Department of Physiology and Biophysics, Cornell University
Medical College, New York, New York 10021
ROMK
channels play a key role in overall K balance by controlling K
secretion across the apical membrane of mammalian cortical collecting
tubule. In contrast to the family of strong inward rectifiers (IRKs),
ROMK channels are markedly sensitive to intracellular pH. Using
Xenopus oocytes, we have confirmed
this pH sensitivity at both the single-channel and whole cell level.
Reduction of oocyte pH from 6.8 to 6.4 (using a permeant acetate
buffer) reduced channel open probability from 0.76 ± 0.02 to near
zero (n = 8), without altering
single-channel conductance. This was due to the appearance of a
long-lived closed state at low internal pH. We have confirmed that a
lysine residue (K61 on ROMK2; K80 on ROMK1), NH2 terminal to the first putative
transmembrane segment (M1), is primarily responsible for conferring a
steep pH sensitivity to ROMK (B. Fakler, J. Schultz, J. Yang, U. Schulte, U. Bråandle, H. P. Zenner, L. Y. Jan, and J. P. Ruppersberg. EMBO J. 15:
4093-4099, 1996). However, the apparent
pKa of ROMK also
depends on another residue in a highly conserved, mildly hydrophobic
area: T51 on ROMK2 (T70 on ROMK1). Replacing this neutral threonine
(T51) with a negatively charged glutamate shifted the apparent
pKa for inward conductance from 6.5 ± 0.01 (n = 8, wild type) to 7.0 ± 0.02 (n = 5, T51E). On the other hand, replacing T51 with a positively charged
lysine shifted the apparent
pKa in the
opposite direction, from 6.5 ± 0.01 (n = 8, wild type) to 6.0 ± 0.02 (n = 9, T51K). The opposite effects of
the glutamate and lysine substitutions at position
51 (ROMK2) are consistent with a model in which T51 is
physically close to K61 and alters either the local pH or the apparent
pKa via an
electrostatic mechanism. In addition to its effects on pH sensitivity,
the mutation T51E also decreased single-channel conductance from 34.0 ± 1.0 pS (n = 8, wild type) to
17.4 ± 1 pS (n = 9, T51E),
reversed the voltage gating of the channel, and significantly increased
open-channel noise. These effects on single-channel currents suggest
that the T51 residue, located in a mildly hydrophobic area of ROMK2,
also interacts with the hydrophobic region of the permeation pathway.
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INTRODUCTION |
POTASSIUM SECRETION in the mammalian cortical
collecting tubule (CCT) is primarily controlled by a mildly inward
rectifying, potassium channel (SK), located at the apical membrane of
CCT principal cells (5, 15). Expression cloning resulted in the putative identification of the SK channel with the ROMK family of mild
inward rectifiers, consisting of the following three splice variants:
ROMK1 (Kir1.1a; see Ref. 8), ROMK2
(Kir1.1b; see Ref. 18), and ROMK3
(Kir1.1c; see Ref.
1). ROMK2 lacks the first 19 amino acids of ROMK1, whereas
ROMK3 contains a 7-amino acid extension.
Potassium channels in the inward rectifier superfamily (ROMK, IRK,
GIRK) are thought to consist of four identical subunits surrounding a central pore (7, 17). Hydropathy analysis of the primary
structure of these subunits suggests a common motif, consisting of two
putative membrane spanning domains (M1 and M2) separated by a
relatively short loop thought to be associated with the permeation path
or pore region. Both the COOH-terminal and
NH2-terminal segments of the
subunits are devoid of long hydrophobic stretches and are presumed to
be cytoplasmic.
Despite similarities in structure among members of the IRK superfamily,
the ROMK channels display a much greater sensitivity to intracellular
pH (pHi) than do the strong
inward rectifiers like IRK1 (4). Reductions in
pHi from 7.4 to 6.8 greatly decrease the open probability
(Po) of native
SK channels (15), as well as ROMK channels expressed in
Xenopus oocytes (10, 11, 14). This
marked sensitivity of the ROMK family to
pHi may be important for K
homeostasis during different metabolic states. Specifically, the
clinical observation that metabolic alkalosis is often accompanied by
enhanced K secretion and hypokalemia could partly be explained by
augmentation of luminal CCT potassium permeability by increases in
pHi (13, 16).
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MATERIALS AND METHODS |
Clones. The primary structure of the
ROMK2 clone (GenBank accession no. L29403) has been previously
described (18). The clone was obtained from a cDNA library made from
rat kidney poly(A)+ RNA in the
plasmid vector pSPORT. To obtain RNA from the clone, plasmid DNA was
purified with the Qiagen anion-exchange column system (Qiagen,
Chatsworth, CA). Plasmid DNA was linearized with Not I and transcribed in vitro with T7
RNA polymerase in the presence of the GpppG cap using mMESSAGE mMACHINE
kit (Ambion, Austin, TX). Synthetic RNA was dissolved in water and
stored at
70°C before use. Mutations were made using the
overlap extension method (9). Nucleotide sequences were checked using
the dideoxy chain termination method with a Sequenase kit (US
Biochemical, Cleveland, OH).
Oocytes. Stage V-VI oocytes were
obtained by partial ovariectomy of female Xenopus
laevis (Xenopus-I, Ann Arbor, MI), anesthetized with
tricaine methanesulfonate (1.5 g/l, adjusted to pH 7.0). Oocytes used
for the whole cell, two-electrode voltage clamp (TEVC) experiments were
defolliculated by incubation with 2 mg/ml collagenase type II and 2 mg/ml hyaluronidase type II (Sigma Chemical, St. Louis, MO) for 60 min
at 23°C and stored overnight at 19°C in modified Leibovitz L-15
medium (18). On the next day, healthy oocytes were selected for RNA
injection.
