The effect of the general anesthetic halothane on the activity of the rat skeletal muscle Ca2+-activated K+ channel in planar lipid bilayers was investigated. Halothane concentrations in the clinical range (1.0-0.2 mM) alter the regulation of the channel by both Ca2+ and membrane potential. At Ca2+ concentrations between 10 and 250 µM and membrane potentials between 0 and -30 mV, halothane significantly decreases the open state probability without changing the channel conductance. The results demonstrate that halothane can act directly on the Ca2+-activated K+ channel or its lipid environment to alter the channel Bating kinetics

Beeler, Troy

Gable, Kenneth

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Uniformed Services University of the Health Sciences U.S. Department of Defense 1993 Effect of the general anesthetic halothane on the activity of the transverse tubule Ca2+-activated K+ channel Troy Beeler Uniformed Services University of the Health Sciences Kenneth Gable Uniformed Services University of the Health Sciences Follow this and additional works at: https://digitalcommons.unl.edu/usuhs  Part of the Medicine and Health Sciences Commons Beeler, Troy and Gable, Kenneth, "Effect of the general anesthetic halothane on the activity of the transverse tubule Ca2+-activated K+ channel" (1993). Uniformed Services University of the Health Sciences. 44. https://digitalcommons.unl.edu/usuhs/44 This Article is brought to you for free and open access by the U.S. Department of Defense at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Uniformed Services University of the Health Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Volume 331, number 3, 207-210 FEBS 13012 0 1993 Federation of European Biochemical Societies 00145793/93/%6.00 October 1993 Effect of the general anesthetic halothane on the activity of the transverse tubule Ca2+-activated K’ channel Troy Beeler* and Kenneth Gable Department of Biochemistry, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20014, USA Received 8 March 1993; revised version received 21 July 1993 The effect of the general anesthetic halothane on the activity of the rat skeletal muscle Ca”-activated K+ channel in planar lipid bilayers was investigated. Halothane concentrations in the clinical range (1.0-0.2 mM) alter the regulation of the channel by both Ca2’ and membrane potential. At Ca*’ concentrations between 10 and 250 ,uM and membrane potentials between 0 and -30 mV, halothane significantly decreases the open state probability without changing the channel conductance. The results demonstrate that halothane can act directly on the Ca*‘-activated K’ channel or its lipid environment to alter the channel Bating kinetics. Ca2+-activated K+ channel; General anesthetic; Halothane; Transverse tubule 1. INTRODUCTION The mechanism by which general anesthetics alter excitable membranes to cause loss of consciousness has not been established. Because the relative potency of general anesthetics is proportional to their ability to partition into oil or into lipid bilayers [1,2], it has been proposed that anesthetics alter signal transmission by excitable membranes by altering membrane fluidity or some other physical property of the membrane. Tas et al. [3] and Pancrazio et al. [4] demonstrated that ha- lothane inhibits the Ca’+-activated K+ channel in rat glioma C6 cells and adrenal ch.romatXn cells respec- tively. Here we describe the effects of halothane on the rat skeletal muscle Ca2+-activated K’ channel inserted into a synthetic planar lipid bilayer. L&I EPC7 Patch clamp (Medical Systems Corp., Greenvale, NY). The data was collected at a rate of 20,000/s. The current distribution was fitted to the Gaussian distribution function to determine the mean current, standard deviation, and the probability of being at each con- ductance level. In the analysis of the gating kinetics, the transfer from one conductance level to another was detined as a change in the current within one standard deviation of the new conductance level for at least 0.25 ms. The membrane potential refers to the trans cham- ber relative to the grounded cis chamber. Negative currents are in the same direction as positive charges moving from the cis to tran.r cham- ber. Halothane and Ca” were applied to the bilayer by perfusion of the cb chamber with solutions containing the desired halothane and Ca2’ concentrations. The chambers were covered with Para8hn to minimize halothane evaporation. 