX) and diluted in RNase-free
ddH2O to desired concentrations before injection. Mature
stage IV Xenopus oocytes were prepared for injection as
described in Wei et al., 1990. Oocytes were injected with 50 nl of cRNA
and incubated at 18°C for 1-5 d in ND96 containing (in
mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES-NaOH, pH 7.5, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 mM sodium
pyruvate. Recording methods used were as published previously in
Covarrubias et al., 1991. Briefly, whole-cell recordings were made 1-5
d after injection at room temperature (~22°C) by conventional
two-microelectrode voltage-clamp techniques. Electrodes ranged from 0.2 to 0.5 M
in resistance and were filled with 3 M KCl. The
standard recording solution consisted of ND96 with 1 mM
4,4-diisothiocyanatostilbene-2,2 disulfonic acid to block native
oocyte chloride currents. Currents were digitally acquired with
CCURRENT using linear leak subtraction, filtered at 1 kHz with an
eight-pole Bessel filter and analyzed with CQUANT. Capacitative
transients were removed by leak subtraction or clipped out.
Stoichiometry calculations. Time constants (see Fig. 7) were
calculated by least-squares fits of double-exponential functions to
traces like those shown in the insets. Because the slowly inactivating jShal1 current could be inactivated by a prepulse to 
90 mV, such a
prepulse was often used to help isolate the fast components produced by
jShal1 + jShal1 heteromultimers in oocytes expressing both currents.
The free-mixing curve shown in Figure 7 assumes a binomial distribution
of channels containing zero to four jShal1 inactivation balls. If
p equals the fraction of jShal1 subunits and q
equals the fraction of jShal1-subunits, then the distribution of
channel types is as follows: p4 (0 balls) + p3q (1 ball) + p2q2 (2 balls) + pq3 (3 balls) + q4 (4 balls). The fractional size of the slowly inactivating current (jShal1
homomultimers, 0 balls) represents p4 and could
thus be used to predict the fraction of each type of channel. The
predicted fast-inactivation time constants were obtained by adjusting
the inactivation rate constant calculated for these currents to the
average number of inactivation balls per channel predicted from the
binomial distribution of channel types. Only those channels containing
one to three balls were included in the calculations of the
fast-inactivation time constants, because channels with four balls
(jShal1 homomultimers) are nonfunctional. Thus, if free mixing
occurs, the fastest inactivation time constants observed in
jShal1-biased currents with no jShal1 homomultimeric fraction (2.69 msec) should be produced primarily by channels with three inactivation
balls. Thus, the time constant of fast N-type inactivation should
approach three times this value as the proportion of jShal1 is
increased. The equations for calculating inactivation rate constants
from the time constants of inactivation and recovery and for
calculating the free mixing curve are from MacKinnon et al., 1993.
Fig. 7.
jShal1 and jShal1 form functional channels in a
single stoichiometry. 1 and 2 show
current traces (normalized in amplitude) for oocytes expressing jShal1 + jShal1 in a jShal1-biased mix (1) or a
jShal1-biased mix (2). The large, slowly inactivating component in 2 has the properties of a jShal1
homomultimer current. On the right, the
fast-inactivating components of 1 and 2
have been normalized to the same amplitude and plotted together,
showing nearly identical inactivation rates. The graph shows a plot of the fast-inactivation time constant versus fraction of the total current with slow inactivation (jShal1 homomultimeric current) for individual experiments in which oocytes were injected with varying
ratios of jShal1 and jShal1 (solid circles). The time constants are normalized to the mean fast-inactivation time constant calculated from the leftmost group of data points (2.69 msec), representing jShal1-biased mixes in which virtually no slowly inactivating current is seen. Inactivation rate was not increased by
further biasing the ratio toward jShal1. The straight
line (Fixed Stoichiometry) indicates the
predicted relationship between the fast-inactivation time constant and
the fraction of current with slow inactivation if only a single
stoichiometry of heteromultimer is functional. The curve
(Free Mixing) represents the prediction of
fast-inactivation time constant if jShal1 and jShal1 form functional
channels in all possible stoichiometries.
