0022-3565/99/2903-0974$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 290:974 –979, 1999
Vol. 290, No. 3
Printed in U.S.A.
Effects of Potassium Channel Blockers on CO2-Induced Slowly
Adapting Pulmonary Stretch Receptor Inhibition
SHIGEJI MATSUMOTO, TOSHIAKI TAKAHASHI, TAKESHI TANIMOTO, CHIKAKO SAIKI, and MAMORU TAKEDA
Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, Tokyo, Japan
Accepted for publication April 28, 1999
This paper is available online at http://www.jpet.org
Inhalation of CO2 gas mixtures inhibits slowly adapting
pulmonary stretch receptor (SAR) activity, and this inhibition is not related to the change in lung mechanics
(Sant’Ambrogio et al., 1974; Coleridge et al., 1978; Matsumoto et al., 1996). After delivery of wood smoke containing a
high concentration of CO2, the SARs decrease their activity,
and the wood smoke-induced SAR decrease is not significantly influenced by pretreatment with a bronchodilator isoproterenol (Lai and Kou, 1998). The administration of acetazolamide, a carbonic anhydrase (CA) inhibitor, blocks or
attenuates the inhibitory responses of SARs to CO2 inhalation (Sant’Ambrogio et al., 1974; Matsumoto et al., 1996) and
wood smoke delivery (Lai and Kou, 1998). From these observations, it is most likely that the inhibitory effect of CO2
inhalation on SAR activity may be related to an increase in
the H1 concentration at the receptor site but does not depend
on the change in lung mechanics.
Blockade of CA-dependent CO2 hydration is thought to
decrease both the rate of change of [H1] and the responsiveness of SARs to rapid changes in CO2, but the exact mechanism by which an increase in the H1 concentration inhibits
the receptor discharge remains to be determined. Nevertheless, in neurons of the marine mollusk Aphysia california,
hyperpolarizing responses of the neural structures to CO2
may be caused by an increase in Cl2 or K1 conductance
Received for publication December 28, 1998.
dependently increased SAR activity during deflation and had no
effect on PT, abolished or attenuated the decrease in SAR
activities induced by CO2 inhalation in a dose-dependent manner. The K1 channel blocker tetraethylammonium (2.0 and 6.0
mg/kg i.v.) that did not significantly alter either basal SAR
discharge or PT had no effect on the inhibitory responses of
receptor activity to CO2 inhalation. These results suggest that
the inhibitory mechanism of CO2 inhalation on SARs may be
involved in the activation of 4-aminopyridine-sensitive K1
channels in the nerve terminals of SARs.
(Brown, 1972). Based on evidence showing that no CA enzymatic reaction is found in the smooth muscle of the bronchi,
a similar mechanism to explain the inhibitory action of CO2
has been suggested by Matsumoto et al. (1996). Because
myelinated afferent fibers in peripheral nerves contain CA
activity (Cammaer and Transey, 1987; Riley et al., 1988;
Szaboks et al., 1989), Matsumoto et al. (1996) also postulated
that the existence of CA enzymes would be expected in airway afferent fibers. It is therefore possible that the blockade
of CA hydration by acetazolamide acts to prevent hyperpolarization of the membrane potential of the SAR terminal.
Concerning the generation of action potentials, K1 conductance is thought to play a significant role in repolarization of
the nerve cell membranes, which regulates the number of
spikes with a cycle Na1 inflow and K1 outflow. We hypothesized that CO2 inhalation may exert an inhibitory effect on
the activity of SARs through the modification of K1 channel
activity; however, no studies have examined this hypothesis.
To elucidate whether there is a correlation between a generalized action of K1 channels and inhibition of the SAR
activity associated with CO2 inhalation, we performed two
different types of experiments in anesthetized, artificially
ventilated rabbits after vagus nerve section. First, the responses of SARs to CO2 inhalation were examined before and
after the administration of 4-aminopyridine (4-AP), a well
known K1 channel blocker. Second, the responses of SARs to
CO2 inhalation before and after the administration of tetra-
ABBREVIATIONS: SAR, slowly adapting pulmonary stretch receptor; PT, tracheal pressure; BP, blood pressure; MBP, mean blood pressure;
4-AP, 4-aminopyridine; TEA, tetraethylammonium; CA, carbonic anhydrase; Ia, fast transient outward K1 current.
