(Received for publication, August 30, 1995; and in revised form, October 10, 1995)
From the
YORK is a newly cloned K channel from yeast.
Unlike all other cloned K
channels, it has two pore
domains instead of one. It displays eight transmembrane segments
arranged like a covalent assembly of a Shaker-type
voltage-dependent K
channel (without S4 transmembrane
segments) with an inward rectifier K
channel. When
expressed in Xenopus oocytes, YORK does not pass inward
currents; it conducts only K
-selective outward
currents. However, the mechanism responsible for this strict outward
rectification is unusual. Like inward rectifiers, its activation
potential threshold closely follows the K
equilibrium
potential. Unlike inward rectifiers, the rectification is not due to a
voltage-dependent Mg
block. The blocking element is
probably intrinsic to the YORK protein itself. YORK activity is
decreased at acidic internal pH, with a pK
of 6.5.
Pharmacological and regulation properties were
analyzed. Ba ions and quinine block YORK currents
through high and low affinity sites, while tetraethylammonium displays
only one affinity for blocking. Activation of protein kinase C
indirectly produces an increase of the current, while protein kinase A
activation has no effect.
A large number of K channel subunits have been
identified in the last few
years(1, 2, 3, 4, 5, 6) .
All of the pore-forming K
channel subunits cloned in
vertebrates as well as in plants, paramecia, and bacteria (7, 8, 9, 10) share a highly
conserved sequence called the pore (or H5) domain (P-domain), (
)which has been shown to be part of the
K
-selective
pore(11, 12, 13, 14) . The presence
of this sequence in a protein is considered the signature of a
K
channel structure. In all K
channels so far cloned, there is never more than one P-domain per
subunit and a variable number of transmembrane segments (TMS).
Voltage-gated K
channels have six TMS, inward
rectifier K
channels have two
TMS(1, 2, 3, 4) , and
Ca
-dependent K
channels have 10
TMS(15, 16) . All six-TMS channels have a positively
charged TMS (S4) that is responsible for voltage sensitivity. The
two-TMS K
channels do not possess such a domain. Their
apparent voltage sensitivity for gating results from a
voltage-dependent block by intracellular Mg
or
polyamines(17, 18, 19, 20, 21, 22) .
YORK, the yeast outward rectifying K
channel described
in this work, presents both novel structural features and novel
functional properties.
For inside-out patch recordings, pipettes were filled with a
high K solution (40 mM KCl, 100 mM potassium methanesulfonate, 1.8 mM CaCl
, 2
mM MgCl
, 5 mM HEPES adjusted to pH 7.4
with KOH, and 10 µM GdCl
) to prevent
endogenous stretch-activated channel activities. The bath solution
contained 140 mM KCl, 3 mM MgCl
, 5 mM EGTA, and 5 mM HEPES adjusted to pH 7.2 with KOH. For
outside-out recordings, pipettes were filled with a solution identical
to that used for the bath solution in the inside-out patch recordings.
The external medium contained 140 mM KCl, 0.3 mM CaCl
, 1 mM MgCl
, and 10 mM HEPES adjusted to pH 7.1 with KOH. The external divalent-free
solution contained 140 mM KCl, 2 mM EGTA, 2 mM EDTA, and 10 mM HEPES adjusted to pH 7.1 with KOH.
Single-channel recording data were filtered at 5 kHz and digitized at
50 kHz.
CO-enriched solution was prepared by bubbling
100% CO
into the standard ND96 solution for 5 min. For
inside-out patch experiments, internal solutions at different pH values
were prepared by adding Na
HPO
and
NaH
PO
at different ratios. To obtain pH values
of 6, 6.4, 6.8, 7.2, 7.6, and 8, the
Na
HPO
/NaH
PO
ratios were
1.2/8.76, 2.6/7.34, 4.8/5.1, 7.2/2.8, 8.6/1.3, and 9.4/0.5 mM,
respectively. The curve was the least-square fit of the following
equation: I = I
/(1 +
(exp(pK - pH))/a), with pK =
6.5, I
= 1.4 pA, and a =
0.38.
