(Received for publication, October 10, 1995; and in revised form, December 20, 1995)
From the
Inwardly rectifying K channel subunits may form
homomeric or heteromeric channels with distinct functional properties.
Hyperpolarizing commands delivered to Xenopus oocytes
expressing homomeric K
4.1 channels evoke inwardly
rectifying K
currents which activate rapidly and
undergo a pronounced decay at more hyperpolarized potentials. In
addition, K
4.1 subunits form heteromeric channels when
coexpressed with several other inward rectifier subunits. However,
coexpression of K
4.1 with K
3.4 causes an
inhibition of the K
4.1 current. We have investigated this
inhibitory effect and show that it is mediated by interactions between
the predicted transmembrane domains of the two subunit classes. Other
subunits within the K
3.0 family also exhibit this
inhibitory effect which can be used to define subgroups of the inward
rectifier family. Further, the mechanism of inhibition is likely due to
the formation of an ``inviable complex'' which becomes
degraded, rather than by formation of stable nonconductive heteromeric
channels. These results provide insight into the assembly and
regulation of inwardly rectifying K
channels and the
domains which define their interactions.
Inwardly rectifying potassium channels (K) (
)are found in a wide variety of tissues and cell types
where they are involved in the maintenance of the resting membrane
potential and control of
excitability(1, 2, 3, 4, 5, 6) .
The diversity of these channels can at least in part be explained by
the growing number of cloned inward rectifier
subunits(7, 8, 9, 10, 11, 12, 13, 14) .
In addition, as with voltage-dependent potassium channels
(K
), diversity is enhanced by the ability of inward
rectifier subunits to form homomeric or heteromeric channels. For
example, coexpression of K
4.1 (BIR10; (10) ) with
K
1.1 (ROMK1; (9) ) or K
5.1 (BIR9)
results in heteromeric channels distinct from either homomeric parental
channel(15) . (
)Also, coexpression of different
members of the K
3.0 subfamily has profound effects. For
instance, coexpression of either K
3.2 (GIRK2; (12) and (14) ), 3.3 (GIRK3; (12) ), or 3.4
(CIR, Refs. 16 and 17) with K
3.1 results in significant
G-protein stimulated channel
activity(16, 18, 19) . Also, an inhibitory
effect of K
3.3 upon K
3.2 channel activity
has been reported although the mechanism has not been
determined(19) .
In this study, we have investigated the
effects of coexpression of K 3.4 with K
4.1.
In this case, the effect is neither a potentiation nor a modification
of channel activity, rather an inhibitory
``dominant-negative'' effect upon K
4.1
currents. We show that this effect on K
4.1 is also
endowed by other members of the K
3.0 family, and that the
TMs are the structural elements which mediate the inhibitory
interactions. Further analysis suggests that the inhibitory
interactions occur shortly after translation and that the resulting
complexes are degraded rather than processed as nonconducting complexes
to the plasma membrane.
Figure 1:
Inhibition
of K 4.1 currents by K
3.4. Currents recorded
from oocytes injected with K
4.1 mRNA (a),
K
3.4 mRNA (b), K
3.4 and K
4.1 mRNAs in a 1:1 ratio (c), and K
3.4 and
K
4.1 mRNAs in a 10:1 ratio (d). The currents
observed following injection of K
3.4 mRNA alone or a 10:1
ratio of K
3.4 to K
4.1 mRNAs were
indistinguishable from mock-injected oocytes. Current families were
evoked by 500-ms voltage steps from a holding potential of -5 mV
to potentials from 40 mV to -100 mV in -10-mV increments. e, averaged current amplitudes were recorded at -100 mV
from oocytes injected with a constant amount of K
4.1 mRNA
and varying ratios of K
3.4 or D2 receptor mRNAs. Currents
were normalized relative to current amplitudes recorded from oocytes
injected with only K
4.1 mRNA. Error bars represent ± S.E.
The reduced K 4.1 currents evoked from
coinjected oocytes were not different from K
4.1 currents
recorded from oocytes injected only with K
4.1 mRNA.
Fitting the time-dependent component of the whole cell current trace
recorded at -100 mV with a double exponential yielded time
constants of
= 57.7 ± 2.5 ms and
= 366.0 ± 11.0 ms (A
= 76.6%, n = 6) for oocytes injected only
with K
4.1, and of
= 50.0
± 1.2 ms and
= 277.2 ± 9.9 ms (A
= 85.1%, n = 6) for
oocytes injected with a 1:1 ratio of K
4.1 and K
3.4 mRNAs. These results suggest a specific inhibitory effect of
K
3.4 upon K
4.1.