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ABSTRACT
In anesthetized, artificially ventilated rabbits with vagus nerve
section, inhalation of CO2 gas mixtures (tracheal CO2 concentration ranging from 8.0 to 10.2%) for 60 s decreased slowly
adapting pulmonary stretch receptor (SAR) activity during both
inflation and deflation. The magnitude of decreased receptor
activity during deflation had a more pronounced effect than that
seen during inflation. CO2 inhalation did not cause any significant change in tracheal pressure (PT) as an index of bronchomotor tone. Intravenous administration of 4-aminopyridine
(0.7 and 2.0 mg/kg i.v.), a K1 channel blocker, which dose-
CO2-Induced SAR Inhibition and K1 Channel
1999
ethylammonium (TEA), a K1 channel blocker, were compared. In the present experiments, control tracheal CO2 concentrations were kept below 4%. Furthermore, we selected
“low-threshold” receptors (Sant’Ambrogio, 1982; Ravi, 1986;
Matsumoto et al., 1996) that were more sensitive to CO2 than
“high-threshold” receptors (Sant’Ambrogio et al., 1974;
Coleridge et al., 1978; Matsumoto et al., 1996).
Materials and Methods
Statistical Analysis. During control conditions, firing rates of
the SARs during inflation and deflation were measured over several
respiratory cycles and expressed as imp/s. The SAR responses to CO2
inhalation for 60 s (tracheal CO2 concentration ranging from 8.2 to
10.2%) were obtained by counting the firing rates of receptors at 10-s
intervals and by performing the measurements over 120 s, and the
average activities of SARs during inflation and deflation were expressed as imp/s. Similarly, control values for PT were averaged over
several respiratory cycles and expressed as cm H2O. The responses of
PT to CO2 inhalation were obtained by measuring the respiratory
parameter at 10-s intervals and by performing the measurements
over 120 s. The statistical significance of the time-dependent effects
of 4-AP and TEA on the responses of SAR activities and PT to CO2
inhalation was first calculated by a one-way ANOVA for repeated
measurements. In addition, the maximum decreases in baseline SAR
activities during inflation and deflation produced by CO2 inhalation
in the absence and presence of 4-AP (0.7 and 2.0 mg/kg) and TEA (2.0
and 6.0 mg/kg) were also analyzed by a paired t test. All values were
expressed as mean 6 S.E. A value of P , .05 was considered statistically significant.
Results
Effect of 4-AP on Responses of SARs to CO2 Inhalation. Inhalation of CO2 gas mixtures caused decreases in
SAR activity during both inflation and deflation. The decrease in SAR activities occurred immediately after the tracheal CO2 concentration began to increase. The magnitude of
the decrease in SAR activity during deflation was greater
than that seen during inflation. The response was not associated with any significant change in PT, as an index of global
bronchomotor tone. After CO2 inhalation stopped, SARs returned to the control activity within 30 s (Fig. 1A). The
inhibitory effect of CO2 inhalation on SAR activities was
abolished by pretreatment with 4-AP (2.0 mg/kg), which produced a significant increase in the SAR activity during deflation and had no significant effect on the receptor activity
during inflation and PT (Fig. 1B). The responses of eight
different SARs to CO2 inhalation before and after pretreatment with 4-AP at 0.7 and 2.0 mg/kg were compared (Fig. 2).