The YORK gene encodes a 691-amino acid-long protein that
does not share significant sequence conservation with previously cloned
K channels, except in the two P-domain sequences (Fig. 1, a and c). The hydropathy profile
shown in Fig. 1b predicts eight membrane-spanning
hydrophobic segments (S1 to S8). The P-domains P1 and P2 are flanked by
TMS S5 and S6 and TMS S7 and S8, respectively. Placing the NH
terminus on the cytoplasmic side, in agreement with the absence
of signal peptide, leads to the topology model proposed in Fig. 1b. In this model, the two P-domains are inserted
into the membrane from the outside, corresponding to the known
orientation of these loops in all K
channels.
Moreover, the overall structural motif of YORK is similar to the motif
that would be obtained by adding a six-TMS subunit of a classic
voltage-dependent K
channel to a two-TMS inward
rectifier subunit. However, none of the YORK TMS shows the highly
conserved charged and regularly spaced residues that are responsible
for the voltage-sensing ability of outward rectifier K
channels. The alignment of P-domain sequences shown in Fig. 1c indicates that the K
-selective
pore regions of YORK are well conserved. In all K
channels, 2 Gly residues are perfectly conserved (Gly-16 and
Gly-18) (Fig. 1c), while nearly exclusive conservative
changes are found for the residues at positions 7 (Phe or Trp, except
for Shal, which has Tyr), 14 (Thr or Ser), and 17 (Tyr or Phe). YORKP2
is perfectly canonical with regard to all of these residues. In the
case of YORKP1, a Leu residue is found in place of Tyr-17, which is
present in all but one of the other K
channels. In
addition to these five positions, the global conservation of YORKP1 and
YORKP2 with other P-domains is high and suggests the participation of
both segments in the K
channel permeation pathway.
Site-directed mutagenesis was used to try to gain experimental
evidences that this is indeed the case. In the mutant YORKP1-L17Y, the
unusual Leu residue was replaced by Tyr, while in YORKP2-Y17L, the
classical Tyr residue was replaced by Leu. Both mutants were found to
be nonfunctional. The absolute necessity for function of the Tyr
residue in the second P-domain was not surprising since several
mutations of the corresponding Tyr in Shaker channels resulted
in production of nonfunctional channels(12) . Conversely, the
loss of function observed for the YORKP1-L17Y mutant was unexpected.
Although improper protein folding or sorting cannot be excluded, this
negative result suggests that the GLG motif is likely to belong to a
functional P-domain. The double mutant YORKP1-L17Y/P2Y17L, in which the
Leu and Tyr residues were mutually exchanged, was also nonfunctional.
Clearly, more mutations will be necessary to definitively establish the
participation of both P-domains in the formation of the K
permeation of YORK. Other relevant features of the YORK protein
are the presence of three N-glycosylation sites located in the
external S1-S2 and S7-P2 loops and several phosphorylation
consensus sites, including seven sites for protein kinase C and one
site for protein kinase A and tyrosine kinase (Fig. 1a).
Figure 1:
Structural properties of
YORK. a, amino acid sequence of YORK. TMS are boxed,
and P-domains are underlined. Consensus sites for
glycosylation () and for protein kinase C (
), protein
kinase A (
), and tyrosine kinase (
) phosphorylation are
shown. These sites have been identified by using the prosite server
(European Bioinformatics Institute) with the ppsearch software that is
derived from the Macpattern program. b, hydropathy profile and
deduced topology for YORK. Hydrophobicity values were calculated
according to the method of Kyte and Doolittle (37) (window size
of 11 amino acids) and are plotted against amino acid position. Shaded hydrophobic peaks correspond to TMS. PHO,
hydrophobicity pH; PHI, hydrophilicity. c, alignment
of the P-domains of YORK and other K
channels.
Identical and conserved residues are boxed in black and gray, respectively.
Figure 2:
Biophysical properties of YORK currents. a, currents recorded under two-electrode voltage clamp in ND96
solution from oocytes injected with 40 ng of cRNA. Superimposed current
traces were induced by voltage steps from a holding potential of
-80 mV. b, mean I-V curves (n = 5) measured at the onset of voltage steps () and at
the end of stimulation (
). Inset, voltage dependence of
the time constant of activation. c, relationships between the
global conductance (G) and the electrochemical potential (V - E
) for different external
K
concentrations. Conductances were calculated from I-V curves using the following equation: G = I/(V - E
). d, outward rectifying currents measured by inside-out patch
single-channel recordings in symmetrical K
solution
(140 mM) (holding potential = -20
mV).
