1Department of Life Science, Kwangju Institute of Science and Technology (K-JIST), Kwangju 500-712; and 2Department of Oral Physiology and Dental Science Research Institute, Chonnam National University Dental School, Kwangju 501-190, Korea
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ABSTRACT |
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Lee, Han Mi,
Young Sun Park,
Wonjae Kim, and
Chul-Seung Park.
Electrophysiological Characteristics of Rat Gustatory Cyclic
Nucleotide-Gated Channel Expressed in Xenopus Oocytes.
J. Neurophysiol. 85: 2335-2349, 2001.
The complementary DNA encoding gustatory cyclic
nucleotide-gated ion channel (or gustCNG channel) cloned from rat
tongue epithelial tissue was expressed in Xenopus oocytes,
and its electrophysiological characteristics were investigated using
tight-seal patch-clamp recordings of single and macroscopic channel
currents. Both cGMP and cAMP directly activated gustCNG channels but
with markedly different affinities. No desensitization or inactivation
of gustCNG channel currents was observed even in the prolonged
application of the cyclic nucleotides. Single-channel conductance of
gustCNG channel was estimated as 28 pS in 130 mM of symmetric
Na+. Single-channel current recordings revealed
fast open-close transitions and longer lasting closure states. The
distribution of both open and closed events could be well fitted with
two exponential components and intracellular cGMP increased the open
probability (Po) of gustCNG channels
mainly by increasing the slower opening rate. Under bi-ionic
conditions, the selectivity order of gustCNG channel among divalent
cations was determined as Na+ ~ K+ > Rb+ > Li+ > Cs+ with the
permeability ratio of 1:0.95:0.74:0.63:0.49. Magnesium ion blocked
Na+ currents through gustCNG channels from both
intracellular and extracellular sides in voltage-dependent manners. The
inhibition constants (Kis) of
intracellular Mg2+ were determined as 360 ± 40 µM at 70 mV and 8.2 ± 1.5 mM at 70 mV with
z
value of 1.04, while
Kis of extracellular
Mg2+ were as 1.1 ± 0.3 mM at 70 mV and
20.0 ± 0.1 µM at
70 mV with z
of 0.94. Although
100 µM l-cis-diltiazem blocked significant portions of outward Na+ currents through both
bovine rod and rat olfactory CNG channels, the gustCNG channel currents
were minimally affected by the same concentration of the drug.
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INTRODUCTION |
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Cyclic
nucleotide-gated channels (or CNG channels) are cation-selective ion
channels that open in response to the direct binding of intracellular
cyclic nucleotides such as cGMP or cAMP. These channels play critical
roles in sensory transduction of vertebrate photoreceptors and
olfactory neurons (for review, Finn et al. 1996;
Li et al. 1997
). Serving as downstream targets of the
signaling pathways, CNG channels mediate the transduction of sensory
stimuli into neuronal activity. CNG channel subtypes differ in their
sensitivity for cyclic nucleotides in that olfactory channel can be
activated by physiological concentrations of both cAMP and cGMP
(Nakamura and Gold 1987
; Zufall et al.
1994
), whereas the photoreceptor channels are activated only by
cGMP (Fesenko et al. 1985
; Yau and Baylor
1989
). The channel subtypes also differ in their relative permeability to physiological Ca2+ such that the
fractional currents carried by Ca2+ in the
olfactory channel are greater than that by the rod channel (Frings et al. 1995
).
CNG channel activities were also detected in neurons other than sensory
receptor cells (Ahmad et al. 1994; Dryer and
Henderson 1993
; Kingston et al. 1996
;
Nawy and Jahr 1990
). Subsequently, several groups
reported the cloning of CNG channel genes from different tissues in
various organisms, strongly suggesting that CNG channels may be
involved in other important physiological processes in different
tissues (for review, Biel et al. 1999
; Finn et
al. 1996
). Thus far, six different genes encoding CNG channels
have been identified in mammals, and these subunits can be classified
as
and
subunits. While the
subunits (CNG 1-3) can form
functional CNG channels expressed in different heterologous expression
systems, the
subunits (CNG 4-6) require the co-expression of
subunits for functional CNG channels.
A complementary DNA (cDNA) of another CNG channel was cloned from rat
tongue epithelial tissues where taste reception takes place
(Misaka et al. 1997). Composed of 611 amino acid
residues and believed as one of the major 5'-splicing variants, the
deduced amino acid sequence of the CNG channel (named "gustCNG
channel") shows 50 to 80% similarities to other CNG channels. While
the rat gustCNG channel is most homologous to mouse cone CNG channel in
its overall amino acid sequence, the predicted amino termini of these
two channels show only 58% sequence identity (Gerstner et al.
2000
). Expressed in HEK293 cells, gustCNG channel gene resulted
in functional CNG channel currents activated by both cAMP and cGMP
(Misaka et al. 1997
). Based on its specific expression in taste bud cells, it was proposed that this CNG channel might be
involved in some types of gustatory signal transduction. Following this
report, it was suggested that taste signal transduction by bitter
tastants such as caffeine and theophylline may be mediated by cGMP
through inhibition of phosphodiesterase (Rosenzweig et al.
1999
). This may be different from a G-protein (or
gustducin)-mediated pathway that inhibits ion channel activity by
decreasing intracellular cAMP and cGMP concentration (Kolesnikov
and Margolskee 1995
). In a recent report, Misaka et al.
reported that the gustCNG channel is also expressed in the outer
segments of rat cone photoreceptor cells and suggested that this CNG
channel is involved in both visual and taste signal transduction
(Misaka et al. 1999
).