3. RESULTS 2. MATERIALS AND METHODS Transverse tubule vesicles were prepared from rat skeletal muscle by a modification of the procedure developed by Barchi and co- workers [5] as previously described [6]. The vesicles were stored in 0.4 M sucrose, 10 mM histidine @H 7.0) at -70°C. Planar lipid bilayers were formed using the procedure and apparatus described by Meissner [A. A 40 mg/ml phospholipid stock solution containing phosphatityle- thanolamine, phosphatitylcholine, phosphatitylserine (purified from bovine brain by Avanti Polar-lipids Inc.) at a ratio of 1:0.75:0.25 (wt:wt:wt) in de-cane was used to form the lipid bilayers. Ca*‘-activated K’ channels were incorporated into the planar lipid bilayer by fusion of transverse tubule vesicles (l-5 pg protein/ml) as described by La- torre and co-workers [8]. The cis chamber (chamber to which the vesicles are added) contained 200 mM KCI, 1.0 mM CaCl,, 10 mM Tris-HEPES, pH 7.0, while the trurt~ chamber contained 50 mM KCl, 10 mM Tris-HEPES, pH 7.0. The membrane potential of the bilayer was clamped to the desired voltage and the current measured with a The skeletal muscle transverse tubule Ca2’-activated K’ channel was incorporated into an artificial planar lipid bilayer membrane by the fusion of transverse tu- bule vesicles using the method developed by Latorre et al. [8]. Because transverse tubule vesicles are cytoplas- mic-side out, the Ca’+-activated K+ channel is expected to be orientated so that the cytoplasmic side of the channel faces the cis chamber. *Corresponding author. Fax: (1) (301) 295-3512. Fig. 1A shows a typical current recording of a single Ca’+-activated K+ channel. The Ca2+ concentration in the cis chamber was 50 PM and the membrane potential was set at -30 mV, -20 mV, or -10 mV (trans side negative). The cis chamber contained 0.2 mM, 0.5 mM or no halothane. These halothane concentrations are within the clinical range [9,10]. Halothane at 0.2 mM and 0.5 mM corresponds to solutions equilibrated with about 0.4% and 1% atm. halothane respectively. The fluctuation in the current recording between two con- ductance levels (o=open, c=closed) is the result of the fluctuation of the Ca’+-activated K’ channel between Published by Elsevier Science Publishers B. K 207 Volume 331, number 3 FEBS LETTERS October 1993 A c 0 C 0 C 0 I : (I.,; IllM IlillOl,lliLI1(! C 0 (: 0 (: 0 -20 mV B -1OmV t -1OmV F -1OmV , No Halothane 0.2 mM Halothdne 0.5 mM Hatothane CURRENT (pAI CURRENT (PA) CURRENT (PA) E x 3 k s 0” 1.00 0.50 0.00 C I4 No Halothane 0.2 mM Halothane 0.5 mM Halothane -0, f? 04 80, SECONDS SECONDS SECONDS 7 Q) Q 2 a -30 -20 -10 0 -20 -10 0 Hold Potential (mV) 208 Volume 33 1, number 3 October 1993 A Control 1mM Halot hane z 8 FEBS LETTERS 10 pM Ca2+ 1 mM Ca2+ _ 1.00 0.50 0.00 0.00 0.50 [Calcium1 (mM) 1.00 Fig. 2. Effect of the Ca*+ concentration on the activity of the Ca2’-activated K+ channel in the presence and absence of 1.0 mM halothane. (A) Current recording of three Ca2+-activated K+ channels at 0 mV in the presence of 0.01 mM Ca” (left column) or 1 .O mM Ca2’ (right column) with (bottom row) or without (top row) 1 .O mM halothane. Other experimental conditions were the same as Fig. 1. (B) The open probability as a function of the Ca*’ concentration in the absence (0) and in the presence (w) of 1 .O mM halothane. the open and closed conformational states. As shown by Latorre and co-workers [8], the open state probability increases as the membrane becomes more polarized (truns side negative). Halothane reduces the time that the channel stays in the open state. The relationship between the membrane potential and the open state probability at different halothane concentrations is shown in Fig. 1B. Halothane reduces the open probability at all potentials measured, but the percent decrease is greater at the lower polarizations. Similar results were obtained in two other experiments. The channel conductance of the open state, 330 pS, (the slope of the line made by plotting the channel cur- rent vs. membrane potential) is not significantly altered by halothane. The effect of 1 mM or less halothane on the gating kinetics was reversed by perfusion of the cis chamber with 10 volumes of a halothane-free