Oocytes destined for patch-clamp experiments were defolliculated by
incubation with 4 mg/ml collagenase type II and 4 mg/ml hyaluronidase
type II (Sigma Chemical) for 60 min at 23°C and then exposed to
hypertonic media (460 mosmol/kgH2O) for 15 min. Only those oocytes exhibiting a clear separation between vitelline and plasma membranes were selected, returned to solutions of normal osmolarity, and saved overnight at 19°C in Leibovitz L-15 medium (2, 3). This modification in the oocyte preparative technique significantly reduced adhesions between the vitelline and plasma membranes and greatly enhanced the probability of forming
high-resistance seals at the time of patching (3). Oocytes used for
either TEVC or patch-clamp were injected with 0.5 to 1 ng cRNA and
incubated at 19°C in Barth's solution supplemented with Leibovitz
medium for 1-3 days before measurements were made.
Electrical. Whole oocyte
current-voltage
(I-V)
relationships were obtained, in intact oocytes, using a TEVC with 3 M
KCl-filled current and voltage electrodes, as previously described (2). Currents were recorded for 50 ms at each voltage, using a pulse protocol in which membrane potential
(Vm)
was stepped by 10 mV from +60 mV to
140 mV, interspersed with a
return to the resting Vm.
The oocytes reserved for single-channel measurements were again
subjected to a hypertonic shrinking solution, thereby allowing the
vitelline membrane to be easily removed. Single channels were studied
using the patch-clamp technique as previously described (2, 18).
The stripped oocytes were placed in a small Lucite chamber where the
bath solution could be exchanged by gravity perfusion. All
measurements were made at room temperature. Methods for data acquisition and analysis have been described in detail elsewhere (6,
12). Pipettes were pulled from hematocrit tubing on a three-stage
puller and were coated with Sylgard prior to use. Currents were
recorded with either List EPC-7 or Dagan 8900 patch-clamp amplifiers
and stored, unfiltered, on videotape. For analysis, current records
were replayed from videotape, sampled at 5 kHz, low-pass filtered at 1 kHz, and analyzed using an Atari-based data acquisition system and TAC
software (Instrutech, Mineola, NY).
Solutions. In the TEVC experiments,
initial measurements of resting potential were performed with (in mM)
105 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), adjusted to pH 7.4 with NaOH. Those experiments examining
the effects of internal oocyte pH on macroscopic conductance utilized
high-K bath solutions consisting of (in mM) 55 KCl, 55 potassium
acetate, 1 MgCl2, and 2 CaCl2, buffered with 10 HEPES, and
adjusted to a final pH between 6.3 and 8.2 with KOH. As previously described (14), internal oocyte pH could be controlled with membrane-permeable potassium acetate solutions. However, this method of
changing oocyte pH required about a 20-min equilibration period for
each new pH, and the measured internal pH was always significantly
lower than bath pH (see below).
In the patch-clamp experiments, oocytes were bathed in a high-K bath
solution, consisting of either 110 mM KCl or 55 mM KCl + 55 potassium
acetate (for the pH studies) and 2 mM
CaCl2, buffered with 10 HEPES.
Pipettes were filled with either 110 mM KCl or 55 mM KCl + 55 mM
potassium acetate (for the pH studies) and 1 mM
MgCl2, buffered with 10 mM HEPES.
In the patch experiments, MgCl2
was omitted from the bath solution, because it accelerates rundown in
excised patches, but it was retained in the pipette solution, because
the single-channel conductance of ROMK is
Mg2+ dependent. All other
chemicals were obtained from Sigma Chemical.
Intracellular pH measurements.
pHi was measured with pH-selective
microelectrodes in a small sample of oocytes to determine how well the
membrane-permeable acetate-buffered solutions controlled pHi. Glass capillaries (model GC
200 F-10; Warner Instruments, Hamden, CT) were pulled on a horizontal
microelectrode puller (model P-97; Sutter Instruments, San Francisco,
CA). pH-selective electrodes were silanized by exposure to 40 µl of
bis(dimethylamino)-dimethylsilane (catalog no. 14755, Fluka Chemical)
in a covered glass container at 200°C, and the tips were coated
with hardened Sylgard (Dow Corning) to reduce electrical noise. The tip
of the pH electrode was filled (from the back) with Hydrogen ionophore
I-Cocktail B (catalog no. 95293, Fluka) and then back-filled with pH 7 solution of 0.04 M
KH2PO4,
0.023 M NaOH, and 0.015 M NaCl and inserted into an Ag/AgCl half-cell
electrode holder. The pH electrodes had resistances of ~5 × 1011
and were calibrated using
commercial standard pH 6 and pH 8 solutions. The mean slope of the
electrodes used in this study was 56.2 ± 0.6 mV/pH unit. Voltage
electrodes were pulled as above but filled with 3 M KCl and had tip
resistances of 30-60 M
. The voltage due to
pHi was obtained by electronically
subtracting the potential recorded by the voltage microelectrode from
the voltage recorded by the pH microelectrode using a high-impedance electrometer (model FD 223; World Precision Instruments, Sarasota, FL).
Intracellular voltage and pH were continuously displayed with an online
computer system. The pHi
measurements reported in this study were conducted at Yale Univ. Dept.
of Physiology in the laboratory of Dr. W. F. Boron, with the expert
assistance of Dr. G. Cooper and Dr. M. Romero.