Although the gustCNG channel gene is likely expressed in several different tissues, the functional characteristics of this channel remain to be elucidated in detail. To reveal the functional differences of those closely related CNG channels, it is required to express the channel genes in a heterologous system and to compare their electrophysiological characteristics. In this study, we expressed the cloned gustCNG channel in Xenopus oocytes and investigated the functional properties of the channel using electrophysiological methods. We determined the single-channel conductance and the selectivity order of the gustCNG channel. The gating properties of gustCNG channel were examined in both single-channel and macroscopic current levels. From a detailed analysis of single-channel recordings, we were able to elucidate that cGMP activates the channel by increasing an opening rate of the channel. We also examined the blockade of gustCNG channel using a divalent cation, Mg2+, and l-cis-diltiazem and compared the effects to two other members of CNG channel family.
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METHODS |
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Materials
Female Xenopus laevis were purchased from Xenopus One (Ann Arbor, MI). Restriction enzymes were purchased from New England Biolabs (Beverly, MA) and Boehringer Mannheim (Mannheim, Germany). T7 RNA Polymerase was purchased from Promega (Madison, WI). Collagenase was from Worthington Biochemical (Freehold, NJ). The cyclic GMP, cyclic AMP, and other chemicals were purchased from either Sigma (St. Louis, MO) or Aldrich Chemical (Milwaukee, WI), unless otherwise specified. The l-cis-diltiazem hydrochloride was purchased from Sigma-RBI (St. Louis, MO). Other molecular biological reagents were purchased from GIBCO BRL (Rockville, MD), DIFCO Laboratories (Grayson, GA), or Bio-Rad laboratories (Hercules, CA).
Subcloning of gustCNG channel gene and in vitro transcription
The cDNA of gustCNG channel in pUC18 vector was provided by Dr.
Keiko Abe of the University of Tokyo, who originally cloned the gene
from rat tongue epithelial tissue (Misaka et al. 1997). The entire coding region of gustCNG channel gene was subcloned into a
modified pGH expression vector, pGH/NBC, for high-level expression in
Xenopus oocytes. Several unique sites of restriction enzymes
were introduced into the multiple cloning site region of pGH/NBC, which
also contains 5'- and 3'-untranslated regions of the Xenopus
-globin gene of the original pGH vector (Liman et al.
1992
). The cDNAs encoding bovine rod and rat olfactory CNG
channels used in this study were described previously (Goulding et al. 1992
; Liman and Buck 1994
).
CNG channels of rat gustatory, bovine retinal, and rat olfactory were expressed in Xenopus oocytes for electrophysiological studies. Complementary RNA (cRNA) was synthesized in vitro from an MluI or Nhe I-linearized plasmid using T7 RNA polymerase. For transcription of cRNA, 10 µg of recombinant DNA was digested with MluI or Nhe I restriction enzymes for 4 h at 37°C. Linearized DNA was extracted once with phenol:sevag (phenol:chloroform:isoamylalcohol = 25:24:1) and subsequently with sevag (chloroform:isoamylalcohol = 24:1). The linearized DNA was added to total 100 µl of reaction mixture containing 1 times transcription buffer, 10 mM dithiothreitol, 100 units RNasin, 1 mM rNTPs, 1.25 units GpppG, and 40 units T7 RNA polymerase (all reagents were from Promega, except GpppG purchased from Pharmacia Biotech). The mixture was incubated at 37°C for 2 h. After transcription, synthesized cRNA was extracted once with phenol:sevag, and twice with sevag. Purified cRNA was dissolved in ~20-40 µL of RNase-free water [autoclaved nanopure-filtered water containing 0.01% (vol/vol) diethyl pyrocarbonate]. Quality and quantity of cRNA were examined by 1% TAE-agarose gel electrophoresis.
Expression in gustCNG channels in Xenopus oocytes
Oocytes harvested from Xenopus laevis were incubated in a solution containing (in mM) 82.5 NaCl, 2.5 KCl, 1.0 MgCl2, and 5.0 HEPES, pH 7.6, and 2-4 mg/ml of collagenase. The oocyte preparation was agitated using a rotating shaker for 90-120 min. It was then rinsed thoroughly and stored in ND-96 solution containing 96 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, and 50 µg/ml gentamicin, adjusted to pH 7.6 with NaOH. Defolliculated oocytes were selected no later than 12 h after collagenase treatment. About 50 ng of cRNA were injected into oocytes for macroscopic, and about 2.5 ng were for single-channel experiments using a microdispenser (VWR Scientific, West Chester, PA). Injected oocytes were incubated at 18°C for 3-5 days in ND-96 solution. For single-channel current recordings the expression of channel protein was allowed only for 18-24 h, and cRNA injection was done every 2 days.
Electrophysiological recordings and data analysis
Ionic currents through the functionally expressed gustCNG
channels were measured with gigaohm-sealed membrane patch-clamp method
in excised inside-out and outside-out configurations with an Axopatch
200B amplifier (Axon Instruments, Foster City, CA). Patch pipettes were
fabricated from borosilicate glass (TW150F-4, World Precision
Instruments) using a Flaming/Brown micropipette puller (Model P-87,
Sutter Instrument). Pipettes were fire-polished with a microforge
(MF-83, Narishige Scientific Instrument) to give the pipette a
resistance of 3-5 M for macropatch recording and 10-20 M
for
single-channel recording, respectively. To reduce noise through
electrodes, patch pipettes for single-channel recording were coated
with beeswax under a stereozoom microscope (World Precision
Instruments). The amplified analog data were filtered at 1 kHz with
80-dB/decade low-pass bessel filter and digitized with a Digidata 1200 (Axon Instruments). Voltage stimulus was delivered to CV202BU headstage
with Digidata 1200 using pClamp6.0 or 7.0 program (Axon Instruments).