The average inspiratory discharges of SARs in the control
and 4-AP (0.7 and 2.0 mg/kg)-treated animals were 59.5 6
2.3, 60.3 6 2.9, and 61.2 6 2.8 imp/s, respectively, and the
average expiratory discharges of receptors in those animals
were 19.7 6 1.6, 25.8 6 1.5, and 28.9 6 1.6 imp/s, respectively. 4-AP treatment at the dose of 2.0 mg/kg caused a
significant increase in the SAR activity during deflation. At
10 s after CO2 inhalation, the inspiratory discharge of SARs
was decreased from 59.5 6 2.3 to 51.2 6 1.7 imp/s, and the
expiratory discharge of receptors was decreased from 19.7 6
1.6 to 10.6 6 1.4 imp/s. The decreases in SAR activities
during inflation and deflation had more pronounced effects at
40 s after CO2 inhalation. The K1 channel blocker 4-AP (0.7
and 2.0 mg/kg) significantly reversed the inhibitory effect of
CO2 inhalation on SAR activities during inflation (percent
inhibition: absence, 32.4 6 1.5, n 5 8; in the presence of 4-AP,
0.7 mg/kg, 18.9 6 0.9, n 5 8, P , .05; 2.0 mg/kg, 3.8 6 1.3, n 5
8, P , .05) and deflation (percent inhibition: absence, 83.5 6
3.2, n 5 8; in the presence of 4-AP, 0.7 mg/kg, 24.8 6 1.1, n 5
8, P , .05; 2.0 mg/kg, 1.5 6 1.4, n 5 8, P , .05). CO2
inhalation before and after pretreatment with 4-AP (0.7 and
2.0 mg/kg) had no significant effect on PT. The increase in BP
occurred after the administration of 4-AP, but this pressor
effect was transient. The mean BP (MBP) values during
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Animal Preparation. Sixteen rabbits, weighing 2.5 to 3.0 kg,
were anesthetized with urethane (1.0 g/kg i.p.). The trachea was
exposed through a middle incision in the neck and cannulated below
the larynx. The trachea and esophagus were dissected free and
retracted rostrally to obtain a wide space for liquid paraffin. Tracheal pressure (PT) was measured by connecting a polyethylene
catheter inserted into the tracheal tube to a pressure transducer.
After the administration of heparin (500 U/kg) into the ear vein, the
femoral artery and vein were cannulated for measurement of blood
pressure (BP) and for administration of anesthetic agents, respectively. Additional doses (0.2– 0.3 g/kg/h i.v.) of urethane were administered as required. A polyethylene catheter was also positioned in
the right atrium through the jugular vein for the administration of
drugs or a 0.9% NaCl solution. Then the vagus nerves were exposed
and sectioned. The rectal temperature was maintained at approximately 37°C by means of a heating pad. The animals were paralyzed
with an initial i.m. administration of suxamethonium (20 mg/kg)
followed by a continuous infusion at 10 mg/kg/min. The stroke volume of the respirator was set at 10 ml/kg, and its frequency ranged
from 35 to 40 cycles/min. Tracheal CO2 was monitored and maintained at approximately 3.5 to 3.9% by adjusting the ventilatory rate.
Measurement of SARs. The peripheral end of the cut left vagus
nerve was desheathed. To record the single-unit activity of SARs,
thin strands containing afferent nerve fibers were separated, placed
on a unipolar silver electrode, and submerged in a pool with warm
liquid paraffin (37–38°C). The SARs were identified, on the basis of
their firing behavior during lung inflation, as follows: 1) the SARs
(low-threshold) increased their discharge during inflation and decreased their discharge during deflation, 2) the increase in SAR
activity was proportional to the increase in the inflation volume of
the respirator, and 3) the discharge of SARs continued as long as the
tracheal tube was occluded in a hyperinflated condition. The SAR
activity was amplified and selected by means of a window discriminator for counting the number of impulses. It was also monitored on
an oscilloscope and recorded on a polygraph. All of the 16 SARs
recorded were low-threshold receptors. The 16 SARs located below
the carina were confirmed in 16 rabbits, by using the same technique
as described in a previous study (Matsumoto et al., 1996).
Experimental Design. The experiments were designed to test
the role of a generalized K1 channel in the responses of SARs to CO2
inhalation. The following experiments were performed: 1) in eight
SAR fibers in eight rabbits, the effects of CO2 inhalation for approximately 60 s on SAR activity were determined. Ten minutes after i.v.
administration of 4-AP (0.7 and 2.0 mg/kg), the same tests were
repeated under the same conditions. The effectiveness of 4-AP was
determined by the presence of increased SAR activity during deflation. 2) In SAR fibers in eight rabbits, the changes in SAR activity in
response to CO2 inhalation were examined. Ten minutes after i.v.
administration of TEA (2.0 and 6.0 mg/kg), the same sets of experiments were repeated. The absence of TEA effects was confirmed by
restoring transient inhibition of SAR activity seen after a hyperinflated condition for several respiratory cycles. Lung compliance was
restored to the control by inflating lungs for several respiratory
cycles with a volume of 30 ml/kg.