To determine if other
members of the K 3.0 subfamily had a similar effect on
K
4.1, K
3.1(8, 24) , and
K
3.2 (14, 12) were coexpressed with
K
4.1. As shown in Fig. 2, both of these K
3.0 subfamily members had similar inhibitory effects upon
K
4.1 current amplitudes. To test whether K
3.4 inhibits other inward rectifier subunits, K
1.1(9) , a subunit closely related to K
4.1,
was coexpressed with K
3.4. Currents evoked following
coexpression of K
1.1 and K
3.4 were reduced
compared to oocytes expressing only K
1.1, similar to the
effects on K
4.1 (currents reduced to <5% of controls;
not shown).
Figure 2:
Inhibition of K 4.1 currents
by other members of the K
3.0 subfamily. Averaged current
amplitudes recorded at -100 mV from oocytes coinjected with a
constant amount of K
4.1 mRNA and varying amounts of
either K
3.1 or K
3.2.
Figure 3:
Chimeras between K 4.1 and
K
3.4 suggest the inhibitory interaction resides within
the TM/pore region. Top, current families recorded from
oocytes injected with mRNAs encoding chimeras 1407, 1408, and 1409; no
currents different from control oocytes were detected following
injection of mRNAs for chimeras 1413, 1414, or 1415 (not shown).
Diagrammatic representations of the chimeric subunits are shown below. Bottom table, activity of the chimeras presented above when
injected alone and their effects upon coexpression with K
4.1 or K
3.4 (N.D. = not
determined).
To determine which domains mediate the inhibitory
effect of K 3.4 upon K
4.1, chimeras 1413,
1414, and 1415 were coexpressed in a 10-fold excess to K
4.1 and chimeras 1407, 1408, and 1409 with a 10-fold excess of
K
3.4 mRNA. The table in Fig. 3shows that those
chimeras with the TM/pore domains of K
3.4 (1413, 1414,
and 1415) had an inhibitory effect on K
4.1, while
chimeras 1407, 1408, and 1409, which contain the TM/pore region of
K
4.1, were inhibited by coexpression with K
3.4. Therefore, the structural elements which mediate inhibition
reside within the TM/pore domain.
To further localize the structural
elements responsible for the inhibitory interactions, three more
chimeras were constructed in which K 4.1 contained either
the first, second, or both transmembrane domains of K
3.4 (Fig. 4a). None of these three chimeras was functional
when expressed alone, and coexpression of K
4.1 and 1417
or 1418, the chimeras containing either one or the other of the
K
3.4 TMs, had no significant effect upon K
4.1 currents. In contrast, coexpression of K
4.1 and
1419, the chimera with both K
3.4 TM domains, inhibited
K
4.1 currents, similar to the inhibition by wild type
K
3.4 (Fig. 4b). These results demonstrate
that both TMs are necessary and sufficient for inhibition of K
4.1 channel activity.
Figure 4:
Both TMs are required for the inhibition. a, chimeras of K 4.1 containing either the first,
second. or both putative TMs of K
3.4. b,
averaged current amplitudes recorded at -100 mV from oocytes
coinjected with K
4.1 mRNA and the chimera mRNAs in the
indicated ratios.
Total membranes were prepared from oocytes injected with
either K 4.1-F mRNA alone or from oocytes coinjected with
K
4.1-F mRNA plus a 10-fold excess of test mRNAs. The
membrane fractions were prepared as a Western blot and probed with the
m2-FLAG antibody (Fig. 5). The K
4.1-F protein was
detected by the m2-FLAG antibody as a protein of approximately 40 kDa,
in close accord with its predicted molecular mass (41.1 kDa); the
fainter bands of higher molecular weight likely represent aggregates of
the K
4.1-F protein. These bands were not detected from
mock injected oocytes (not shown) or oocytes coinjected with an excess
of K
3.4, 1419, K
3.1, or K
3.2
mRNAs, in which K
4.1-F currents were completely inhibited
(<6% of the control current). However, the K
4.1-F
protein was detected in oocytes coinjected with mRNAs encoding K
4.1-F and the D2 receptor, K
1.1, or the two
transmembrane chimeras which do not inhibit K
4.1 (1417
and 1418); currents from these oocytes were not reduced compared to
control oocytes expressing K
4.1-F (Fig. 5).
Figure 5:
Coexpression of K 4.1 with
K
3.0 results in K
4.1 subunit degradation.
Western blot of total oocyte membranes probed with the m2-FLAG
antibody. Oocytes were coinjected with a constant amount of K
4.1-F mRNA and a 10-fold excess of the indicated test mRNAs. The
immunoreactive higher molecular mass bands likely represent aggregates
of K
4.1-F because they are not detected in control
oocytes and because heating of the samples above 45 °C prior to
loading results in disappearance of the major 40-kDa band and increased
intensity of the higher molecular mass bands. Shown below each lane is
the normalized current amplitude recorded at -100 mV, from each
group of oocytes prior to membrane
preparation.