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RESULTS |
Control of oocyte pH with acetate
solutions. To determine the time course and
effectiveness of membrane-permeable, acetate-buffered solutions at
controlling oocyte pH, pH-selective (resin-filled) microelectrodes were
used to measure pHi in a small
group of oocytes having either high or low K conductance (see
MATERIALS AND METHODS). When
initially placed in a 2 mM K-Ringer solution, those oocytes expressing large amounts of wild-type ROMK2 (WT-ROMK2) channels for
several days had generally more negative resting potentials (
90 ± 5 mV) and slightly lower initial
pHi (7.13 ± 0.03) than low K
conductance, water-injected oocytes (
73 ± 6 mV, initial pHi = 7.32 ± 0.02).
Immersion in 55 mM KCl + 55 mM potassium acetate depolarized both high
and low K conductance oocytes to resting potentials near zero mV.
Progressive exposure of these oocytes to potassium acetate-chloride
solutions buffered at pH values between 8.2 and 6.3 decreased oocyte
pHi from 7.2 to 6.1 where
steady-state pHi values were
generally obtained at 20-25 min after a change in external pH.
These results are summarized in Table 1 for
the five bath pH values tested on either ROMK2- or water-injected oocytes from three frogs.
In all oocytes, steady-state pHi
was always lower than the bath pH, although the degree of deviation
depended on extracellular pH. Furthermore, exposure to acetate
solutions always decreased pHi
below its resting value, as initially measured in 2 mM K Ringer solution. It was not possible to alkalinize the oocytes with
extracellular potassium acetate solutions.
Effect of pH on single-channel ROMK2
currents. Results of the present study confirm the pH
sensitivity of ROMK at both the single channel and whole cell level.
Expression of ROMK2 in Xenopus oocytes
yielded channels with high open probability and mild inward rectification as previously described (2). In cell-attached patches
with one channel in the patch, intracellular acidification (via
permeant acetate buffers) consistently reduced
Po.
A complete time course illustrating the effect of lowering
pHi on ROMK2 is given in Fig.
1. The cell-attached patch contained only
one channel, and the oocyte was maintained at
100 mV relative to
the pipette throughout the 20-min recording. In this experiment, extracellular pH was changed from 7.4 to 6.8 using the
membrane-permeable acetate buffer described (MATERIALS
AND METHODS). According to the
pHi measurements (Table 1), this
would correspond to a steady-state change in oocyte
pHi from 6.8 to 6.4. Although
pHi was not directly measured in
this particular experiment, the decline in
pHi roughly paralleled the
decrease in channel activity. Comparison of the time
course of pHi and inward current
is described below. About 10 min after the change in bath pH from 7.4 to 6.8, there was a noticeable increase in long closures characterized
by the appearance of a third closed state in the kinetic scheme (Fig.
2B).
This effect could be reversed by returning the bath pH to 7.4. In five single-channel recordings (each on a separate oocyte), the average lifetime of this third long-closed state was 2.6 ± 0.5 s (Table 2).

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Fig. 1.
Effect of changing internal pH from 7.4 to 6.8 on wild-type ROMK2
(WT-ROMK2) currents recorded from a single channel in a cell-attached
patch on an oocyte depolarized by 55 mM KCl + 55 mM potassium acetate.
Oocyte was maintained at 100 mV relative to pipette throughout
the changes in internal pH, which were accomplished with a
membrane-permeable acetate buffer. Bath pH values of 7.4 and 6.8 correspond, respectively, to intracellular pH
(pHi) values of 6.8 and 6.4. Downward deflections from the closed state (dotted line) denote inward
currents. Circled events denote subconductances, appearing before
channel shutdown and shortly after reactivation.
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Fig. 2.
Effect of decreasing pHi on
closed-time histograms of WT-ROMK: analysis of cell-attached patch,
containing a single channel (records shown in Fig. 1). Oocyte was
maintained at 100 mV relative to pipette throughout the changes
in internal pH, which were accomplished with a permeant buffer.
A: closed-time distribution for
initial recording at a bath pH of 7.4 (pHi = 6.8),
showing 2 discrete closed states with time constants of 1.6 ms and 85 ms and open probability
(Po) = 0.8. B: 6 min after changing external,
acetate-buffered solution to pH 6.8 (pHi = 6.4). Note
appearance of very long closed state;
Po = 0.2. C: recovery of channel activity 3 min
after return of bath to pH 7.4 (pHi = 6.8),
showing 2 closed states with time constants of 1.5 ms and 81 ms. Note
disappearance of very long closed state.
D: single-channel conductance (39 pS
in this experiment) was unaffected by changes in pH.
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In five experiments at a bath pH of 7.4 and a
Vm
of
100 mV, the average open probability for
WT-ROMK2 was Po = 0.76 ± 0.02. Reduction of bath pH to 6.8 decreased
Po in all five
cases. Analysis of cell-attached current records 30 s preceding
shutdown of the channel yielded the kinetic data shown in Table 2,
middle. This decrease in open
probability to Po = 0.38 ± 0.11 (n = 5) could be
completely explained by the appearance of a very long closed state
(Fig. 2B). In five recordings, return to a bath pH of 7.4 (steady-state pHi = 6.8) caused a
disappearance of the this long closed state and restored the open
probability to Po = 0.72 ± 0.06.
As indicated by the example of Figs. 1 and 2, the period of long
closures persisted for ~20-30 s until a complete closure occurred, producing an apparent cessation of channel activity. It was
possible to restore channel activity if bath pH were returned to 7.4 within about 45 s of channel shutdown. Allowing channels to remain for
more than 45 s in the closed state at low
pHi prevented reactivation upon return to bath pH 7.4. In addition, the long closures
prior to complete shutdown were often preceded by the random appearance
of discrete substates (circled events in Fig. 1). These also appeared
at the beginning of channel recovery following return to a bath pH of
7.4. These substates were too few in number to study, and their
significance is not understood.