Drugs. 4-AP and TEA were obtained from Sigma Chemical Co.
(St. Louis, MO). 4-AP (20 mg) and TEA (40 mg) were dissolved and
diluted with a 0.9% NaCl solution.
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Matsumoto et al.
Vol. 290
Fig. 2. Changes in PT and SAR during
both inflation and deflation in response
to CO2 inhalation before (F) and after
i.v. administration of 4-AP at 0.7 (Œ) and
2.0 (f) mg/kg. 0, the onset of increased
tracheal CO2 concentration. Values are
mean 6 S.E.; n 5 8. *P , .05, significant
difference from control values.
control and after 4-AP treatment were 98.4 6 3.6, 98.7 6 3.5,
and 99.2 6 3.5 mm Hg with 0.7 and 2.0 mg/kg, respectively,
and 118.5 6 4.3, 117.8 6 4.5, and 117.3 6 3.9 mm Hg after
CO2 inhalation, respectively. The maximal changes in MBP
in response to CO2 inhalation were not significantly altered
by 4-AP treatment.
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Fig. 1. Effect of 4-AP on the responses of PT, SAR
activity, and BP to CO2 inhalation. A, control. B, after
i.v. administration of 4-AP (2.0 mg/kg). Straight line,
period of increased tracheal CO2 concentration.
1999
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Discussion
The present study provided evidence that the inhibitory
responses of SAR activity to CO2 inhalation were diminished
by a well known K1 channel blocker, 4-AP, whereas TEA, a
K1 channel blocker, had no effect on CO2-induced SAR inhibition. Because blockade of CA-dependent hydration due to
acetazolamide is known to attenuate the inhibitory responses
of SAR activity to CO2 inhalation (Sant’Ambrogio et al., 1974;
Matsumoto et al., 1996) and wood smoke delivery (Lai and
Kou, 1998), it is most likely that an increase in the H1
concentration at the receptor site inhibits the activation of
4-AP-sensitive K1 channels in the SAR terminals.
The rapid inhibition of SAR activity was seen after CO2
inhalation, and the magnitude of decreased receptor activity
was more prominent in deflation than in inflation. These
significant characteristics were compatible with previous observations (Sant’Ambrogio et al., 1972; Coleridge et al., 1978;
Matsumoto et al., 1996). At a higher transmural pressure,
the inhibitory action of CO2 on SAR activity becomes weaker
(Mustafa and Purves, 1972; Bartlett and Sant’Ambrogio,
1976). It is therefore possible that the different effect of CO2
on SAR activity may be due to the difference between transmural pressures on inflation and deflation, passing through
the nerve endings of receptors.
CO2 inhalation usually induced a pressor effect. This effect
was not significantly altered by pretreatment with either
4-AP or TEA. Hargreaves et al. (1991) reported that during
Fig. 3. Effect of TEA on the responses of PT, SAR
activity, and BP to CO2 inhalation. A, control. B, after
i.v. administration of TEA (6.0 mg/kg). Straight line,
period of increased tracheal CO2 concentration.