solution, however irreversible alterations in the gating kinetics were ob- served after treating the membrane with halothane above 5 mM (data not shown). The rate constants for the open to closed state transi- t Fig. 1. Effect of halothane on the activity of the Ca’+-activated K+ channel. (A) Single channel current recordings of the Ca*+-activated channel at -30 mV (top row), -20 mV (middle row), and -10 mV (bottom row) in the presence of 0 (left column), 0.2 mM (center column), or 0.5 mM (right column) halothane. The cis chamber contained 200 mM KCl, 10 mM Tris-HEPES (PH 7.2), and 50 ,uM Ca*’ and trans chamber contained 50 mM KCl, 10 mM Tris-HEPES (pH 7.2). Current histograms for the channel at -10 mV membrane potential are shown below the current traces along with the Gaussian distribution fit (bold line). (B) Open probability as a function of the membrane potential for the control without halothane (o), with 0.2 mM halothane (m), or 0.5 mM halothane (A). (C) Probability distribution curves for the closed (no conductance) state (C > 0) and the open state (0 > C) of the channel at -30 mV membrane potential. The data is indicated by the bold line and the double exponential fit is shown by the thin line. The close. state probability distribution curves were fitted to the the following equations: ‘no halothane’, P(t) = 0.57eva”+0.43em2”‘; ‘0.2 mM halothane’, P(t) = 0.57e-a”+0.43e-2”‘; 0.2 mM halothane P(t = 0.57e-48”+0.43e- et; 0.5 mM halothane P(t) = 0.56e-‘5”+0.44F’s’. The open state probability distribution curves were fit to the the following equations: ‘no halothane’, P(r) = 0.52e-“‘+0.48e-‘5’; ‘0.2 mM halothane’, P(t)=0.53e-‘57’+0.47e-2’r; ‘ 0.5 mM halothane’, P(t)=0.55e-273’+0.45e-571. (D) The effect of membrane potential on the rate constant, k2c+o, for the closed to open transition without halothane (o), or with 0.2 mM halothane (H) or 0.5 mM halothane (A). (E) The effect of membrane potential on the rate constant, k20+c, for the open to closed transition without halothane (o), or with 0.2 mM halothane @) or 0.5 mM halothane (A). 209 Volume 331, number 3 FEBSLE-ITERS October 1993 tion (ko+J and the closed to open state transition (kc+o) were obtained by analyzing the curve obtained by potting the probability (P) of the open or closed state lasting for time t against t. The probability distribution curves for both O+C and C+O transitions are de- scribed by a double exponential equation (P(t) = A,e-kl’+A,e-k2’) indicating the existence of at least 2 open states and 2 closed states with different transition rate constants (klo+c and k20_,c for the O+C transi- tion, klC+O and k2c+0 for the C+O transition) (Fig. 1C). A, is the fraction of transitions that occur with the rate constant k,, and A, is the fraction of transitions that occur with the rate constant of k2. A, and A, are both approximately 0.5 for both O+C and C-+0 transitions. The higher rate constants for the O+C (k,,,,) and C+O G&o) t ransitions are above 200 s-l; too rapid to determined accurately under these experimental con- ditions. The lower rate constants for the O+C (k20+c) and the C+O (k 2c4) transitions in the presence of 50 PM Ca2’ ranged from 7 s-l to 200 s-’ depending on the membrane potential and the halothane concentration (Fig. 1D and E). The rate constant, k2C+0, for the C+O transition is dependent on the membrane polarization in the absence of halothane, but following the addition of 0.5 mM halothane, the transition rate was not signif- icantly affected by the membrane potential. Halothane increases the open to closed rate constant, k20_,c, but doesn’t significantly alter the effect of membrane poten- tial on the k20_+c. The effect of halothane on the channel activity at different Ca2’ concentrations is shown in Fig. 2. In this experiment, the planar lipid bilayer contained three Ca2+-activated K’ channels. The membrane potential was held at 0 mV potential and the Ca2’ concentration in the cis chamber was varied between 0.01 and 1 .O mM. As shown by Latorre and co-workers [8], the open state probability increases as the Ca2+ concentration in the cis chamber is raised. At 1 mM Ca2’, the probability of the open state with or without 1 mM halothane is about 0.8. However, at 10 PM Ca2’, 1 mM halothane caused a 96% decrease in the open state probability. The Ca2’ concentration that gives a 50% open state probability was 10 ,uM in the absence of halothane and 130 PM in the presence of 1 mM halothane. Similar results were observed when the membrane potential was held at - 10, -20 and -30 mV. These results were also obtained in three other experiments. 