The pH-dependent shutdown of the channel did not alter the lifetime of
either the open state or the two shorter closed states. This is
illustrated in Fig. 2, and the kinetic data are summarized in Table 2.
Single-channel conductance also remained the same during the
pH-dependent shutdown of the channel (Fig.
2D). The decrease in
Po at low pH can be attributed to the appearance
of a long closed state.
Channels in excised (inside-out) patches had a similar, but somewhat
faster, response to alterations in cytoplasmic-side pH (Fig.
3) than was observed with cell-attached
patches. ROMK2 channel activity in the excised patch of Fig. 3 was
completely blocked by a bath pH of 6.4, although this required 47 s
after the start of the bath solution change. Since bath exchange was
complete within 10 s, pH shutdown of the channel is not immediate, even in excised patches. Furthermore, channel activity in the excised patch
did not recover until more than a minute after return of the bath pH to
7.4 (Fig. 3).

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Fig. 3.
Effect of changing cytosolic side pH from 7.4 to 6.4 on WT-ROMK2
currents recorded from a single channel in an excised (inside-out)
patch in a high-K bath (55 mM KCl + 55 mM potassium acetate).
Cytoplasmic side of the membrane was maintained at 100 mV
relative to pipette throughout the changes in cytoplasmic-side pH.
Downward deflections from the closed state (dotted line) denote inward
currents.
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Similar to the results with cell-attached patches, a complete absence
of channel activity for more than 45 s at low pH prevented recovery,
even after a return of bath pH to 7.4. In no case did a reduction in pH
cause significant changes in current amplitude, although the rundown
that often occurred with excised patches sometimes reduced current
amplitude before complete disappearance of channel activity. The reason
for this is not known, but, to avoid confusion, average kinetic
parameters were computed only for cell-attached patches.
pHi dependence of
inward rectifier macroscopic currents.
Macroscopic whole cell currents from WT-ROMK display a marked
sensitivity to pHi (14), analogous
to the pH sensitivity of the native small K channel in native rat CCT
(15). Our studies confirmed this steep dependence of inward conductance
on pHi (solid line of Fig.
4). For WT-ROMK2 the average
pKa was 6.5 ± 0.01 (8 oocytes), and the Hill coefficient was 5.2 ± 0.6, indicating that permeation through this channel is turned on and off
over a relatively narrow range of pH.

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Fig. 4.
pH dependence of WT-ROMK2, IRK1, and K61M mutants.
Ordinate: normalized inward
conductance (at 80 mV) of oocytes expressing either WT-ROMK2
( , solid line), IRK1 (, dotted line) or K61M ( , dashed line).
Abscissa:
pHi controlled with extracellular
potassium acetate solutions (see text). Conductances at different
values of internal pH were normalized to the maximum inward conductance
for that oocyte. Data were fit to a modified Hill equation (see
text).
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In the experiments used to construct Fig. 4, inward conductances were
measured at different pH values and normalized to the maximum inward
conductance for each oocyte. Intracellular pH was controlled with
extracellular potassium acetate solutions, where stepwise decreases in
extracellular pH from 8.2 to 6.3 reduced pHi from 7.2 to 6.1 (see
MATERIALS AND METHODS Table 1).
The steep dependence of ROMK2 currents on
pHi was not shared by other
members of the Kir family (IRK1),
which were essentially insensitive to
pHi between 7.2 and 6.2. As
indicated in Fig. 4, the strong inward rectifier (IRK1) exhibited
nearly constant inward conductance until
pHi dropped below 6.1. Conductance
block at this pHi may involve a
completely different process whereby hydrogen ions directly interfere
with the permeation path (4).
Although internal oocyte pH was not measured during the TEVC
experiments of Fig. 4, the time course for the change in oocyte pHi was determined in a separate
batch of oocytes from several frogs (see MATERIALS AND
METHODS). The results are illustrated in Fig. 5,
which compares the time course of oocyte
pHi and inward current during a
decrease in bath pH from 7.4 to 6.8 (corresponding to a change in
steady-state pHi from 6.8 to 6.4).
The squares (and solid line) of Fig. 5
depict inward current, measured at an oocyte potential of
60
mV and normalized to its maximum value at time
0. The triangles (dashed line) in Fig. 5 represent the oocyte pH measured with pH-selective microelectrodes. As shown in Fig.
5, the average time course for the reduction in
pHi was slightly longer (7.6 ± 0.6 min
1) than the time
course for reduction of inward current (5.6 ± 0.2 min
1). In any case,
macroscopic oocyte currents were routinely measured 25-30 min
after a change in bath solution. At these times the currents, and
presumably pHi, should both be in
a steady state.

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Fig. 5.
Comparison of macroscopic current and intracellular pH
(pHi) time course for WT-ROMK2.
For both curves the bath pH was changed from 7.4 to 6.8 using potassium
acetate solutions. This produced a decline in steady-state
pHi from 6.8 to 6.4 in 20 min.
Solid line ( ) are the measured normalized macroscopic inward
currents at 60 mV during the change in bath pH. This was fit to
a single exponential with a time constant of 5.6 ± 0.2 min 1. Dashed line ( ),
time course for pHi during this
same period. This was fit to a single exponential with a time constant
of 7.6 ± 0.6 min 1.
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Recently, it was discovered that a single lysine (K61 on ROMK2, K80 on
ROMK1), NH2 terminal to the first
hydrophobic segment (M1), is essential for conferring steep pH
sensitivity to ROMK (4). This result was confirmed in the present study
in which replacing the lysine at position
61 with methionine (K61M) abolished the pH sensitivity
of ROMK2 (K61M mutant in Fig. 4).
pHi dependence of
T51 mutant macroscopic currents.