The data were stored in a pentium computer and analyzed using programs
such as pClamp 6.0 or 7.0 and Origin 4.1 (Microcal). Collected data
were stored in Axon binary format with pClamp7, which was
converted into a relevant format with pClamp6 afterward and analyzed
with Origin4.1 for macroscopic channel current recording and pSTAT for
single-channel current recording.
Both intracellular (bath for inside-out and pipette for outside-out)
and extracellular (pipette for inside-out and bath for outside-out)
solutions contained 130 mM NaOH, 3 mM HEPES, and 0.5 mM
Na2-EDTA and were adjusted to pH 7.6 with HCl,
unless otherwise specified. Na-cGMP (freely dissolved in water) was
added to bath solution for inside-out patch and to pipette solution for
outside-out patch before the final pH adjustment. For
Mg2+ blocking experiments,
MgCl2 was added to give the desired free concentration calculated by Martell and Smith
(1974). The calculation also took pH into account.
Blockade was measured by perfusing the intracellular or extracellular
face of the membrane patch with the solutions containing indicated free
concentrations of blockers. After the control recordings were obtained
in the absence and presence of 10 mM Mg2+ (for
intracellular side) or 20 mM Mg2+ (for
extracellular side) to achieve maximum blockade (Park and MacKinnon 1995
), each patch was perfused with various
concentrations of blockers to investigate intracellular or
extracellular Mg2+ effects on channel permeation.
To investigate the selectivity of gustCNG channel, we analyzed current-voltage (I-V) relationship under symmetrical bi-ionic conditions. In the test solutions, 130 mM Na+ was substituted with the same concentrations of other monovalent cations, K+, Li+, Rb+, or Cs+. The free acid form of EDTA (Sigma E-6758) was used instead of Na2EDTA to prevent asymmetric Na+ concentration and anomalous mole fraction effect. All the test ions are the hydroxylated forms, which remain symmetric to both intracellular and extracellular solutions after pH adjustment to pH 7.6 with HCl (KOH, Sigma P-5958; LiOH · H2O, Sigma L-4256; RbOH, Aldrich Chemical, 24,389-2; CsOH: Aldrich Chemical 23,204-1). To activate channel currents, 50 µM of cGMP was added into intracellular solutions. Rapid and complete solution changes were obtained by moving a linear array of micorcapillary tubes (1 µL, 64 mm length; Drummond), each containing different concentrations of ligand or blocker sequentially.
To use ramp pulse for macroscopic current recordings, an independent protocol of voltage steps lasting 150 ms was preceded to ensure that the steady state was achieved throughout the ramp. We used only step pulses to record the blocked currents by l-cis-diltiazem, since the current did not reach steady-state level until tens of milliseconds after voltage pulses.
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RESULTS |
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Macroscopic current-voltage relationship of gustCNG channels
The cDNA of gustCNG channel was transcribed in vitro and
functionally expressed in Xenopus oocytes. Ionic currents
through gustCNG channels in excised membrane were investigated in
symmetrical 130 mM Na+ conditions using various
concentrations of cGMP perfused onto the intracellular side of the
membrane to activate channels. The membrane was held at 0 mV in the
bath solution containing cGMP and stepped from 100 to 100 mV with
20-mV increments (Fig. 1). As the
concentration of intracellular cGMP ([cGMP]int)
was increased from 0.1 to 100 µM, the currents evoked by cGMP were
also increased. However, the channel currents were saturated at around
100 µM, and the higher [cGMP]int did not
further increase the channel currents significantly. The channel
currents were rapidly activated and reached steady-state current levels
on stepping to various voltages. No apparent inactivation or
desensitization was observed during the 150-ms voltage pulses. We thus
used a ramp pulse protocol to study I-V relationship of this
channel in subsequent experiments.
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The membranes containing many channels (usually 1,000-2,000 channels)
were applied with a ramp pulse from 100 to 100 mV, and currents
evoked by the voltage pulse were recorded (Fig.
2A). The macroscopic
I-V relationship was roughly linear in most of the voltage
range. At extreme depolarizing membrane potentials, slight outward
rectifications were observed in some recordings. From five such
experiments of macroscopic current recordings, the dose-response
relationship and the effects of membrane voltage on the activation of
gustCNG channels were analyzed. Since the membrane potential of rat
taste cell is usually around
70 mV or even more negative under
physiological conditions (Akabas et al. 1988
)
and becomes positive when excited, the dose-response relationships were
analyzed at two different membrane voltages,
80 and 80 mV. After
partially activated currents were normalized to fully activated
currents at 100 µM cGMP, the data were plotted against cGMP
concentrations and fitted to the Hill equation (Fig. 2B).
The results show no significant difference in the concentration for
half-maximal activation, K1/2, and the
Hill coefficient, n, both at
80 mV, 3.3 ± 0.1 (SE) µM cGMP and 1.4 ± 0.1, and at 80 mV, 2.9 ± 0.2 µM cGMP and 1.4 ± 0.1, respectively. The
K1/2 value of 3.3 µM at
80 mV is
consistent with that of the previous report obtained from gustCNG
channel expressed in HEK293 cells (Misaka et al. 1997
).