Figure 7:
Voltage dependence of gating kinetics of
YORK currents. a, absence of tail current following a voltage
pulse to +30 mV. Inset, voltage clamp protocol. b, same outside-out patch in symmetrical K solution (140 mM). Average currents were obtained from
30 successive voltage pulses to +60 mV from holding potentials (HP) of -60 mV (left trace) and 0 mV (right
trace) (n = 10). c and d,
voltage dependence of the recovery of the slow component of the YORK
current obtained after a short interruption (40 ms) of the test pulse
to potentials from -200 to +30 mV. c, superimposed
current traces obtained with the pulse protocol illustrated in the inset of d (1-min interval between each pulse). d, plot of the percentage of recovery for the slow component
as a function of membrane potential; same experiment as in c. e and f, effect of the time interval between two
successive test pulses on the recovery of the slow component. e, superimposed current traces at increasing pulse intervals. f, plot of the percentage of recovery for the slow component
as a function of the time between two successive pulses; same
experiment as in e. g, proposed model for YORK
channel gatings with an intrinsic blocking particle. Em,
membrane potential.
In inside-out patches containing several YORK channels, responses to depolarizing voltage steps, from a holding potential of -20 mV, were consistent with those obtained in whole cell measurements, i.e. an instantaneous activity followed by a progressive increase in the number of open channels (Fig. 2d). Single-channel recordings showed that the current was strictly outward, with a unitary conductance of 26 ± 2 picosiemens (n = 5), and had a high tendency to flicker, with a mean open time of 0.19 ± 0.01 ms (six inside-out patches) (Fig. 3, a and b).
Figure 3:
Outward
rectification of YORK currents in excised membrane patches. a,
steady-state single-channel activities at the indicated potentials;
inside-out (I/O) patch mode in symmetrical K solution. b, I-V plot of the mean
single-channel currents recorded as in a. c, absence
of inward current in divalent-free external solution (n = 7). YORK channels were recorded in outside-out (O/O) patch mode at -60 or +60 mV in symmetrical
K
solution. The same patch was bathed in solution
containing either 1 mM Mg
(left
traces) or 2 mM EGTA and EDTA and no added divalent
cations (right traces).
The absence of an S4 voltage sensor structure,
associated with striking similarities to the biophysical properties of
inward rectifier K channels, suggested that the gating
mechanism for the outwardly rectifying YORK channel might be analogous
to that of inward rectifiers, i.e. a voltage-dependent block
by penetration of an impermeant ion in the pore. Mg
or polyamine cations play this role for inward rectifier channels (17, 18, 19, 20, 21, 22) .
Surprisingly, Mg
and Ca
removal
from the external solution has no effect on the outward rectifying
properties of YORK whole cell currents (data not shown). This lack of
effect of divalent cations on YORK rectification was fully confirmed on
excised patches. In outside-out configuration, the complete chelation
of divalent cations by EGTA and EDTA in the external bathing solution
did not allow inward currents to be recorded (Fig. 3c).
This finding has been validated on seven independent patches and
differs from the results of Ketchum et al.(27) that
appeared while this paper was submitted for publication. Therefore, it
cannot be concluded, as these authors did, that the mechanism leading
to YORK outward rectification is strictly the same as in inward
rectifiers, but in the opposite direction. The blocking element
responsible for outward rectification probably belongs to the channel
protein itself and obstructs the permeation pathway when the membrane
potential is below E
.
Figure 4:
Pharmacological properties of YORK
channel. a and b, concentration dependence of the
blocking effects of external Ba on the two components
of the YORK current. a, steady-state effects of increasing
concentrations of Ba
on currents elicited by
depolarizing pulses to +30 mV from a holding potential of
-80 mV. b, dose-response relation of the Ba
effects on the instantaneous component (
) and the total
current at the end of the pulse (
). The difference between the
two dose-response relations gives the sensitivity to Ba
of the slow component (
). c and d, same
protocol as in a. c, superimposed current traces
obtained in the absence and presence of 10 mM tetraethylammonium (TEA). d, superimposed
current traces obtained in the absence and presence of 100 µM quinine.