Both IRK and ROMK channels possess a highly conserved, mildly
hydrophobic area (designated the "Q" region) that is located immediately NH2 terminal to the
first transmembrane segment (M1) of both inward rectifiers (Fig.
6). The "Q regions" of the inward rectifier superfamily show a high degree of homology. The boldface residues of Fig. 7 indicate amino acids that are identical to those of
ROMK2. A cursory inspection of Fig. 7
reveals that many of the nonidentical residues in the same column have
similar properties. Finally, the hydrophobicity and proximity of the Q
region to the M1 transmembrane segment raises the possibility that this
region might be involved in the permeation process.

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Fig. 6.
Hydropathy analysis of the predicted amino acid sequence of ROMK2,
based on the Kyte-Doolittle algorithm.
Abscissa: amino acid sequence number.
Ordinate: positive numbers denote
increasing hydrophobicity. M1 and M2, putative membrane spanning
region; P, pore region; and Q, area of mild hydrophobicity, explored in
the present treatment.
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Fig. 7.
Homologies within the "Q" regions of ROMK2 and the inward
rectifier family. Boldface letters indicate residues identical to those
of ROMK2. Boxed residues depict the locus of the T51E and T52E
mutations. Numbers 44, 51, and 59 refer to amino acid locations along
the ROMK2 sequence.
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Two completely conserved residues of the Q region are the pair of
threonines at positions 51 and
52 (indicated by the rectangle in Fig.
7). Hence, we chose to examine the effect of mutations at this site on
the conduction properties of ROMK2. As shown below, this did indeed
produce alterations in the K conductance of the channel. Unexpectedly,
the normally permeant cation NH4 blocked the mutant
channels. Since extracellular NH4 is known to reduce pHi in the oocyte,
we tested whether this phenomenon could be explained by an enhanced pH
sensitivity of the channel.
As illustrated in Fig. 8 (and Table 3),
substitution of glutamate for threonine at position
51 shifted the apparent
pKa for inward
conductance from 6.5 ± 0.01 for WT-ROMK2 to 7.0 ± 0.02 for
T51E. The analogous substitution at position T52 had no significant effect on the apparent
pKa (T52E, dotted
line, Fig. 8). In contrast, the introduction of a positively charged
lysine at location T51 shifted the apparent
pKa in the
opposite direction to 6.0 ± 0.02 (T51K, Fig. 8 and Table
3).

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Fig. 8.
pH dependence of WT-ROMK2 and T51 mutants.
Ordinate: normalized inward
conductance (at 80 mV) of oocytes expressing either WT-ROMK (WT,
solid line, ) or mutants T51E (dashed line, ) or T52E (dotted
line, ), or T52K (broken line, ). Conductances at different
values of internal pH were normalized to the maximum inward conductance
for that oocyte. Data were fit to a modified Hill equation (see text).
Abscissa:
pHi controlled with extracellular
potassium acetate solutions. Relationship between internal and external
pH is given in Table 1.
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The curves in Figs. 4 and 8 were constructed by fitting the data to the
Hill equation for normalized inward conductance:
G/Gmax = 1/[1 + ([H]/K)n],
where [H] is the internal hydrogen ion concentration,
n is the Hill coefficient, and
pKa =
log10K.
Results of the parameter fit are summarized in Table 3. The Hill
coefficient for WT-ROMK was n = 5.2 ± 0.6, indicating that the process underlying the pH regulation is
highly cooperative. The mutants T51E and T51K had significantly lower
Hill coefficients (Table 3). No acceptable fit to the Hill equation
could be obtained for K61M or IRK1.
We also examined whether the greater sensitivity to protons of T51E
could still be observed in the absence of the critical lysine (K61).
Evaluation of the double mutant (K61M/T51E) indicated that it had no pH
sensitivity in the range between 6 and 8, similar to the result
obtained with K61M alone and IRK1 (Fig. 4). This suggests that K61 is
essential for the pH sensitivity of ROMK2, whereas the (charge of the)
residue at position 51 modulates this pH sensitivity and helps to determine the
pKa of the
channel.
Single-channel properties of T51E. The
"P" region (Fig. 6) is a hydrophobic stretch of amino acids that
are thought to form at least part of the pore in both voltage-gated and
inward rectifier K channels. The similarity of the Q and P regions with
regard to hydrophobicity and proximity to the transmembrane domain
raises the possibility that the Q region also contributes to the
formation of the pore. This was assessed by comparing single-channel
records from T51E mutants and WT-ROMK2 channels. Figure
9A
illustrates a typical cell-attached recording of inward K currents
through T51E. Both bath and pipette contained 110 mM KCl, and the
average resting potential of the oocytes was about zero
mV, implying an oocyte K concentration close to that of
the bath. To maximize the signal-to-noise ratio for T51E, the voltage
clamp was set so that the oocyte interior was effectively
200 mV
relative to the pipette. Downward deflections from the closed state
(dotted line, Fig. 9A) correspond to
K current from pipette to oocyte. The single-channel conductance in the
inward direction for this experiment was 16 pS, as determined from
currents at voltages between 0 and
200 mV.

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Fig. 9.
K currents through a single T51E mutant ROMK2 channel, expressed in an
oocyte, depolarized by 110 mM KCl (pH 7.4).
A: cell-attached patch with oocyte
potential of 200 mV relative to pipette. Downward deflections
from the closed state (dotted line) denote inward currents. Inward
single-channel conductance was 16 pS.
B: closed time distribution for T51E,
showing 2 discrete closed states with time constants of 1.0 and 25 ms.
C: open time distribution for T51E
currents, showing 1 open state with time constant of 5.2 ms.
Po ( 200
mV) = 0.74.