[View Larger Version of this Image (17K GIF file)]
The time constants (see Fig. 8) were determined by triple
exponential fits to current traces like those shown in the insets, because two time constants of slow inactivation (from jShal1 + jShal1(T) heteromultimers) contributed to the accuracy of the fit.
The curve for the prediction ofchannels containing three jShal1-subunits assumes that four types exist, all heteromultimers with three jShal1-based-subunits and one jShal1-subunit, but with
zero to three inactivation balls. Because of the high ratio of
jShal1 and jShal1(T) to jShal1 used in these experiments, few
jShal1 homomultimers are predicted to exist, allowing these channels to
be excluded from the calculations. If p equals the fraction
of jShal1(T)-subunits and q equals the fraction of
jShal1-subunits, then the distribution of channel types is:
p3 (0 balls) + p2q (1 ball) + pq2 (2 balls) + q3 (3 balls). The fraction of slowly inactivating current
(p3) was used to calculate the fraction
of each channel type. For the prediction of two jShal1-subunits in
each channel, the distribution is simplified to three possible channel
types: p2 (0 balls) + pq (1 ball) + q2 (2 balls). In this case, the slowly
inactivating current fraction equals p2. For the
prediction of a single jShal1-subunit, just two channel types will
exist, p and q, and the slowly inactivating
current fraction equals p. The curves were again calculated
by the method of MacKinnon et al. (1993) but with the binomial
distributions explained above.
Fig. 8.
Functional jShal1-jShal1 heteromultimers have
a 2:2 stoichiometry. 1 and 2 show
currents (normalized in amplitude) from an oocyte expressing only
jShal1 + jShal1 (1) and an oocyte expressing jShal1 + jShal1 + jShal1(T) (2) [jShal1 and
jShal1(T) are in a 1:1 ratio]. The slowly inactivating current
fraction is almost completely produced by jShal1 + jShal1(T)
heteromultimers. jShal1 homomultimers contribute little to this slow
current, because cRNA mixes were biased toward jShal1 + jShal1(T). Therefore, the inactivation time constants and
steady-state inactivation curves for slowly inactivating fractions
closely matched the inactivation time constants of jShal1 + jShal1(T) heteromultimers. The traces on the right
show a comparison of the fast-inactivating fractions of
1 and 2 normalized to the same amplitude.
This shows that adding jShal1(T) slows N-type inactivation. The
graph shows predictions of the fast-inactivation time constant versus
fraction of slowly inactivating current, in which the functional
heteromultimeric channels contain either 1, 2, or 3 jShal1-based-subunits. Solid circles represent data
from individual experiments; the fit assumes two jShal1-subunits in
the functional heteromultimers.
[View Larger Version of this Image (20K GIF file)]
RESULTS
Cloning and conservation
A previous study of the evolutionary origins of voltage-gated
K+ channels showed that homologs of mammalian channels are
present in the jellyfish Polyorchis penicillatus, which is
among the simplest extant metazoans to have an organized nervous system
(Jegla et al., 1995). We infer from this result that a similar set of
voltage-gated K+ channels play an indispensable role in the
nervous systems of all metazoans. Shal is the most conserved among
these channels in the higher triploblastic metazoa, and here we
describe highly conserved Polyorchis Shal homologs,
jShal1 and jShal1. Because regulation of Shal
channels may be a key element in the generation of patterned neuronal
output, we have now focused attention on the mechanism of this
regulation, which may be conserved as well.
jShal1 and jShal1 gene fragments were initially isolated by a PCR
screen of Polyorchis penicillatus genomic DNA. Degenerate primers for this screen were based on regions of the P-domain (MTTVGYGD) and S6 transmembrane domain (GVL(T/V)IAL) that are highly
conserved among voltage-gated K+ channels. The P-domain-S6
fragments of jShal1 and jShal1 were used to
isolate complete coding sequences for these genes both by hybridization
and PCR techniques, as described in Materials and Methods. Figure
1 shows the deduced amino acid sequences of the jShal1
and jShal1 proteins compared with the sequences of Shal proteins
from the fruit fly Drosophila (dShal) and mouse (mShal,
Kv4.1). Each contains the characteristic structural features of
voltage-gated K+ channel -subunits.