Figure 5:
pH regulation of YORK currents. a, blocking effect of internal acidosis on YORK currents by
superfusion with a CO-bubbled solution.
, control
currents;
, currents in the presence of CO
-bubbled
solution. b, I-V relationships derived from
the experiment shown in a. c, inside-out patch
single-channel activities at +80 mV at different internal pH
values (pH
). d, open
probability (NPo) as a function of internal pH. Open
probability values were calculated from steady-state recordings lasting
1 min.
Activation of protein kinase C by PMA (30
nM) increased the YORK current (Fig. 6, a and b). The time course of activation (Fig. 6c)
and the range of PMA concentrations needed to observe the effect
(between 1 and 60 nM) (Fig. 6e) are those
expected for the protein kinase C activation in Xenopus oocytes(28, 29) . Moreover, the inactive phorbol
ester 4-phorbol 12,13-didecanoate was without effect (Fig. 6c), as was PMA after preincubation with the
protein kinase C inhibitor staurosporine (30 µM) (Fig. 6e). Activation of protein kinase C can also be
achieved in a more physiological way by activating a receptor coupled
to phospholipase C through G proteins. When the cloned 5-HT
serotonin receptor (30) was coexpressed with YORK,
serotonin, similarly to PMA, induced an increase of the K
current (Fig. 6d). All these results taken
together clearly indicate that protein kinase C is involved in YORK
regulation. To our knowledge, this is the first description of
activation of a cloned K
channel by protein kinase C.
However, when protein kinase C was directly applied on excised patches,
it did not increase channel activity (data not shown). It is therefore
likely that the protein kinase C regulation is indirect and requires
the involvement of soluble cytoplasmic components. Elevation of
[Ca
]
by application of A23187
(1 µM) or microinjection of inositol triphosphate (1
µM) did not affect YORK activity. Therefore, YORK seems
insensitive to variations of internal Ca
. Protein
kinase A activation by application of 8-chloro-cAMP (300
µM) or forskolin (10 µM) had no effect on
channel function.
Figure 6:
Protein kinase C regulation of YORK
currents. a, superfusion for 10 min with the protein kinase C
activator PMA (30 nM) enhanced the YORK currents. b, I-V relationship derived from the experiment shown in a. , control currents;
, currents in the presence
of PMA. c, absence of effect of 100 nM 4
-phorbol
12,13-didecanoate (PDA) and time course of the effect of 30
nM PMA. d, increase of the YORK current after the
activation of coexpressed 5-HT
serotonin receptors with 3
µM serotonin. e, average K
currents expressed in percent of the control; effects of 1 nM PMA, 60 nM PMA (alone or after a 45-min preincubation
with 30 µM staurosporine), 100 µM synthetic
diacylglycerol analog (OAG), and 3 µM serotonin (5HT). Data are from three to eight oocytes under each
condition.
The model presented in Fig. 7g takes into account
all the properties of YORK channels described in this study. (i) The
currents are outwardly rectifying. (ii) As for inward rectifiers, the
activation potential is coupled to E. (iii) The
rectification persists in the absence of external divalent cations.
(iv) Two kinetically distinct current components are observed.
In
this model, an endogenous blocking particle such as an external peptide
domain of the channel enters the permeation pathway upon
hyperpolarization (Fig. 7g). The presence of two
binding sites is hypothesized to account for the instantaneous and the
delayed slow currents. A ``deep'' binding site for the
blocking particle is favored in hyperpolarization against a
``shallow'' binding site. Under polarized conditions, YORK
channels are either in the deep blocked state (C1) or in the shallow
blocked state (C2). Upon depolarization, the channels undergo either a
``delayed'' conformational change from C2 to C1 or an
``instantaneous'' transition from C1 to the open state (O).
The transition C1 O will give the instantaneous component of the
current, while the transitions C1
C2
O will impose the
delayed component. Upon repolarization, the only
``observable'' transition is O
C1, which is
instantaneous. The model involves two binding sites for the putative
blocking element. This view is supported by the fact that both
Ba
and quinine display two affinities for YORK
blockade (Fig. 4).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U37254[GenBank].