In Fig. 2C, K1/2 values
were plotted against membrane voltages. Although
K1/2 values show slight voltage
dependence, i.e., higher affinity at more positive voltages, the
tendency is not significant enough to argue that the cGMP-dependent
channel activation is voltage dependent. We also examined the
activation of gustCNG channel by intracellular cAMP (Fig.
3). K1/2
for cAMP was 250 ± 20 µM at
80 mV with n of
1.61 ± 0.1. Fully activated currents by 10 mM cAMP was about 90%
of the currents by a saturating concentration of cGMP at 100 µM.
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Single-channel characteristics of gustCNG channels
To investigate the detailed characteristics of gustCNG channel, we
obtained single-channel currents of gustCNG channel at various membrane
voltages and cGMP concentrations. Figure
4 shows a continuous current recording of
a single gustCNG channel at 80 mV in the presence of 3 µM of cGMP.
Several single-channel characteristics of gustCNG channels were
evident. Single-channel current amplitude was ~2.2-2.3 pA at
80
mV, and stochastic long closed states along with fast open-close
transition were observed. Consistent with the macroscopic current
recordings, single-channel activity of gustCNG channel did not show any
desensitization or inactivation even at 100 µM cGMP (data not shown).
Since the long gaps observed in single-channel recordings did not seem
to have any particular pattern in its frequency and duration, we
excluded those events from the analyses of single-channel
characteristics (see DISCUSSION).
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The effects of cGMP on channel gating were investigated at the
single-channel level under various cGMP concentrations. Since we
already showed that the gating of gustCNG channel was not strongly influenced by membrane voltages, the single-channel currents were measured at a constant membrane voltage, 80 mV. As shown in Fig. 5A, the frequency of the
channel opening was increased as a function of cGMP concentration. Open
probability (Popen) of a single
channel was analyzed as following, and the results provided relative
times during the channel spent in the open state:
Popen =
o/
i (where,
o, total open time for the level under
consideration;
i, total interval over which
Popen is measured).
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As we expected, the analysis result showed that open probabilities of
this channel followed sigmoidal curve when the data were plotted
against cGMP concentrations and the curve was fitted to the Hill
equation (Fig. 5B). The two parameters,
K1/2 of 3.9 ± 0.7 µM and
n of 1.3 ± 0.3, obtained from single-channel analysis, were in good agreement with those values obtained from macroscopic dose-response relationship at 80 mV. These results strongly support the idea that the cGMP-evoked macroscopic currents of gustCNG channel
are due to the increase in the open probability and cGMP is a direct
agonist of the channel.
Single-channel conductance of gustCNG channel
To determine the single-channel conductance, we measured the
single-channel current amplitudes at different membrane voltages. The
raw traces of Fig. 6A show the
current levels of a single gustCNG channel increase as membrane
potentials increase from 80 to 20 mV. Precise current levels of
single-channel currents were determined by analyzing the histogram of
current amplitudes. The total areas under the histogram were normalized
to give a unity, and the amplitude histograms were fitted to the sum of two Gaussian functions (Fig. 6B). Increases in current
amplitudes following membrane potentials can be clearly seen. The peaks
of the Gaussian functions representing the closed level were used to
subtract the leak currents so that the closed level at each membrane
potential could be lined. From several single-channel recordings,
average single-channel currents at various membrane potentials were
obtained, and the current values were plotted against membrane voltages
(Fig. 6C). The I-V relationship shows a linearity
throughout the membrane voltages tested, consistent with the
macroscopic I-V relationship shown in Fig. 2A.
The linear slope in Fig. 6C gave rise to the single-channel
conductance of 28 pS.
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Single-channel kinetics of gustCNG channels: two exponentially fitted open and closed events
Answering the question regarding how gustCNG channel may open and
close in response to cGMP requires the kinetic analysis of open and
closed events. In an expanded time scale as shown in Fig.
7A, two distinct populations
of dwell-times, short and long, were observed in both open and closed
events of single gustCNG channels. In addition, the channels also
exhibited flickering openings in most of membrane voltages. When the
closed dwell-time and open dwell-time were measured and distributed in
an exponential time scale, the sum of two decaying exponential
functions well described the histograms of both dwell-times. In Fig.
7B, both open and closed events are represented with log
dwell-time function to show more clearly the two exponentially
distributed components of a single-channel current trace recorded with
3 µM cGMP at 80 mV. The opening and closing rates can be obtained
from the reciprocal of closed and open time constants (
),
respectively. After analyzing the single-channel dwell-times, opening
and closing rate constants were obtained and plotted against cGMP
concentrations in Fig. 7C. The analysis of closed events
shows that there are two opening rates: one is fast and independent of
[cGMP]int, 2 × 103 ~ 3 × 103
s
1, and the other is
slower and changes in response to [cGMP]int, from 10 s
1 at 1 µM cGMP
to 300 s
1 at 100 µM
cGMP. Open event analysis shows that both of the fast and the slow
closing rates are insensitive to [cGMP]int,
about 2 × 103
s
1 and 10 ~ 50 s
1, respectively. Thus
the gustCNG channel goes to open state with high propensity as cGMP
concentration increases without much effects on the closing process.
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Selectivity of gustCNG channel among monovalent cations
Cyclic GMP-gated channels are nonselective cation channels
(Bastian and Fain 1982; Capovilla et al.