jShal1 is very clearly a direct homolog of triploblastic
Shal genes, because the jShal1 protein shares high conservation to triploblastic Shal -subunits over virtually its entire length. It is
~65% identical to dShal and mShal across the membrane-spanning channel core (Table 1). Interspecies homologs of
specific voltage-gated K+ channel genes typically share at
least 50% amino acid identity over this region, and jellyfish and
triploblastic Shaker homologs share only this 50% amino acid identity
(Jegla et al., 1995). The higher level of conservation in jShal1 is
consistent with previous observations that Shal is the most highly
conserved subfamily of voltage-gated K+ channels (Salkoff,
1992). Conservation is nearly as high in the T1 domain, which mediates
subfamily-specific channel assembly (Li et al., 1992; Shen et al.,
1993; Shen and Pfaffinger, 1995). jShal1 also has a
conserved genomic structure; the positions of two introns, one in the
P-domain motif and one in the C-terminal cytoplasmic domain, are
perfectly conserved among jShal1, dShal, and
mShal (Fig. 1, arrows).
In contrast, jShal1 is less well conserved and shares barely >40%
amino acid identity from S1 to S6 (Table 1). Despite this lower
conservation, several lines of evidence lead us to put
jShal1 in the Shal K+ channel subfamily. As
with all Shal -subunits, jShal1 contains an intron at
the "Shal-specific" site in the P domain. Introns are not found at
this position in genes from the Shaker, Shab, or Shaw subfamilies.
Secondly, Shal-specific conservation is high (>50%) in the T1
subfamily-specific assembly domain. Finally, phylogenetic analysis of
jShal1, jShal1, and 15 other voltage-gated K+ channel
proteins representing the Shaker, Shab, Shal, and Shaw subfamilies
unequivocally places jShal1 within the Shal subfamily. A consensus
of the 18 shortest trees found in a heuristic search using maximum
parsimony (PAUP, Swofford, 1993) is shown in Figure 2.
jShal1 is placed within the Shal subfamily in all of these trees.
The order of branching between channel subfamilies shown in Figure 2 is
not consistent in these 18 trees and should not be taken to indicate
the evolutionary relationships of these subfamilies.
The lower conservation shared between jShal1 and Shal -subunits
is especially evident in the S4 voltage sensor (Papazian et al., 1991),
which is perfectly conserved between dShal and mShal and has only two
substitutions of 22 residues in jShal1. In contrast, there are 12 substitutions in the same 22 residues of jShal1 (Fig. 1).
Interestingly, these S4 differences are found in the hydrophobic
residues, whereas the string of positively charged residues
characteristic of S4 is identical.
Two other key features distinguish jShal1 from jShal1 and other Shal
homologs. One is that jShal1 has a group of seven positively charged
residues at near its N terminal that is not found in other Shal
homologs (Fig. 1). This motif is reminiscent of an inactivation "`ball," similar to those responsible for rapid inactivation in both triplobastic and diploblastic Shaker channels (Hoshi et al., 1990;
Zagotta et al., 1990; Jegla et al., 1995). Later, we will show that
these charges are indeed part of a functional inactivation ball in
channels containing jShal1. The second feature is that after S6,
jShal1 has a segment of just seven amino acids and thus lacks a long
conserved C-terminal cytoplasmic segment found even in jShal1 (Fig. 1).
Expression
Shal1 expresses a rapidly activating transient current in
Xenopus oocytes that resembles Shal currents from
Drosophila and mammals. Figure 3 shows a
comparison of the jShal1 current and the Drosophila Shal
current dShal. The biophysical properties of these two currents are
summarized in Table 2. Both currents share features of
classic subthreshold A-currents that distinguish Shal currents from
other voltage-dependent K+ currents. These include a
hyperpolarized steady-state inactivation curve and an inactivation time
course that is relatively insensitive to voltage. However, the jShal1
current differs in two important ways. First, its inactivation rate is
severalfold slower than that of dShal. Whereas the fastest inactivation
time constant is ~140 msec for jShal1 at +50 mV, it is ~40 msec for
dShal. Second, jShal1's activation and steady-state inactivation
curves are shifted to even more hyperpolarized voltages. The
V50 for activation of jShal1 is approximately
35 mV more hyperpolarized than that of dShal (Fig. 3C).