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Effect of TEA on Responses of SARs to CO2 Inhalation. Typical examples of the effect of TEA (6.0 mg/kg), a K1
channel blocker, on the responses of SAR activity, PT, and BP
to CO2 inhalation are shown in Fig. 3, A and B. Pretreatment
with TEA did not significantly alter either the control activity of SARs or the CO2-induced SAR inhibition. The effects of
TEA at different doses (2.0 and 6.0 mg/kg) on the responses
of SAR activities and PT to CO2 inhalation in eight different
SAR fibers in eight rabbits are summarized in Fig. 4. TEA
(2.0 and 6.0 mg/kg) did not significantly attenuate the inhibitory effect of CO2 inhalation on SAR activities during inflation (percent inhibition; absence, 29.3 6 1.3, n 5 8; in the
presence of TEA, 2.0 mg/kg, 28.6 6 1.4, n 5 8, P . .05; 6.0
mg/kg, 27.8 6 1.3, n 5 8, P . .05) and deflation (percent
inhibition: absence, 90.8 6 2.8, n 5 8; in the presence of TEA,
2.0 mg/kg, 90.9 6 2.6, n 5 8, P . .05; 6.0 mg/kg, 88.4 6 3.1,
n 5 8, P . .05). Inhalation of CO2 gas mixtures in the
absence and presence of TEA (2.0 and 6.0 mg/kg) had no
effect on PT. The administration of TEA caused a hypotensive
effect, but the hypotension evoked by TEA was restored to
the control level within 5 min. The MBP values were 95.4 6
3.7, 95.6 6 4.2, and 96.2 6 4.5 mm Hg during control and
after TEA treatment at 2.0 and 6.0 mg/kg, respectively, and
114.3 6 3.9, 114.2 6 4.1, and 116.3 6 4.4 mm Hg during CO2
inhalation, respectively. TEA treatment had no significant
effect on the maximal changes in MBP induced by CO2 inhalation.
CO2-Induced SAR Inhibition and K1 Channel
978
Matsumoto et al.
Vol. 290
Fig. 4. Changes in PT and SAR activity
during both inflation and deflation in
response to CO2 inhalation before (F)
and after i.v. administration of TEA at
2.0 (Œ) and 6.0 (f) mg/kg. 0, the onset
of increased tracheal CO2 concentration. Values are mean 6 S.E.; n 5 8.
*P , .05, significant difference from
control values.
other investigators (Yanagisawa and Taira, 1979; Chung et
al., 1996), but the latter effect was very short lasting. The
former effect is probably explained by evidence demonstrating that 4-AP can elicit both membrane depolarization and
repetitive firing in squid axons (Yeh et al., 1976a,b). Presumably, specific actions of 4-AP on the expiratory discharge of
SARs readily appear in the condition in which there is no
mechanical deformation due to lung inflation. In the wholecell patch-clamp study with nerve terminals of the rat posterior pituitary, which are acutely dissociated and identified
by both morphological and immunohistochemical techniques,
4-AP and cesium block Ias in a dose-dependent manner, but
TEA (100 mM) and charybdotoxin at a concentration (4 mg/
ml) that blocks Ca21-activated K1 currents (Miller et al.,
1985) have no effect on Ia (Thorn et al., 1991). They also
found no evidence of a delayed rectifier K1 current (Thorn et
al., 1991). In this study, 4-AP at a relatively smaller dose of
0.7 mg/kg significantly suppressed the inhibitory responses
of SAR activity to CO2 inhalation, and the administration of
4-AP up to 2.0 mg/kg abolished CO2-induced SAR inhibition.
Because pretreatment with a CA inhibitor prevents CO2induced SAR inhibition (Sant’Ambrogio et al., 1974; Matsumoto et al., 1996), the results of this study led us to suggest
that an increase in [H1] at the receptor site may involve
inhibition of Ias of the SAR terminals, which are the major
currents responsible for terminal repolarization after a spike.
In other words, the blocking effect of K1 efflux attenuates the
repolarization of the membrane potential of SAR terminals
and, as a result, decreases the number of action potentials. In
other neuronal structures, a decrease in extracellular pH
induced by CO2 would cause hyperpolarization in accordance
with increases in Cl2 or K1 conductances (Brown, 1972).
However, further studies are needed to define the relationship between the Cl2 transport coupled to Cl2/Cl2 and/or
Cl2/HCO32 exchanger systems and the inhibition of SAR
activity associated with CO2 inhalation.
In the nodal membrane of rat myelinated nerve fibers, the
slow TEA-sensitive K1 conductance is prevalent (Roeper and
Schwarz, 1989). A similar localization of TEA-sensitive channels was reported by Baker et al. (1987). In the sucrose gas
recordings obtained from sciatic nerves of immature and
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graded increases in mean left atrial pressure in the rabbit,
there was a small but statistically significant increase in
SAR activity during inflation. Pulmonary venous congestion
is known to increase the pressure in both the left atrium and
the pulmonary veins (Braunwald, 1988). If CO2 inhalation is
permitted to cause the development of pulmonary venous
congestion, one can expect that an increase in SAR activity
during inflation actually occurs during CO2 inhalation, but
such no effect was observed in this study.