1983
; Hodgkin et al. 1984
; Woodruff et
al. 1982
; Yau et al. 1981
) permeating both
Na+ and K+. These channels
are also highly selective for divalent cations as permeant blockers
(Capovilla et al. 1983
; Hodgkin et al.
1984
; Yau et al. 1981
). Although the
similarity of amino acid residues known to form ion-conducting
pathway is high among CNG channels, their selectivity for monovalent
cations can be different as revealed in the case of CNG channels of
bovine rod and catfish olfactory neurons (Goulding et al.
1993
). We examined the selectivity of gustCNG channel among
monovalent cations. Under bi-ionic conditions, i.e., pipette solution
contains 130 mM Na+ and bath solutions contain
130 mM Na+, K+,
Li+, Rb+, or
Cs+, reversal potentials are determined in
accordance to their permeability. Then we can obtain the relative
ratios of the permeability coefficients according to the GHK-derived
equation. From the four independent experiments, we obtained reversal
potential values of gustCNG channel under bi-ionic condition, as
follows, in millivolts (Fig. 8):
Na+,
0.4 ± 0.5; K+,
3.1 ± 0.7; Li+, 10.2 ± 1.4;
Rb+, 10.0 ± 1.9; Cs+,
20.4 ± 2.6.
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After liquid junction potential was taken into account (Barry
and Lynch 1991; Neher 1992
), the selectivity
order of gustCNG channel was determined that
Na+:K+:Rb+:Li+:Cs+
equals 1:0.95:0.74:0.63:0.49. As a control, the same experiments were
performed with the
subunit of bovine rod CNG channel expressed in
Xenopus oocytes and the selectivity order was obtained as
Na+ ~ K+ > Rb+ > Li+ > Cs+, identical to that of the previous report
(Goulding et al. 1993
).
Effects of Mg2+ ions on gustCNG channel permeation from intracellular sides
Magnesium ion is a ubiquitous and abundant divalent cation in both
intra- and extracellular milieu and affects the permeation of several
different ion channels. CNG channels are known to be blocked by
both intracellular and extracellular
Mg2+ at a physiological concentration range. We
thus investigated the effects of Mg2+ on gustCNG
channel using inside-out and outside-out patch configurations. In the
inside-out patch configuration, 100 µM cGMP was used to fully
activate gustCNG channels, and different concentrations of
Mg2+ were perfused to block the channel currents
(Fig. 9A). Intracellular Mg2+ blocked the outward currents with higher
affinity than the inward currents. Due to the voltage-dependent
blockade, 10 mM Mg2+ almost completely blocked
the outward currents, while large inward currents are still detected in
an identical Mg2+ concentration. In Fig.
9B, the fraction of unblocked currents normalized to fully
activated currents (I/Io)
were plotted as a function of Mg2+ concentrations
and fitted to the Langmuir isotherm. The Mg2+
concentrations inhibiting the channel currents to one-half
(Ki) were quite different at different
voltages such as 8.2 ± 1.5 mM at 70 mV and 360 ± 40 µM
at 70 mV. As the membrane voltage was increased from
70 to 70 mV, the
affinity of intracellular Mg2+ was increased
about 23-fold. The Hill coefficient (n) of
Mg2+ binding near unity (e.g., 1.1 at 70 mV and
0.7 at
70 mV) suggests that a single Mg2+ may
be enough to block the channel currents.
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For more detailed analysis of voltage-dependent
Mg2+ blockade on gustCNG channel currents,
Ki values at various membrane voltages were obtained from five independent experiments and plotted as a
function of membrane voltages (Fig. 9C). The linear
relationship appears in negative membrane voltages, and thus data
points were fitted to the equation, Ki = Ki(0 mV)
exp(zFV/RT). The slope gave a
z
value of 1.04, which indicates that the electrical
distance of internal Mg2+ binding site is about
52% across the membrane potential from inside. At positive voltage
range, this voltage dependency disappeared. Voltage dependency observed
in negative membrane potential indicated that the internal
Mg2+ binding site might be located in the
conducting pathway as suggested for another CNG channel in a previous
study (Root and MacKinnon 1993
). At highly depolarized
voltage range, Mg2+ "pops through"
the channel, and the voltage dependence of Mg2+
blockade would deviate from the above relationship to membrane voltages. The affinities (Ki) and the
electrical distances (
) of rat gustatory, bovine rod, and rat
olfactory CNG channels were compared in Fig. 9D. Three
different channels showed very similar Ki values, 6.6 ± 0.4 mM for
gustatory (n = 5), 8.8 ± 2.6 mM for rod
(n = 4), and 8.9 ± 1.9 mM for olfactory
(n = 4) at
60 mV and
values, 0.52 for gustatory,
0.64 for rod, and 0.52 for olfactory CNG channels. These results also
reflect the high similarity in their amino acid sequence of the
pore-forming region (see DISCUSSION and Fig. 12). In
addition, six or seven negatively charged amino acids are also found in
the putative intracellular vestibule of all three different CNG
channels, further suggesting that the local electrostatic environment
of channel entryways for the binding of intracellular
Mg2+ may be quite similar.
Effects of Mg2+ ions on gustCNG channel permeation from extracellular sides
The effects of extracellular Mg2+ on the
permeation of gustCNG channel were studied in outside-out patch
configuration. To determine the effects of Mg2+
from the extracellular side, 100 µM cGMP was used to activate gustCNG
channel and indicated concentrations of Mg2+ were
applied to block the channel currents from the outside surface (Fig.