The V50 for steady-state inactivation for jShal1 is approximately 106 mV compared with 62 mV for dShal
(Fig. 3D).
Fig. 3.
Comparison of the jShal1 and dShal currents.
Families of outward currents are shown for Xenopus
oocytes expressing either jShal1 (A) or dShal
(B). Currents were recorded in response to 1 sec test
pulses from 90 mV to +50 mV in 20 mV increments. Five second
prepulses to 140 mV (from a holding potential of 90 mV) preceded
each test pulse to ensure complete recovery from inactivation. C, Conductance versus voltage curves are shown for
jShal1 (solid circles) and dShal (open
circles). Error bars indicate SEM, and solid
curves represent Boltzmann fits of the data
(G/Gmax = 1/(1 + exp(
(V V50)/a), where
G is the conductance at voltage V, Gmax is the maximal conductance,
V50 is the voltage at which
G = 0.5 × Gmax,
and a is the slope factor. D,
Steady-state inactivation curves for jShal1 and dShal. Currents were
obtained by measuring the peak current during test pulses to +40 mV,
after 10 sec prepulses to the voltage shown on the
x-axis. Holding potentials were 90 mV with 5 sec
prepulses to 140 mV preceding test pulses. The curves
represent best fits of the data to the Boltzmann function I/Imax = 1/(1 + exp((V V50)/a), where
I is the peak current measured during the test pulse
after a prepulse to voltage V, Imax is the maximal current measured,
V50 is the prepulse voltage at which
I = 0.5 × Imax, and
a is the slope factor.
[View Larger Version of this Image (26K GIF file)]
jShal1 is unlike an -subunit in that it does not form
functional voltage-dependent channels when expressed as a homomultimer in Xenopus oocytes (Fig.
4A). Instead, jShal1 functions only in combination with Shal -subunits. Figure 4B
shows currents resulting from its co-expression with jShal1 in
Xenopus oocytes. Because this current is distinct from
jShal1 currents, it is assumed that it results from the
heteromultimeric assembly of jShal1 and jShal1. Relative to jShal1
homomultimers, jShal1 + jShal1 heteromultimers inactivate much more
rapidly with a time constant of <3 msec at +50 mV. This represents a
several hundredfold increase in the inactivation rate from jShal1
currents (Table 2). These heteromultimeric jellyfish Shal currents
resemble the majority of Shal currents expressed in
Drosophila embryonic neurons, which inactivate with time
constants near 5 msec (Tsunoda and Salkoff, 1995a). A second major
change produced by the co-expression of jShal1 with jShal1 is a
large depolarizing shift in the activation and steady-state
inactivation curves compared with jShal1 (~+30 mV for activation and
~+35 mV for steady-state inactivation) (Fig. 4C,D, Table 2). Unlike jShal1
homomultimers, these heteromeric channels are much more typical of Shal
channels from triploblasts with regard to their voltage range of
activation.
Fig. 4.
jShal1 modifies the inactivation and activation
range of jShal1. A, Currents recorded from a
Xenopus oocyte expressing only jShal1. Test pulses
(400 msec) from 70 mV to +50 mV were as described in Figure
3A. B, Rapidly inactivating outward
currents recorded from an oocyte co-expressing jShal1 and jShal1.
The same voltage protocol was used, except that test pulses were 100 msec in duration. The thin dotted line is a jShal1
homomultimeric current (+50 mV) scaled to the same amplitude for
comparison of inactivation rate. C, Conductance versus
voltage curves for jShal1 homomultimers (solid circles),
dShal homomultimers (open circles), and jShal1 + jShal1 heteromultimers (solid triangles).