When injected at a 10-fold excess, all members of the K 3.0 family tested, as well as chimera 1419, abolish the K
4.1 current and result in undetectable levels of the K
4.1-F protein, suggesting that they act through a common
mechanism. Because the membrane preparations contained intracellular as
well as plasma membrane compartments, it is likely that coexpression of
K
4.1 and K
3.4 results in degradation of
heteromeric complexes. If this is the case, then temporally separating
the expression of K
4.1 and K
3.4 might
separate coassembly of these subunits and allow K
4.1
channels to reach the plasma membrane. To test this hypothesis, oocytes
were injected with a 10-fold excess of K
3.4 mRNA either
12 h before or 12 h after injection of a constant amount of K
4.1 mRNA. Fig. 6shows that if the expression of either
subunit is delayed by 12 h then approximately 50% of the control
K
4.1 current is observed. However, if the two are
simultaneously coinjected, then K
4.1 currents are
abolished. These results suggest that coassembly of K
4.1
and K
3.4 subunits occurs shortly after translation, and
temporally separating translation of the two classes of mRNAs reduces
the likelihood of heteromer formation.
Figure 6:
Temporal separation of K 4.1
and K
3.4 expression results in reduced inhibition.
Normalized current amplitudes recorded from oocytes injected with
K
4.1 and a 10-fold excess of K
3.4; the
injection order at either time 0 or 12 h later is shown below each bar.
Coexpression of members of the K 3.0 family with
K
4.1 inhibits K
4.1 currents, an effect
which is likely due to cotranslational subunit assembly and subsequent
degradation of the heteromeric complexes. The structural motifs which
mediate the inhibitory interactions of K
3.4 with K
4.1 reside within the putative TMs, and, while these are the only
necessary structural motifs, both are required for inhibition.
The
inhibition of K 4.1 currents by K
3.4 is not
due to nonspecific effects on translation, because coexpression of
K
4.1 with an equivalent amount of additional mRNA
encoding another membrane protein, such as the dopamine D2 receptor, is
without effect. Further, K
3.4 has a similar inhibitory
effect upon K
1.1(9) , another inward rectifier
which is closely related to K
4.1. The inhibition of
K
4.1 and related subunits by members of the K
3.0 family thus provides an additional criterion for inward
rectifier subunit classification(25) .
The observation that
both TMs are required for the inhibitory interaction suggests that when
a single TM is swapped for that of another family, the subunits cannot
be coassembled. Chimera 1419 which has both K 3.4 TMs is
capable of interacting with K
4.1 subunits and inhibiting
the current similar to K
3.4. Although the structural
domains which mediate inhibitory interactions between members of the
K
3.4 subfamily and K
4.1 are different from
the domains which mediate coassembly of distinct K
subunits, both K
and K
subunit
coassembly may occur cotranslationally(26) .
The mechanism
of inhibition appears to act through sequestration of heteromeric
complexes into a degradative pathway soon after translation and before
insertion in the plasma membrane. If heteromeric channels are processed
to the plasma membrane as inactive complexes or if subunit coassembly
occurs by association within the plasma membrane, then the K 4.1-F subunit should be detected in oocytes coinjected with
K
4.1-F and K
3.4 mRNAs. However, K
4.1-F subunits were not detected by Western blot, even though the
preparations did not separate intracellular and plasma membrane
compartments. In addition, the inhibitory interactions were uncoupled
by temporally separating expression of the two different subunit types.
Thus, when K
4.1 subunits are allowed to assemble together
before interference from K
3.4 subunits, they are
processed to the plasma membrane as functional channels, and,
conversely, when K
3.4 subunits have already assembled
together, subsequent inhibition of K
4.1 is reduced.
However, when they are simultaneously expressed, as shown above,
K
4.1 currents are abolished. The fact that other members
of the K
3.0 family also had the same effect implies that
the mechanism of inhibition by members of this family is the same.
There are several tissues, including the heart and brain, where
members of the K 3.0 family and the subunits they inhibit
are coexpressed. In atrial myocytes for example, K
3.4 and
K
3.1 coassemble to form the channel underlying
I
(16) , but K
4.1 is also expressed
in this tissue(10) . It is possible that the inhibitory
interaction described here between the K
3.4 and K
4.1 provides a way for the cell to prevent either inactive or
possibly disruptive heteromeric complexes from reaching the plasma
membrane, reflecting an additional physiological mechanism which
regulates the array of distinct inward rectifier subtypes.