10A). The inward currents
were blocked almost completely in 1 mM Mg2+,
while the outward currents were not blocked completely even at 20 mM in
extremely positive voltage ranges. The current blockade was also
voltage dependent in that the inward currents were more sensitive to
Mg2+ than the outward currents. Under
physiological conditions, retinal CNG channel shows a strong outward
rectification due to largely voltage-dependent blockades by divalent
cations (Yau and Baylor 1989), and the blockade is known
to reduce the signal-to-noise ratio of the rod cells. The gustCNG
channel seems to share these characteristics. The fractions of
unblocked currents were plotted against Mg2+
concentrations and fitted with a Langmuir function in Fig.
10B, where Ki was 1.1 ± 0.3 mM at 70 mV and 20 ± 14 µM at
70 mV, an almost 50 times higher affinity at
70 mV than at 70 mV.
Ki values obtained from seven
independent experiments for external Mg2+ at
various membrane voltages were plotted as a function of membrane voltages (Fig. 10C). The linear relationship appearing in
the positive voltage range resulted in the slope, z
value, of 0.94. In other words, the electrical distance of external
Mg2+ binding site across the membrane potential
is about 47% from the outside. The affinity of external
Mg2+ on macroscopic currents among gustatory,
rod, and olfactory CNG channels showed somewhat different results (Fig.
9D). While the voltage dependence of external
Mg2+ blockade are almost identical among three
different channels leading to the same electrical distance of external
Mg2+ binding site of about 0.47, the apparent
affinity of Mg2+ shifted in parallel where
olfactory CNG channel showed the highest affinity and gust CNG channel
the lowest. Their half-maximal blocking concentrations at 60 mV are
760 ± 190 µM for gust (n = 7), 540 ± 30 µM for rod (n = 5), and 370 ± 30 µM for
olfactory (n = 5).
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Effects of intracellular l-cis-diltiazem on gustCNG channels
A benzothiazepine, l-cis-diltiazem,
has been known to block many types of CNG channels from intracellular
side of an excised patch in a voltage-dependent manner (Haynes
1992; McLatchie and Matthews 1992
;
Stern et al. 1986
; Yau and Baylor 1989
).
Since the affinity of intracellular Mg2+ to
gustCNG channel was similar to the other two CNG channels, we wondered
whether l-cis-diltiazem also blocks gustCNG
channel with a similar affinity compared with other CNG channels.
Assuming that l-cis-diltiazem blocks the channel
current by occluding the ion conduction pathway from intracellular
side, the binding site of l-cis-diltiazem within
gustCNG channel may remain similar to other CNG channels. Three
different CNG channels were fully activated using 500 µM cGMP, and
100 µM l-cis-diltiazem was used to block the
channel currents. Since it took tens of milliseconds to reach the
steady-state current levels after step-pulse to positive voltages (Fig.
11A), the blockade was
studied using step-pulse protocols instead of voltage ramps. The
significant blockade of gustCNG channel currents by 100 µM
l-cis-diltiazem was detected only at depolarizing
voltage steps >80 mV. Quite different from gustCNG channel, the
steady-state currents of rat olfactory CNG channels were greatly
reduced by 100 µM of intracellular
l-cis-diltiazem, especially in positive voltage
range. The potency of l-cis-diltiazem for bovine
retinal channel seemed to be lower than that of rat olfactory but
higher than gustatory CNG channels. From several such experiments, we
obtained a normalized I-V relationship where blocked
currents were normalized to fully activated currents at indicated
membrane voltages (Fig. 11B). The inward currents of gustCNG
channel did not show any current blockage, and a slight reduction of
channel currents was observed in extreme positive voltages
(n = 4). The steady-state outward currents of bovine rod CNG and rat olfactory CNG channels, however, showed significant reductions in all voltages. The blockade by 100 µM
l-cis-diltiazem was highly voltage dependent, and
more than one-half of the currents by rod and olfactory CNG channels
were blocked by 100 µM at 100 mV (n = 5 for each
channel).
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DISCUSSION |
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In this study, we investigated the functional characteristics of a
putative gustCNG channel cloned from rat tongue epithelial cells
(Misaka et al. 1997). The homomeric gustCNG channels
were expressed at the level of single and macroscopic currents in
Xenopus oocytes by injecting cRNA transcribed in vitro. The
gating and permeation properties of gustCNG channels were studied using
electrophysiological means.
Although the channels can be directly activated using either cGMP or
cAMP, cGMP is a better agonist in terms of both affinity and efficacy
for channel opening. K1/2 value of
cGMP for the activation of gustCNG channel was relatively insensitive
to transmembrane voltages and was determined as 3.3 ± 0.1 µM.
This affinity is similar to that of rat olfactory CNG channel at
~1-2 µM (Dhallan et al. 1990) but significantly
different from that of bovine rod CNG channel (about 50 µM) under
physiological conditions (Nakatani and Yau 1988
). Cyclic
AMP can activate the gustCNG channel with K1/2 of 250 µM and to about 90% of
the maximum current level activated by saturating cGMP. Both of the
affinity and the efficacy of gustCNG channel to cAMP are markedly
different from those of homomeric cone CNG channels (Gerstner et
al. 2000
). Since the amino acid sequence of the cyclic
nucleotide binding domain in rat gustatory and mouse cone channels are
virtually identical, these differences in gating behavior might come
from the differences in their amino-terminal regions. It was shown that
the amino-terminal domain participates in the allosteric gating
transition in previous studies using other CNG channels
(Goulding et al. 1994
; Tibbs et al.