D, Steady-state inactivation curves for jShal1, dShal,
and jShal1 + jShal1 co-injection. Data collection, analysis, and
curve fitting for C and D are as in
Figure 3, C and D.
[View Larger Version of this Image (22K GIF file)]
An N-type inactivation mechanism in jShal1
Because jShal1 is unique among Shal homologs in containing a
positively charged inactivation ball-like motif at its N terminal, we
tested the possibility that jShal1 causes rapid inactivation in
jShal1 currents by contributing an N-type inactivation mechanism. To
investigate this, we removed the positively charged N terminal from
jShal1; this truncated construct is referred to here as jShal1(T). This N-terminal truncation does indeed result in removal of fast inactivation from the heteromultimeric current (Fig.
5A, Table 2), providing the first clear
example of rapid N- type inactivation in Shal channels. The mechanism
of N-type inactivation conferred by jShal1 is independent of the
shift in the voltage range of activation. Thus, the activation and
steady-state inactivation curves of jShal1 + jShal1(T)
heteromultimers resemble those of jShal1 + jShal1 heteromultimers;
both have a depolarizing shift relative to that of jShal1 homomultimers
(Fig. 5B,C). Hence, this depolarized voltage range is likely to be conferred by regions other
than the N terminal such as the poorly conserved regions of the
jShal1 core. For instance, it was demonstrated that changes in the
hydrophobic residues of the S4 region of Shaker can cause large shifts
in the activation range (Lopez et al., 1991). Although fast N-type
inactivation is absent in jShal1 + jShal1(T) heteromultimers, slower
inactivation remains that is accelerated with respect to the
inactivation of jShal1 homomultimers (Fig. 5A, Table 2). The
residues responsible for this increased rate of residual inactivation are not known. Because N-type inactivation has been removed from jShal1(T), the remaining inactivation may occur by a mechanism similar to C-type inactivation in Shaker channels, which depends on
residues in the P-domain and S6 (Hoshi et al., 1991, Lopez-Barneo et
al., 1993).
Fig. 5.
jShal1 confers rapid inactivation by an N-type
mechanism. An N-terminal truncated form of jShal1, jShal1(T), was
constructed by replacing the N-terminal amino acids
MYSVTSTATYFLTRKVKNRHRTNVTRNK with an initiator Met. Seven positively charge residues
(bold) that distinguish jShal1 from Shal -subunits
were removed. A, Current traces from oocytes expressing
jShal1, or jShal1 + jShal1, or jShal1 + jShal1(T) recorded in
response to depolarizations to +50 mV. Traces are scaled to the same
amplitude for comparison of inactivation rate. B,
Conductance voltage curves for jShal1 (solid circles),
jShal1 + jShal1 (solid triangles), and jShal1 + jShal1(T) (open triangles). C,
Steady-state inactivation curves for jShal1, jShal1 + jShal1, and
jShal1 + jShal1(T). D, Time course of recovery from
inactivation for jShal1, jShal1 + jShal1, and jShal1 + jShal1(T).
Recovery rates were determined by a double-pulse protocol in which test
pulses to +40 mV were separated by a recovery pulse of increasing
duration. Recovery was at either 100 mV or 120 mV
(jShal1). Because the recovery rate of all Shal channels increases with increasing hyperpolarization (T. Jegla and L. Salkoff, unpublished observations), the difference in recovery rate between jShal1(T) heteromultimers and jShal1 homomultimers at an identical voltage is likely to be even greater than the difference we show here.
Pulse series were preceded by 5 sec prepulses to 140 mV to ensure
complete recovery from inactivation. Test pulse durations were 1 sec
for jShal1 and jShal1 + jShal1(T) and 200 msec for jShal1 + jShal1. Curves were generated using the equation
It = {a × (1 exp(t/
1))} + {b × (1 exp(t/2))}, where
It is the current amplitude of the second
+40 mV pulse divided by the current amplitude of the first pulse,
t is the interval of the recovery step at 100 mV, and
a and b are the components of current recovering exponentially with time constants of 1 and 2,
respectively.