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The T51E records of Fig. 9 can be contrasted with those from WT-ROMK2,
which exhibited low open-channel noise and an inward conductance of 33 pS (Fig. 10). Although both Figs. 9 and
10 depict current records with the oocyte clamped at
200 mV relative to pipette, qualitatively similar results were
obtained at more physiological Vm values of
80 mV (oocyte relative to pipette). At all
voltages and over a pHi range from
6.1 to 7.2, T51E channels consistently exhibited smaller current
amplitudes and greater open-channel noise than wild-type currents.

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Fig. 10.
K currents through a single WT-ROMK2 channel, expressed in an oocyte,
depolarized by 110 mM KCl (pH 7.4). A:
cell-attached patch with oocyte potential of 200 mV relative to
pipette. Downward deflections from the closed state (dotted line)
denote inward currents. Inward single-channel conductance was 33 pS.
B: closed time distribution for
WT-ROMK, showing 2 discrete closed states with time constants of 0.9 and 17 ms. C: open time distribution
for WT-ROMK, showing 1 open state with time constant of 4.8 ms.
Po ( 200
mV) = 0.72.
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Since the current records in Figs. 9 and 10 were obtained from
single-channel patches, the kinetics of T51E and WT-ROMK2 could be
readily compared. Figure 9, B and
C, vs. Fig. 10,
B and
C, indicates that T51E (at
Vm =
200
mV) has a somewhat greater long closed time than wild type (25 vs. 17 ms), but this is offset by a longer open time (5.2 vs. 4.8 ms). The
combined effect leaves the open probability similar for mutant
(Po = 0.72) and
wild type (Po = 0.74) at Vm
values of
200 mV. In contrast (discussed below), the Po of T51E
decreases dramatically with depolarization, so that at normal cell
potentials of
70 mV, T51E has a much lower Po than WT-ROMK2.
Single-channel
I-V
relations (Fig. 11) were constructed from
cell-attached current records at different holding potentials. In all
cases, the patch pipette contained 1 mM
MgCl2 + 110 mM KCl, and the bath
was maintained at 110 mM KCl, which depolarized the oocyte resting
potential to zero. Both wild type and T51E mutants showed mild inward
rectification at the single channel level, characteristic of ROMK
channels. Consistent with the current records of Figs. 9 and 10, the
average inward single-channel conductance of T51E was 17 ± 1 pS in
eight oocytes, about one-half the single-channel conductance of
WT-ROMK2 (34.8 ± 0.5 pS in 5 oocytes).

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Fig. 11.
Comparison of single-channel rectification between T51E and WT-ROMK2.
Cell-attached patches on oocytes expressing either T51E mutant (dashed
line, ) or WT-ROMK (solid line, ). Pipette and bath solutions
consisted of 110 mM KCl buffered to pH 7.4. Pipette also contained 1 mM
MgCl2. Mean inward single-channel
conductance for T51E was 17 ± 1 pS
(n = 9). Mean inward single-channel
conductance for wild type was 34 ± 1 pS
(n = 8).
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If the wild type and mutant current amplitudes are normalized by their
respective inward conductances, then the single-channel I-V
relations are practically superimposable (Fig.
12). When viewed in this way, there is no
difference in single-channel rectification between wild type and the
T51E mutant. This is in contrast to the normalized macroscopic currents
in which T51E displayed 40% more rectification than WT-ROMK (Fig. 13).
In Fig. 13, all currents were normalized
to the maximum inward current at
80 mV. Similar results were
obtained at bath pH values of 7.4 and 7.8. When 10 mM K rather than 110 mM K was used in the bath, reversal potentials of the macroscopic
I-V
relations indicated no difference in ion selectivity between WT-ROMK
and T51E.

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Fig. 12.
Normalized single-channel current-voltage
(I-V)
relations for T51E and wild type. Cell-attached patches on oocytes
expressing either T51E mutant (dashed line, ) or WT-ROMK (solid
line, ). Pipette and bath solutions consisted of 110 mM KCl buffered
to pH 7.4. Pipette also contained 1 mM
MgCl2. Data are from Fig. 11,
where T51E currents have been scaled by the WT/T51E ratio of inward
conductances at 80 mV.
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Fig. 13.
T51E enhances macroscopic inward rectification. Comparison of the
2-electrode
I-V
relations for the mutant T51E and WT-ROMK2. All currents were
normalized to maximum inward current at 80 mV. Bathing solution
contained 110 mM KCl (pH = 7.4), and the average oocyte resting
potential was zero mV.
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The apparent effect of the T51E mutation on macroscopic (but not
single-channel) rectification can be attributed to its effect on the
voltage-dependent gating of the channel. At normal resting potentials
(Vm =
70
mV) and a bath pH between 7.4 and 7.8, WT-ROMK2 exhibits a high open
probability (Po = 0.8) that increases slightly with membrane depolarization (solid line,
Fig. 14). Although the Po of T51E is
similar to wild type at large negative potentials (see Figs. 9 and 10),
it declines with depolarization, and at normal cell potentials it is
only ~0.25 compared with 0.8 for wild type (dashed line, Fig. 14).
This voltage dependence of the
Po (Fig. 14) can
explain the apparent increased inward rectification of the T51E
macroscopic currents (Fig. 13), since macroscopic conductance (G) is the product of single-channel
conductance (g), number of channels
(N), and
Po.

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Fig. 14.
Voltage dependence of open probability for T51E and wild type.
Cell-attached patches on oocytes expressing either T51E mutant (dashed
line, ) or WT-ROMK2 (solid line, ). Pipette and bath solutions
consisted of 110 mM KCl buffered to pH 7.4. Pipette also contained 1 mM
MgCl2.