1997
). It is known that relatively low affinity of rod CNG
channel for cGMP is essential for phototransduction by allowing the
channels to stay open in the steady presence of cGMP in darkness and to be closed only by light (Yau and Chen 1995
). The high
affinity of gustCNG channel for cGMP may reflect the mechanistic
similarity between gustatory and olfactory signal transductions, an
increase in cGMP concentration on the stimulatory signal, and the
opening of CNG channels. Since it is still not clear whether taste
receptor cells also express a second (or
) subunit of CNG channel,
however, the physiological affinity for cGMP to gustCNG channel may be significantly altered in vivo. We observed that the Hill coefficient for the channel activation of about 1.4 for cGMP, strongly suggesting that the cooperative bindings of more than two molecules of cGMP might
be required to activate gustCNG channels. The functional CNG channels
are generally believed to be tetramers (Liu et al. 1996
;
Shammat and Gordon 1999
; Shapiro and Zagotta
1998
; Varnum and Zagotta 1996
). Like other
members of CNG channel family (Sunderman and Zagotta
1999
), the cloned gustCNG channel does not exhibit any
desensitization or inactivation during a prolonged exposure of
intracellular cyclic nucleotides both at single and macroscopic current levels.
The I-V relationship of gustCNG channel shows near linearity
except at extreme positive voltages. The lack of desensitization allowed us to investigate the gating properties of gustCNG channels in
detail using single-channel current recordings. The single-channel gating characteristics were similar to other CNG channels, such as
homomeric rod or cone CNG channel expressed in Xenopus
oocytes. The single-channel currents of gustCNG channel showed rapid
transitions between open and closed current levels occasionally
interrupted by long closures lasting on the average of about 1 s.
The long closures were also observed in single-channel recordings of
bovine rod CNG channels expressed in Xenopus oocytes, and
they were considered as a separate state apart from the typical open
and closed transitions (Sunderman and Zagotta 1999).
Even at the saturating concentration of cGMP, 100 µM, the gustCNG
channels remained open and closed at a constant rate. Two distinct
dwell-times, short and long, were readily identifiable in both open and
closed events of single gustCNG channels, and the channels also
exhibited flickering openings in most membrane voltages. At this
concentration of agonist, the bursts of channel openings were often no
longer discrete and fused into continuous rapid flickers. As far as
single-channel kinetic analysis is concerned, we could not properly fit
the dwell time histograms of both open-state and closed-state using
single exponential functions. After the process of various trials for
more accurate analysis, we were able to fit both open and closed states
with a sum of two exponential functions. In a previous study, the
single-channel recordings of olfactory CNG channels in the membrane of
the dendrite and soma of isolated Salamander olfactory neurons were
best fitted with a single open state and two closed states
(Zufall et al. 1991
). In another study using the
subunit of the cGMP-gated channel from rod photoreceptor expressed in
Xenopus oocytes, however, Benndorf et al. reported the
kinetic analysis of single channel, which revealed two exponentially
distributed open and closed time histograms (Benndorf et al.
1999
). Using the single-channel kinetic analysis of gustCNG
channel at various concentration of cGMP, we were able to show that the
cGMP increases the open probability of the channel by increasing solely
the slower component of the opening rate without much effect on the
faster opening rate or the components of the two closing rates.
The single-channel conductance of gustCNG channel was determined as 28 pS, which is smaller than those of olfactory CNG channel from the
salamander olfactory receptor neuron, 45 pS (Zufall et al.
1991) and the catfish olfactory neuron, 55 pS
(Goulding et al. 1992
), respectively. However, it is
similar to the single-channel conductance of the
subunit of the CNG
channel from bovine rod, ~25-28 pS (Kaupp et al.
1989
; Nizzari et al. 1993
). One of the characteristic gating properties of CNG channels is flickering opening,
i.e., open and close transitions are too fast to be analyzed when
converted to digitized data. The deviation of current amplitude histogram from the theoretical Gaussian functions seen in the analysis
of single gustCNG channel recordings (asterisks in Fig. 6B)
may be caused by the flickering openings. Some single-channel current
traces showed brief openings at sub-levels of full open state (data not
shown). Since this sublevel conductance state was not always seen,
however, further experiments are needed to support the preliminary
observation using D2O instead of
H2O in recording solutions, which markedly slows
down the gating (Root and MacKinnon 1994
).
CNG channels are nonselective cation channels permeating different
monovalent cations. The gustCNG also showed weak selectivity among
monovalent cations. The selectivity order of gustCNG channel was
determined as Na+ ~ K+ > Rb+ > Li+ > Cs+, which was similar to that of bovine retinal
CNG channel (Goulding et al. 1993). It is intriguing to
find similarities of permeation characteristics between gustCNG channel
and rod CNG channel despite a significant difference in the affinity
for cGMP.
We probed the structure of ion conduction pathway of gustCNG channel by
investigating the effects of CNG channel blockers, Mg2+ and l-cis-diltazem, and by
comparing the results with two other CNG channels. Both intracellular
and extracellular Mg2+ blocked
Na+ currents through gustCNG channel in a
voltage-dependent manner. Voltage-dependent blockade and
"pop-through" phenomenon at extreme voltages are the
characteristics of Mg2+ blockade observed in
other CNG channels (Root and MacKinnon 1993). Likewise,
gustCNG channel also contains Mg2+-binding sites
located in the conducting pathway, and Mg2+ can
permeate the conducting pathway by extreme membrane potentials. The
electrical distances of Mg2+ binding sites (
)
estimated for gustCNG channel, 0.52 from intracellular side and 0.47 from extracellular side, indicate that internal Mg2+ and external Mg2+
binding sites may be located closely within the electrical field. Comparison of
values among rat gustatory, bovine rod, and rat olfactory CNG channels revealed that the binding affinity as well as
the electrical location of the intracellular Mg2+
binding are quite similar to each other. Although the exact location of
the internal Mg2+ binding site in CNG channels
has not been identified yet, it is not too surprising to find this
similarity since the amino acid sequence comprising the ion-conduction
pathway (or S5-pore-S6 region) among these channels show a high
sequence homology.