4. DISCUSSION The main conclusion of this study is that halothane alters the gating kinetics of the skeletal muscle Ca2+- activated K’ at clinically relevant concentrations. Ha- lothane increases the open to closed state transition rate and decreases the closed to open state transition rate by destabilizing the open state and stabilizing the closed state. Perhaps the closed state contains more exposed hydrophobic surfaces than the open state, and ha- lothane interacts with these surfaces to stabilize the closed state conformation. In the absence of halothane, the closed to open state transition rate is strongly dependent on the membrane potential, but in the presence of 0.5 mM halothane, this transition rate is not affected by the membrane poten- tial. The interaction of the voltage sensor domain of the channel with halothane may block its voltage-sensing action. On the other hand, the sensitivity of the open to closed state transition rate constant to the membrane potential is not altered by halothane suggesting the closed to open state transition and the open to closed state transition may not share a common voltage sen- sor. The role of halothane mediated inhibition of the Ca2+-activated K+ channel in the physiological response to halothane is not known. Inhibition of a K’ channel would seem to increase the excitability of membranes by decreasing the hyperpolarization which accompanies the opening of the channel. However increasing the ex- citability of some cells may lead to the decrease activity of other cells that are linked through a negative feed- back. Acknowledgements: This work was supported by Grants GM 29300 and GM 46495 from the National Institutes of Health and Grant CO7lBK the Uniformed University of the Health Sciences. The opin- ions or assertions contained herein are private ones of the authors and are not to be construed as official or reflecting the views of the Depart- ment of Defense or the Uniformed Services University of the Health Sciences. REFERENCES [l] Meyer, H. (1899) Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmak. 42, 109-l 18. [2] Overton, E. (1896) Z. Phys. Chem. 22, 189-209. [3] Tas, P.W.L., Kress, H.-G. and Koschel, K. (1989) Biochim. Bi- opys. Acta 983, 264-268. [4] Pancrazio, J.J., Park, W.K. and Lynch, C. (1993) Mol. Pharmo- col. 43, 783-794. [5] Barchi, R.L., Wiegele, J.B., Chalikian, D.M. and Murphy, L.E. (1979) Biochim. Biophys. Acta 550, 59-76. [6] Beeler, T.J., Wang, T., Gable, K. and Lee, S. (1985) Arch. Bio- them. Biophys. 243, 644654. [7] Smith, J.S., Coronado, R. and Meissner, G. (1988) Methods Enzymol. 157,480489 [8] Latorre, R., Vergara, C. and Hidalgo, C. (1982) Proc. Natl. Acad. Sci. USA 79, 805-809. [9] Steward, A., Allott, P.R., Cowles, A.L. and Mapleson, W.W. (1973) Br. J. Anesthesiol. 45, 282-293. [lo] Larson, M.D., Eger, E.I. and Severinghaus, J.W. (1962) Anesthe- siology 23, 349-355. 210 

Effect of the general anesthetic halothane on the activity of the transverse tubule Ca\u3csup\u3e2+\u3c/sup\u3e-activated K\u3csup\u3e+\u3c/sup\u3e channel

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Uniformed Services University of the Health 
Sciences U.S. Department of Defense 
1993 
Effect of the general anesthetic halothane on the activity of the 
transverse tubule Ca2+-activated K+ channel 
Troy Beeler 
Uniformed Services University of the Health Sciences 
Kenneth Gable 
Uniformed Services University of the Health Sciences 
Follow this and additional works at: https://digitalcommons.unl.edu/usuhs 
 Part of the Medicine and Health Sciences Commons 
Beeler, Troy and Gable, Kenneth, "Effect of the general anesthetic halothane on the activity of the 
transverse tubule Ca2+-activated K+ channel" (1993). Uniformed Services University of the Health 
Sciences. 44. 
https://digitalcommons.unl.edu/usuhs/44 
This Article is brought to you for free and open access by the U.S. Department of Defense at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Uniformed Services 
University of the Health Sciences by an authorized administrator of DigitalCommons@University of Nebraska - 
Lincoln. 