[View Larger Version of this Image (26K GIF file)]
The highly charged N-terminal domain of jShal1 had a profound effect
on recovery from inactivation as well as on the inactivation rate.
Recovery from inactivation in jShal1 + jShal1 heteromultimers was
measured and compared with recovery of jShal1 + jShal1(T) heteromultimers and jShal1 homomultimers (Fig. 5D). jShal1 + jShal1 heteromultimers recovered slowest, with a time constant of
several seconds at 100 mV. However, in jShal1 + jShal1(T)
heteromultimers, recovery is significantly more rapid and is complete
within a few hundred milliseconds (Fig. 5D). Thus, the very
slow phase of recovery from inactivation is attributable to fast N-type
inactivation. On the other hand, recovery from slow "C-type"
inactivation is actually faster in jShal1 + jShal1(T)
heteromultimers than in jShal1 homomultimers (Fig. 5D).
jShal1 recovery was measured at 120 mV instead of 100 mV because,
as Figure 5C illustrates, most jShal1 channels are
inactivated at 100 mV. jShal1 + jShal1 heteromultimers have a
small but faster component to recovery, which may be caused by a few
channels entering an alternative "C-type" inactivated state before
N-type inactivation can occur. The very slow recovery from N-type
inactivation indicates that the association between the jShal1
inactivation ball and its blocking site on jShal1 + jShal1
heteromultimers is very strong.
jShal1 heteromultimerizes with dShal and mShal
jShal1 does not appear to form functional heteromultimers with
-subunits from subfamilies of voltage-gated K+ channels
other than Shal. In co-expression experiments, we found no evidence
that jShal1 alters any biophysical properties of channels formed by
the Polyorchis Shaker homologs jShak1 and jShak2 (data not
shown). However, the ability of jShal1 to form heteromultimers with
Shal -subunits from other species is conserved. Evidence for this is
presented in Figure 6, which shows currents resulting from the co-expression of jShal1 with dShal or mShal. Inactivation rates are increased when dShal and mShal are co-expressed with jShal1 in Xenopus oocytes. However, co-expression of
jShal1 with dShal and mShal has only minor effects on the activation range, probably because homomultimeric dShal and mShal channels already
operate in the voltage range in which jShal1 biases activation (data
not shown).
Fig. 6.
Heteromultimerization of jShal1 with dShal and
mShal. A, Current traces recorded in response to a test
pulse to +50 mV for Xenopus oocytes expressing dShal,
dShal + jShal1, and dShal + jShal1(T) are shown normalized in
amplitude for comparison of inactivation rates. B,
Similar traces for oocytes expressing mShal, mShal + jShal1, and
mShal + jShal1(T). C, Comparison of currents produced
by co-expression of jShal1 + jShal1, jShal1 + dShal, and
jShal1 + mShal. Although jShal1(T) appears to have opposite effects on the inactivation rates of dShal and mShal relative to jShal1
(Fig. 5A), the absolute inactivation rates of all three heteromultimers are actually quite similar.
[View Larger Version of this Image (16K GIF file)]
Several observations suggest that the inactivation ball binding site on
heteromultimers containing dShal or mShal has lower affinity for the
jShal1 inactivation ball than the binding site on heteromultimers
containing jShal1. The increase in inactivation rate in the
interspecies heteromultimers is clearly caused by the N-terminal
inactivation ball of jShal1, because inactivation is slowed if this
N-terminal ball is removed (Fig.
6A,B). Nevertheless, N-type
inactivation in these heteromultimers is not nearly as fast or complete
as when jShal1 is mixed with jShal1 (Fig. 6C, Table
3). This shows that the Shal -subunits, which do not
have a high affinity inactivation ball themselves, contribute to the binding affinity of the jShal1 inactivation ball. This result is
consistent with observations of the Drosophila Shaker
channel showing that only one inactivation ball at a time is able to
block the channel (MacKinnon et al., 1993). Taken together, these
results suggest that N-type inactivation particles may bind to a single site formed by all four subunits (Murrell-Lagnado and Aldrich, 1993).