Regarding several K1 channels with different kinetic and
pharmacological properties, the three most prevalent types of
K1 channels have been classified as “delayed rectifier”
(Hodgkin and Huxley, 1952), “Ca21-activated K1 ” (Meech
and Standen, 1975), and “fast transient outward” (Connor
and Stevens, 1971) currents. The fast transient outward K1
currents (Ias) are identified in invertebrates (Hagiwara et al.,
1961; Connor and Stevens, 1971; Neher, 1971) as well as in
vertebrate neurons (Adams et al., 1982; Gustafsson et al.,
1982). Furthermore, frog myelinated axons have at least
three different and distinct types of K1 channels, which are
characterized by the two fast and slow K1 conductances;
4-AP blocks the fast conductances and TEA blocks the fast
and slow conductances (Dubois, 1981; Grissmer, 1986). In
mammalian myelinated axons, the two pharmacologically
different types of K1 channels are also identified in the
peripheral (Baker et al., 1987; Kocsis et al., 1987) and central
(Kocsis et al., 1986; Gordon et al., 1988; Thorn et al., 1991)
nervous systems: one is sensitive to 4-AP, but the other is
sensitive to TEA. There are functional differences between
4-AP- and TEA-sensitive K1 channels in the myelinated axons of the rat sciatic nerve fibers because the 4-AP-sensitive
K1 channels are related to action potential repolarization,
but the TEA-sensitive K1 currents cause the afterhyperpolization after repetitive activity (Kocsis et al., 1987). Although
the application of 4-AP results in the broad spike of action
potentials (Kocsis et al., 1987; Thorn et al., 1991; Poulter and
Padjen, 1995), such an effect could not be confirmed in this
study because we were measuring extracellular action potentials. In addition, we found that the administration of 4-AP
increased the discharge of SARs during deflation in a dosedependent manner and caused a pressor effect reported by
1999
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mature rats, TEA (10 mM) application alone has little effect
on the wave form of the compound action potential at any age
but blocks 4-AP-induced postspike positivity (hyperpolarization; Eng et al., 1988). The results indicate that the slow K1
channels sensitive to TEA are not responsible for repolarization after single action potentials. Indeed, in this study, pretreatment with TEA (2.0 and 6.0 mg/kg) that did not significantly alter the basal activity of SARs had no effect on
CO2-induced SAR inhibition. Furthermore, there is evidence
that TEA only slightly reduces Ias. TEA-sensitive K1 channels might, therefore, not play a significant role in the inhibitory response of SARs to CO2 inhalation, but the possibility
that TEA did not reach the SAR endings at concentrations
sufficient to block K1 channels cannot be completely excluded.
TEA blocks voltage-dependent K1 conductances as well as
some Ca21-dependent K1 conductances (Rudy, 1988). In particular, the large conductance Ca21-activated K1 channels
(Miller et al., 1985) are known to be blocked by TEA (Blatz
and Magleby, 1987), and this blocking effect acts more effectively at the external membrane surface than at the internal
membrane surface (Adams et al., 1982; Latorre et al., 1982).
In this study, however, the administration of TEA at a higher
dose (6 mg/kg) did not significantly modify the inhibitory
responses of SAR activity to CO2 inhalation, so the large
conductance Ca21-activated K1 channels sensitive to TEA
might not contribute greatly to the mechanism of CO2-induced SAR inhibition. Further studies are necessary to determine whether the inhibitory effect of CO2 on SAR activity
is related to the functioning of the stretch-activated channels
on the receptor endings.
In conclusion, the inhibitory responses of SAR activity to
CO2 inhalation were blocked by pretreatment with 4-AP, but
TEA treatment did not significantly alter the CO2-induced
inhibition of SAR activity. The results suggest that inhibition
of SARs by CO2 inhalation may be mediated by the stimulating action of 4-AP-sensitive K1 currents in the nerve terminals of SARs.
CO2-Induced SAR Inhibition and K1 Channel