Volume 331, number 3, 207-210 FEBS 13012 
0 1993 Federation of European Biochemical Societies 00145793/93/%6.00 
October 1993 
Effect of the general anesthetic halothane on the activity of the transverse 
tubule Ca2+-activated K’ channel 
Troy Beeler* and Kenneth Gable 
Department of Biochemistry, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20014, 
USA 
Received 8 March 1993; revised version received 21 July 1993 
The effect of the general anesthetic halothane on the activity of the rat skeletal muscle Ca”-activated K+ channel in planar lipid bilayers was 
investigated. Halothane concentrations in the clinical range (1.0-0.2 mM) alter the regulation of the channel by both Ca2’ and membrane potential. 
At Ca*’ concentrations between 10 and 250 ,uM and membrane potentials between 0 and -30 mV, halothane significantly decreases the open state 
probability without changing the channel conductance. The results demonstrate that halothane can act directly on the Ca*‘-activated K’ channel 
or its lipid environment to alter the channel Bating kinetics. 
Ca2+-activated K+ channel; General anesthetic; Halothane; Transverse tubule 
1. INTRODUCTION 
The mechanism by which general anesthetics alter 
excitable membranes to cause loss of consciousness has 
not been established. Because the relative potency of 
general anesthetics is proportional to their ability to 
partition into oil or into lipid bilayers [1,2], it has been 
proposed that anesthetics alter signal transmission by 
excitable membranes by altering membrane fluidity or 
some other physical property of the membrane. Tas et 
al. [3] and Pancrazio et al. [4] demonstrated that ha- 
lothane inhibits the Ca’+-activated K+ channel in rat 
glioma C6 cells and adrenal ch.romatXn cells respec- 
tively. Here we describe the effects of halothane on the 
rat skeletal muscle Ca2+-activated K’ channel inserted 
into a synthetic planar lipid bilayer. 
L&I EPC7 Patch clamp (Medical Systems Corp., Greenvale, NY). 
The data was collected at a rate of 20,000/s. The current distribution 
was fitted to the Gaussian distribution function to determine the mean 
current, standard deviation, and the probability of being at each con- 
ductance level. In the analysis of the gating kinetics, the transfer from 
one conductance level to another was detined as a change in the 
current within one standard deviation of the new conductance level 
for at least 0.25 ms. The membrane potential refers to the trans cham- 
ber relative to the grounded cis chamber. Negative currents are in the 
same direction as positive charges moving from the cis to tran.r cham- 
ber. Halothane and Ca” were applied to the bilayer by perfusion of 
the cb chamber with solutions containing the desired halothane and 
Ca2’ concentrations. The chambers were covered with Para8hn to 
minimize halothane evaporation. 
3. RESULTS 
2. MATERIALS AND METHODS 
Transverse tubule vesicles were prepared from rat skeletal muscle 
by a modification of the procedure developed by Barchi and co- 
workers [5] as previously described [6]. The vesicles were stored in 0.4 
M sucrose, 10 mM histidine @H 7.0) at -70°C. Planar lipid bilayers 
were formed using the procedure and apparatus described by Meissner 
[A. A 40 mg/ml phospholipid stock solution containing phosphatityle- 
thanolamine, phosphatitylcholine, phosphatitylserine (purified from 
bovine brain by Avanti Polar-lipids Inc.) at a ratio of 1:0.75:0.25 
(wt:wt:wt) in de-cane was used to form the lipid bilayers. Ca*‘-activated 
K’ channels were incorporated into the planar lipid bilayer by fusion 
of transverse tubule vesicles (l-5 pg protein/ml) as described by La- 
torre and co-workers [8]. The cis chamber (chamber to which the 
vesicles are added) contained 200 mM KCI, 1.0 mM CaCl,, 10 mM 
Tris-HEPES, pH 7.0, while the trurt~ chamber contained 50 mM KCl, 
10 mM Tris-HEPES, pH 7.0. The membrane potential of the bilayer 
was clamped to the desired voltage and the current measured with a 
The skeletal muscle transverse tubule Ca2’-activated 
K’ channel was incorporated into an artificial planar 
lipid bilayer membrane by the fusion of transverse tu- 
bule vesicles using the method developed by Latorre et 
al. [8]. Because transverse tubule vesicles are cytoplas- 
mic-side out, the Ca’+-activated K+ channel is expected 
to be orientated so that the cytoplasmic side of the 
channel faces the cis chamber. 
*Corresponding author. Fax: (1) (301) 295-3512. 