Stoichiometry of jShal1 + jShal1 heteromultimers
We have exploited the presence of the N-terminal inactivation ball
on jShal1 to estimate the subunit stoichiometry of jShal1 + jShal1 heteromultimers. Our strategy was based on two assumptions. (1) The functional mechanism of N-terminal inactivation provided by
jShal1 is the same as in Shaker N-type inactivation (MacKinnon et
al., 1993). (2) jShal1 and jShal1 form tetrameric channels. This
latter assumption is based on the structural similarity of Shal
-subunits to Shaker -subunits, which are known to form tetramers
(MacKinnon, 1991). In Shaker, the inactivation ball contributed by each
of the four subunits functions independently (MacKinnon et al., 1993).
This independence results in a simple additive effect of each ball on
the inactivation rate. Thus, the inactivation rate of channels that
have only one inactivation ball would be roughly four times slower than
the inactivation rate for channels containing four inactivation balls.
This result was shown by mixing increasing proportions of Shaker
subunits lacking an inactivation ball with Shaker subunits having an
inactivation ball. In these experiments, the inactivation rate is
progressively slowed, because the number of inactivation balls per
channel is progressively reduced.
Similarly, if jShal1 and jShal1 mix freely to produce functional
heteromultimers of several stoichiometries, then increasing the
proportion of jShal1 in an oocyte will progressively reduce the average
number of inactivation balls on each heteromultimeric channel. This
should result in slower N-type inactivation. If, on the other hand,
there is only a single functional stoichiometry of jShal1 + jShal1
heteromultimer, then the rate of N-type inactivation should remain
constant as the proportion of jShal1 is increased (while the relative
amount of slowly inactivating current increases). Figure
7 illustrates this unchanging rate of fast inactivation and suggests that there is only a single functional stoichiometry in
jShal1 + jShal1 heteromultimers. As predicted, when the amount of
jShal1 is increased relative to the amount of jShal1, the fast-inactivating component of the current is progressively reduced, leaving a larger slow component (corresponding to the jShal1
homomultimeric current). Significantly, the time constant of fast
inactivation remains unchanged.
To further investigate the stoichiometry of jShal1 + jShal1
heteromultimers, we co-injected jShal1(T), which has the
inactivation ball removed, with jShal1 and jShal1. Unlike the
previous experiment, increasing proportions of jShal1(T) should
progressively reduce the average number of inactivation balls per
heteromultimeric channel, even if the functional stoichiometry of
jShal1 + jShal1 heteromultimers is fixed. In contrast to the
experiments shown in Figure 7, the fast-inactivation rate should
progressively slow. Oocytes injected with jShal1(T), jShal1, and
jShal1 again had both fast- and slow-inactivating current fractions
(Fig. 8). Most importantly, the rate of fast N-type
inactivation is slowed almost twofold by the addition of increasing
amounts of jShal1(T). By analogy to the Shaker results, the twofold
slowing of N-type inactivation suggests that the number of inactivation
balls is reduced by half when jShal1(T) is added. These results can
be explained by postulating three significant current components: a
very fast component produced by channels with two jShal1 and two
jShal1-subunits, a slower "fast" component produced by channels
with two jShal1, one jShal1, and one jShal1(T)-subunits, and the
slow component produced by channels with two jShal1 and two
jShal1(T)-subunits. A second slow component produced by jShal1
homomultimers is of insignificant size in these mixes, because of the
high ratio of jShal1 and jShal1(T) to jShal1. Thus, it appears
that heteromultimers are limited to a single stoichiometry of two
jShal1-subunits and two jShal1-subunits (Fig. 8). The fact that we
can progressively reduce the number of inactivation balls without
completely removing N-type inactivation strongly supports our initial
assumption that the jShal1 inactivation balls function independently
of each other, exactly like Shaker inactivation balls.
DISCUSSION
Significance of the conservation of Shal in diploblasts
Connor and Stevens (1971b) first recognized that
subthreshold A-currents have the capacity to influence neuronal firing
frequency because of their active role during interspike intervals.