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Single-channel properties of other substitutions at
T51. The striking effect on voltage gating produced by
replacing the threonine at position 51 with a negatively charged glutamate raised the possibility that other
amino acid substitutions at this location might also affect channel
gating and/or conductance. In this regard, Table
4 compares currents for wild type, T51E,
T51H, and T51K, all obtained from one-channel, cell-attached patches on
oocytes clamped at
100 mV relative to the pipette.
At
100 mV, T51E currents had lower amplitude and lower open
probability than wild type, analogous to results obtained at
200
mV (Fig. 9). On the other hand, replacing the threonine at position 51 with either a histidine
(T51H) or a positively charged lysine (T51K) produced K currents that
exhibited many of the features of WT-ROMK, such as clear channel
openings and low open-channel noise. However, T51K and T51H currents
also showed some important differences. First of all, T51H had a
significantly higher single-channel conductance than WT-ROMK2,
although its Po
was iden- tical to that of WT-ROMK (Table 4). Second, T51K had
a significantly lower Po than either
wild type or T51H, although its single-channel conductance was
indistinguishable from wild type. Finally, depolarization from
100 mV to
50 mV reduced the
Po of T51K from
0.32 to 0.22. This type of voltage gating is opposite to
that of WT-ROMK but similar to the voltage gating of T51E.
 |
DISCUSSION |
Modulation of macroscopic pH
sensitivity. Patch-clamp studies on cut-open rat CCT
initially established the regulation of secretory SK potassium channels
by pHi (15). This dependence on
pHi was also observed with ROMK
channels expressed in oocytes and studied either at the single channel
(10, 11) or the whole cell level (14). In the latter experiments,
simultaneous measurement of macroscopic current and
pHi indicated a steep dependence
of ROMK1 conductance on internal pH, involving a highly cooperative process with four H+ binding sites
and a pKa of 6.8 (14). This is in contrast to the pH insensitivity of the strong inward
rectifiers like IRK1 (4).
Recently, it was discovered that a single lysine (K61 on ROMK2, K80 on
ROMK1), NH2 terminal to the first
hydrophobic segment (M1), is necessary for conferring
pHi sensitivity to ROMK (4). This
result was confirmed in the present study, where replacing the lysine
at position 61 with methionine (K61M)
abolished the pHi sensitivity of
ROMK2 between 7.2 and 6.2. In addition to the critical residue K61, a
second site (position 51 on
ROMK2; 70 on ROMK1) was also found to affect pH sensitivity of the
channel. This might indicate a physical proximity between
positions 51 and
61 (on ROMK2) since changing the
charge of residue 51 shifted the
apparent pKa of
the channel. Replacing the uncharged threonine by a negatively charged
glutamate shifted the
pKa in an
alkaline direction by 0.5 pH units, from 6.5 ± 0.01 (WT) to 7.0 ± 0.02 (T51E). Replacing threonine by a positively charged lysine
shifted the pKa
in an acidic direction by 0.5 pH units, from 6.5 ± 0.01 (WT) to 6.0 ± 0.02 (T51K). However, the modulatory effects of position 51 were not observed after
removal of the critical lysine (K61); i.e., the double mutant T51E/K61M
had the same insensitivity to pH as the single-point mutant (K61M,
ROMK2). This suggests that the T51 locus modulates the pH sensitivity
of the K61 site rather than functioning as an additional pH sensor.
Since the pKa of
lysine in free solution is 10.5, it was suggested that residue K61
confers pH sensitivity to the channel in the physiological range
because the local chemical environment within the protein shifts the
pKa of lysine
from 10.5 to ~6.5, (4). How residue T51 modulates this pH sensitivity
is not known. One hypothesis consistent with our results is that the
mildly hydrophobic region at T51 is sufficiently close to the
hydrophobic region of the first transmembrane segment (M1) to
physically interact with K61, which is just
NH2 terminal to M1. The
interaction could be electrostatic in nature, since positive and
negative charges had opposite effects.
We believe that intracellular rather than extracellular pH modulates
both the K61 and the T51 sites on ROMK2. We observed no obvious effect
on ROMK macroscopic inward currents when bath pH was changed with
membrane-impermeable KCl-HEPES solutions. A lack of effect of
extracellular pH was also reported for ROMK1, where changes in bath pH
via membrane-impermeable biphthalate had no effect on macroscopic
inward currents (14).
Effect of pH on single-channel
currents. The mechanism whereby decreases in
cytoplasmic-side pH shut down channel activity is not well understood.
In cell-attached patches, no ostensible change in channel kinetics
occurs until ~10-15 min after reducing bath pH to 6.8. Presumably, this is the time required for the interior of the oocyte to
reach a critical internal pH of ~6.4. At this
pHi a very long closed state
appears (Fig. 2B), which eventually
becomes a complete closure. For reasons we do not understand, complete
channel closure at low pHi for
more than 45 s seems to interfere with reactivation upon return to a pH
7.4 bath solution. Although channels may continue to remain closed for
a variable time after return to normal bath pH, the time spent at low
pH seems to be a critical factor for restoration of channel activity.
Recordings from excised patches support the hypothesis that lowering
cytoplasmic-side pH initiates very long closures, followed by complete
cessation of channel activity. In excised patches, the decline in
channel activity is slower than the decline in bath pH. It is unclear
whether this represents a delay in diffusion of new solution to the
patch of membrane within the pipette or simply a delayed effect of pH
on channel gating.
Upon return of the bath to pH 7.4, ROMK2 channel activity recovered
with a latency that varied between about a minute for excised patches
to more than 15 min for cell-attached recordings. The delay for the
cell-attached experiments presumably represents the time required to
elevate oocyte pH back to normal with the membrane-permeable acetate
solutions. However, a delay in recovery of channel activity was also
observed in excised patches, suggesting that the reversal of the
inhibited state is slow compared with the actual change in pH.