On the contrary, however, extracellular Mg2+
binding affinities were significantly different among the three CNG
channels: the strongest for olfactory CNG channel and the weakest for
gustCNG channel with similar values. The blockade of CNG channels
by external Mg2+ is mediated by a conserved
glutamate in the pore-forming region, Glu333 of the catfish olfactory
channel (Root and MacKinnon 1993
) and Glu363 in the
bovine retinal channel (Eismann et al. 1994
). The
corresponding glutamate residue (Glu369) and adjacent amino acid
residues of the pore-forming region are also highly conserved in
gustCNG channel. The Mg2+ affinity can be altered
without too much effect on
value by those charged amino acid
residues nearby the Mg2+-binding site affecting
the local concentration of Mg2+. We compared the
total net charges in putative extracellular vestibule regions, S5-P
linker and P-S6 linker (Fig. 12). It
was intriguing to find that the total net charge of homo-tetrameric gustatory, rod, and olfactory CNG channels were 0,
8, and
20, respectively, at a neural pH. Thus the apparent difference in the
affinities of extracellular Mg2+ may simply be
the result of the differences in local Mg2+
concentration influenced by the electrostatic attraction due to net
negative charges in the extracellular vestibule. Although the affinity
for extracellular Mg2+ is lower than that of rod
or olfactory CNG channels, the I-V relationship of gustCNG
channel in physiological conditions would also be strongly rectified
toward outward due to the Mg2+ blockade as seen
in other CNG channels (Yau and Baylor 1989
). The
significance of extracellular divalent cation blockade on gustCNG
channel function and taste signal transduction remains to be
elucidated.
|
Although the ionic currents through rat gustatory, bovine retinal, and
rat olfactory CNG channels were all blocked by intracellular Mg2+ with almost an identical affinity, another
intracellular blocker of CNG channels,
l-cis-diltiazem, showed markedly different
effects on these channels. In the case of bovine rod and rat olfactory CNG channels, 100 µM l-cis-diltiazem blocked
almost one-half of the maximum currents. However, the same
concentration of l-cis-diltiazem did not inhibit
the gustCNG channel currents significantly even at positive voltages.
This finding is intriguing since the mechanism of
l-cis-diltiazem action on CNG channels is still
controversial and may even be varied for different channels. Block of
the rod channel by l-cis-diltiazem showed a pH
dependence suggesting that the charged form of
l-cis-diltiazem blocks the channel
(McLatchie and Matthews 1994). Thus
l-cis-diltiazem may block the channel currents by
directly binding to a site at the inner vestibule of CNG channels,
although its binding site has not been localized. In contrast, the
blockade of cone CNG channel by l-cis-diltiazem is not pH dependent (Haynes 1992
), and it was suggested
that l-cis-diltiazem may reduce the channel
currents by altering the CNG channel gating and the hydrophobic
interaction rather than the charge interaction is important for the
interaction. Thus the binding site for
l-cis-diltiazem may not even be in the conducting
pathway despite its strong voltage dependence in blocking CNG channel
currents. Since gustatory, retinal, and olfactory CNG channels exhibit
markedly different apparent affinities for
l-cis-diltiazem despite their overall amino acid
sequence similarity, it would be possible to localize the binding site
of l-cis-diltiazem in CNG channels using chimeric studies and to determine the mechanism of action at molecular levels.
In conclusion, we elucidated the electrophysiological properties of the gustatory CNG channel expressed in Xenopus oocytes. While the gustCNG channel shares many common characteristics with other members of the CNG channel family, several properties of both gating and permeation distinguish the gustCNG channels from the other CNG channels. Since it is still elusive whether any gustatory signaling pathway involves the cyclic nucleotide-mediated increase in cation permeability and this channel is also involved in the visual signal transduction in retinal cone cells, it would be critical to characterize the channel activity in the membrane of mammalian taste bud cells. Therefore the functional properties of cloned gustCNG channel investigated in this study will guide such studies in the future.
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ACKNOWLEDGMENTS |
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The authors thank Dr. Keiko Abe (University of Tokyo) for generously providing us with the cDNA of gustatory CNG channel. We also thank the other members of the Neuro-biochemistry Laboratory at K-JIST for timely help throughout the work.
This work was supported by grants to C.-S. Park from Korea Science and Technology Foundation (1999-2-210-001-3 and 2000-2-21400-002-2) and the Ministry of Education of Korea (BK 21 Project), and to W. Kim from Korea Research Foundation (1998-021-F00310).
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FOOTNOTES |
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Address for reprint requests: C.-S. Park, Dept. of Life Science, Kwangju Institute of Science and Technology (K-JIST), 1 Oryong-dong, Puk-gu, Kwangju 500-712, Korea (E-mail: cspark{at}kjist.ac.kr).
Received 2 October 2000; accepted in final form 12 February 2001.
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REFERENCES |
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