Patterned neural output from single neurons appears to be highly
conserved among animals. Neurons of diploblastic
coelenterates such as Polyorchis are capable of
firing trains of action potentials or rhythmic bursts of action
potentials that are virtually indistinguishable from those of
vertebrates (Anderson, 1979; Przysiezniak and Spencer, 1989). The high
conservation of Shal channels is likely to be part of the reason. The
conservation of several additional classes of voltage-gated ion
channels also contributes to this conservation of intrinsic electrical
properties between diploblastic and triploblastic neurons (Anderson and
McKay, 1987; Dunlap et al., 1987; Holman and Anderson, 1991;
Przysiezniak and Spencer, 1992, 1994; Anderson et al., 1993; Meech and
Mackie, 1993; Jegla et al., 1995). This growing set of channels known
to be shared by diploblasts and triploblasts may represent an essential
set for the patterning of signals in all nervous systems and suggests
that the intrinsic electrical properties of neurons were optimized
early in the evolution of the nervous system.
Regulation of Shal inactivation by jShal1
Our finding that jShal1, a functionally novel
Shal-subunit, regulates the inactivation rate of Shal currents
parallels the discovery that -subunits can induce rapid inactivation
in Shaker currents (Rettig et al., 1994). In contrast to
jShal1, these -subunits are cytosolic proteins homologous to
members of the NAD(P)H-dependent oxidoreductase superfamily (McCormack
and McCormack, 1994) and are not structurally homologous to
voltage-gated K+ channel -subunits. Although both
jShal1 and Shaker -subunits cause rapid inactivation by N-type
mechanisms, jShal1 has additional effects on the voltage range of
channel activation. This is, perhaps, attributable to the fact that
jShal1 forms an integral part of the voltage-sensing mechanism of
the channel.
Although jShal1 is similar in structure to the -subunits of Shal
and other voltage-gated K+ channels, it is unique in that
it does not form functional homomultimeric channels. Instead, it
modifies the gating properties of Shal -subunits, functioning only
as a heteromultimer. This distinct functional role has led to an
interesting pattern of conservation between Shal -subunits and
jShal1. Regions involved in subfamily-specific assembly (T1) and ion
selectivity (pore region) are highly conserved, whereas regions
involved in determining gating properties (N terminal, S1-S4) differ
substantially.
The reason that jShal1 does not form functional homomultimers is not
clear, but it is not exclusively attributable to constitutive inactivation resulting from the presence of four high-affinity inactivation balls. This is demonstrated by the fact that the N-terminal truncated construct jShal1(T) also fails to express a
voltage-dependent current as a homomultimer. The unusually short C-terminal cytoplasmic domain is also unlikely to be responsible for
the failure of homomultimer formation. This was shown by substituting the longer C-terminal domain of jShal1 for the shorter C-terminal domain of jShal1; expression of voltage-dependent currents was not
recovered (data not shown). These experiments strongly imply that
jShal1 is specifically designed to function only in a
heteromultimeric configuration.
The restriction of functional jShal1 + jShal1 heteromultimers
to a single 2:2 functional stoichiometry may be necessary to precisely
fix the voltage range and inactivation rate of the current rather than
to allow gradations of these properties. Because the jShal1
inactivation ball binding site appears to involve both the jShal1 and
jShal1-subunits, a precise arrangement of these two types of
subunits may be necessary to produce a high-affinity binding site. If
so, only one of two possible arrangements of the two jShal1-subunits
within the 2:2 heteromultimers (side by side or opposite) may produce
functional fast-inactivating channels. It may be possible to address
this question in the future by using tandem constructs of jShal1 and
jShal1. If both arrangements are functional, fast-inactivating
heteromultimers should be obtained by expression of jShal1 + jShal1
dimers (opposite arrangement) as well as by co-expression of jShal1 + jShal1 and jShal1 + jShal1 dimers (side by side arrangement). We
do not know whether 3:1 and 1:3 stoichiometries of jShal1 + jShal1
heteromultimers assemble but are nonfunctional or simply do not
15%), virtually instantaneous component to jShal1 recovery fit is not listed, because it is probably caused by channels that did not inactivate during the test pulse and did not close during the shortest recovery periods.