Fig. 1A shows a typical current recording of a single 
Ca’+-activated K+ channel. The Ca2+ concentration in 
the cis chamber was 50 PM and the membrane potential 
was set at -30 mV, -20 mV, or -10 mV (trans side 
negative). The cis chamber contained 0.2 mM, 0.5 mM 
or no halothane. These halothane concentrations are 
within the clinical range [9,10]. Halothane at 0.2 mM 
and 0.5 mM corresponds to solutions equilibrated with 
about 0.4% and 1% atm. halothane respectively. The 
fluctuation in the current recording between two con- 
ductance levels (o=open, c=closed) is the result of the 
fluctuation of the Ca’+-activated K’ channel between 
Published by Elsevier Science Publishers B. K 207 
Volume 331, number 3 FEBS LETTERS October 1993 
A 
c 
0 
C 
0 
C 
0 
I : 
(I.,; IllM IlillOl,lliLI1(! 
C 
0 
(: 
0 
(: 
0 
-20 mV 
B 
-1OmV 
t 
-1OmV 
F 
-1OmV 
, No Halothane 0.2 mM Halothdne 0.5 mM Hatothane 
CURRENT (pAI CURRENT (PA) CURRENT (PA) 
E 
x 
3 
k 
s 
0” 
1.00 
0.50 
0.00 
C 
I4 No Halothane 0.2 mM Halothane 0.5 mM Halothane 
-0, 
f? 04 
80, 
SECONDS SECONDS SECONDS 7 
Q) 
Q 
2 a 
-30 -20 -10 0 -20 -10 0 
Hold Potential (mV) 
208 
Volume 33 1, number 3 October 1993 
A 
Control 
1mM 
Halot hane 
z 
8 
FEBS LETTERS 
10 pM Ca2+ 1 mM Ca2+ _ 
1.00 
0.50 
0.00 
0.00 0.50 
[Calcium1 (mM) 
1.00 
Fig. 2. Effect of the Ca*+ concentration on the activity of the Ca2’-activated K+ channel in the presence and absence of 1.0 mM halothane. (A) 
Current recording of three Ca2+-activated K+ channels at 0 mV in the presence of 0.01 mM Ca” (left column) or 1 .O mM Ca2’ (right column) with 
(bottom row) or without (top row) 1 .O mM halothane. Other experimental conditions were the same as Fig. 1. (B) The open probability as a function 
of the Ca*’ concentration in the absence (0) and in the presence (w) of 1 .O mM halothane. 
the open and closed conformational states. As shown by 
Latorre and co-workers [8], the open state probability 
increases as the membrane becomes more polarized 
(truns side negative). Halothane reduces the time that 
the channel stays in the open state. 
The relationship between the membrane potential 
and the open state probability at different halothane 
concentrations is shown in Fig. 1B. Halothane reduces 
the open probability at all potentials measured, but the 
percent decrease is greater at the lower polarizations. 
Similar results were obtained in two other experiments. 
The channel conductance of the open state, 330 pS, 
(the slope of the line made by plotting the channel cur- 
rent vs. membrane potential) is not significantly altered 
by halothane. 
The effect of 1 mM or less halothane on the gating 
kinetics was reversed by perfusion of the cis chamber 
with 10 volumes of a halothane-free solution, however 
irreversible alterations in the gating kinetics were ob- 
served after treating the membrane with halothane 
above 5 mM (data not shown). 