Exposure of either excised or cell-attached patches for longer than 45 s prevented restoration of channel activity following a return to pH
7.4. The relatively narrow time window (45 s) beyond which it became
impossible to reactivate individual single channels differs from the
reversibility of WT-ROMK macroscopic conductance measured with the
TEVC. In these latter experiments, inward conductance essentially
returned to control values even after a 10-min exposure to a pH 6.8 bath. The reason for this difference in microscopic vs. macroscopic
reversibility is not known.
Single-channel properties of the T51
mutants. The position of residue T51 (ROMK2) relative
to the first transmembrane segment raised the possibility that this
site might also interact with the K permeation path of the channel. In
fact, the T51E mutation reduces apparent single-channel conductance by
50% and dramatically increases open-channel noise. These changes are
consistent with an interaction between residue
51 and the permeation path of the channel.
The persistence of T51E-specific open-channel noise at all voltages and
over a range of pHi from 6.1 to
7.4 seems to rule out cytoplasmic proton block as a cause of the
increased noise level. However, it is still possible that the T51E
mutation introduces a fast-flicker open state that is
beyond the resolution of the data acquisition system. In this case,
T51E could appear to have a much reduced conductance when records are
filtered at 1 kHz.
Although the reason for the decrease in apparent conductance produced
by T51E is not known, it is unlikely that it arises from a simple
electrostatic effect, since replacing threonine with a positively
charged basic lysine residue (T51K) did not produce an opposite effect
on conductance. In fact, the single-channel conductance of T51K was
indistinguishable from that of WT-ROMK2 (Table 4). Furthermore, when
the T51 residue was replaced by histidine (which should be less
protonated than lysine at pH = 7.4), single-channel conductance
actually increased (Table 4).
We have no hypothesis for the dramatic effect of the glutamate
substitution (T51E) on single-channel voltage gating (Fig. 14). Other
amino acid substitutions at this position, T51H for example, leave
Po virtually
unchanged from wild type (Table 4). A simple electrostatic mechanism
seems an even less likely explanation for changes in voltage gating,
since the T51K mutant (which introduces a positive charge at
position 51) also decreases the
Po of the channel, in a manner similar to that seen with (the negatively charged)
T51E.
Comparison with previous studies. The
present study confirms previous findings on the marked dependence of
ROMK current on pHi
(4, 10, 11, 14, 15). We have also confirmed that the lysine residue
(K61 on ROMK2, K80 on ROMK1), NH2
terminal to the first putative transmembrane segment (M1), is primarily responsible for conferring a steep pH sensitivity to ROMK that is not
seen with IRK1 (4).
The principal difference between our pH dependence of WT-ROMK and that
reported by both Tsai et al. (14) and Fakler et al. (4) is the value of
apparent pKa for
ROMK. In our studies the apparent
pKa for ROMK2 was
6.5 ± 0.01. This is significantly lower than either the
pKa of 6.8 reported for ROMK1 by Tsai et al. (14) or the
pKa of 6.9 reported for ROMK2 by Fakler et al. (4). The precise reason for this
disagreement is not known but probably arises from the larger
discrepancy between intracellular and extracellular pH measured in our
study, compared with previous reports (14).
In our experiments (Table 1), decreasing bath pH from 7.4 to 6.8 reduced oocyte pH from 6.8 to 6.4 compared with the decrease in
pHi of 7.2 to 6.7 reported by Tsai
et al. (14). Furthermore, we were never able to alkalinize oocytes
above their resting pH (about 7.2) using acetate-buffered solutions, so
that it was only possible (with this technique) to study oocytes with
an internal pH less than or equal to 7.2.
The disparity in
pKa values is of
interest, since it determines whether ROMK2 would be regulated by
normal variations in
pHi. The results of
our study suggest that ROMK2 is probably not significantly regulated by
pH under physiological conditions, since it is unlikely that renal
cells would normally possess a
pHi below 6.7. Hence, only those cells with impaired acid extrusion processes would have a sufficiently low pHi to
shut down the secretory ROMK channel. This might serve as a protective
mechanism to conserve intracellular K during energetically restricted
conditions in which
pHi regulation was
compromised.
Summary. The present study confirms
that a lysine residue (K61 on ROMK2; K80 on ROMK1) is the primary locus
of the pH sensitivity present in ROMK but not in IRK1. However, a
specific residue in a mildly hydrophobic region of ROMK (T51 on ROMK2;
T70 on ROMK1) appears to modulate this pH sensitivity. Replacing the
neutral threonine at this site with a negatively charged residue shifts the apparent pKa
in an alkaline direction, whereas substitution with a positively
charged residue shifts the
pKa in an acid
direction. Substitutions at position
51 also significantly affected single-channel conductance and voltage-dependent gating of the channel. In contrast, mutations in a neighboring residue T52 (T71 on ROMK1) had no effect on
pKa or
single-channel conductance. This is consistent with a model in which
T51 is sufficiently close to the primary pH site (K61) to alter ROMK pH
sensitivity while also exerting effects on the permeation pathway in
the pore region.
We gratefully acknowledge the expert assistance of Dr. Gordon
Cooper, Dr. Michael Romero, and Dr. Walter Boron with the intracellular measurements of oocyte pH, which were all performed in the Dept. of
Cellular and Molecular Physiology at Yale University School of
Medicine.
This work was supported by a grant-in-aid from the American Heart
Association/New York City Affiliate (to L. G. Palmer) and by National
Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46950
(to H. Sackin).
Address for reprint requests: H. Sackin, Yale University School of
Medicine, 2073 LMP, 333 Cedar St., New Haven, CT 06510.