The rate constants for the open to closed state transi- 
t 
Fig. 1. Effect of halothane on the activity of the Ca’+-activated K+ channel. (A) Single channel current recordings of the Ca*+-activated channel 
at -30 mV (top row), -20 mV (middle row), and -10 mV (bottom row) in the presence of 0 (left column), 0.2 mM (center column), or 0.5 mM 
(right column) halothane. The cis chamber contained 200 mM KCl, 10 mM Tris-HEPES (PH 7.2), and 50 ,uM Ca*’ and trans chamber contained 
50 mM KCl, 10 mM Tris-HEPES (pH 7.2). Current histograms for the channel at -10 mV membrane potential are shown below the current traces 
along with the Gaussian distribution fit (bold line). (B) Open probability as a function of the membrane potential for the control without halothane 
(o), with 0.2 mM halothane (m), or 0.5 mM halothane (A). (C) Probability distribution curves for the closed (no conductance) state (C > 0) and 
the open state (0 > C) of the channel at -30 mV membrane potential. The data is indicated by the bold line and the double exponential fit is shown 
by the thin line. The close. state probability distribution curves were fitted to the the following equations: ‘no halothane’, P(t) = 0.57eva”+0.43em2”‘; 
‘0.2 mM halothane’, P(t) = 0.57e-a”+0.43e-2”‘; 0.2 mM halothane P(t = 0.57e-48”+0.43e- et; 0.5 mM halothane P(t) = 0.56e-‘5”+0.44F’s’. The 
open state probability distribution curves were fit to the the following equations: ‘no halothane’, P(r) = 0.52e-“‘+0.48e-‘5’; ‘0.2 mM halothane’, 
P(t)=0.53e-‘57’+0.47e-2’r; ‘ 0.5 mM halothane’, P(t)=0.55e-273’+0.45e-571. (D) The effect of membrane potential on the rate constant, k2c+o, for 
the closed to open transition without halothane (o), or with 0.2 mM halothane (H) or 0.5 mM halothane (A). (E) The effect of membrane potential 
on the rate constant, k20+c, for the open to closed transition without halothane (o), or with 0.2 mM halothane @) or 0.5 mM halothane (A). 
209 
Volume 331, number 3 FEBSLE-ITERS October 1993 
tion (ko+J and the closed to open state transition 
(kc+o) were obtained by analyzing the curve obtained 
by potting the probability (P) of the open or closed state 
lasting for time t against t. The probability distribution 
curves for both O+C and C+O transitions are de- 
scribed by a double exponential equation (P(t) = 
A,e-kl’+A,e-k2’) indicating the existence of at least 2 
open states and 2 closed states with different transition 
rate constants (klo+c and k20_,c for the O+C transi- 
tion, klC+O and k2c+0 for the C+O transition) (Fig. 
1C). A, is the fraction of transitions that occur with the 
rate constant k,, and A, is the fraction of transitions that 
occur with the rate constant of k2. A, and A, are both 
approximately 0.5 for both O+C and C-+0 transitions. 
The higher rate constants for the O+C (k,,,,) and 
C+O G&o) t ransitions are above 200 s-l; too rapid 
to determined accurately under these experimental con- 
ditions. The lower rate constants for the O+C (k20+c) 
and the C+O (k 2c4) transitions in the presence of 50 
PM Ca2’ ranged from 7 s-l to 200 s-’ depending on the 
membrane potential and the halothane concentration 
(Fig. 1D and E). The rate constant, k2C+0, for the C+O 
transition is dependent on the membrane polarization 
in the absence of halothane, but following the addition 
of 0.5 mM halothane, the transition rate was not signif- 
icantly affected by the membrane potential. Halothane 
increases the open to closed rate constant, k20_,c, but 
doesn’t significantly alter the effect of membrane poten- 
tial on the k20_+c. 
The effect of halothane on the channel activity at 
different Ca2’ concentrations is shown in Fig. 2. In this 
experiment, the planar lipid bilayer contained three 
Ca2+-activated K’ channels. The membrane potential 
was held at 0 mV potential and the Ca2’ concentration 
in the cis chamber was varied between 0.01 and 1 .O mM. 
As shown by Latorre and co-workers [8], the open state 
probability increases as the Ca2+ concentration in the cis 
chamber is raised. At 1 mM Ca2’, the probability of the 
open state with or without 1 mM halothane is about 0.8. 
However, at 10 PM Ca2’, 1 mM halothane caused a 
96% decrease in the open state probability. The Ca2’ 
concentration that gives a 50% open state probability 
was 10 ,uM in the absence of halothane and 130 PM in 
the presence of 1 mM halothane. Similar results were 
observed when the membrane potential was held at - 10, 
-20 and -30 mV. These results were also obtained in 
three other experiments. 
4. DISCUSSION 
The main conclusion of this study is that halothane 
alters the gating kinetics of the skeletal muscle Ca2+- 
activated K’ at clinically relevant concentrations. Ha- 
lothane increases the open to closed state transition rate 
and decreases the closed to open state transition rate by 
destabilizing the open state and stabilizing the closed 
state. Perhaps the closed state contains more exposed 
hydrophobic surfaces than the open state, and ha- 
lothane interacts with these surfaces to stabilize the 
closed state conformation. 
In the absence of halothane, the closed to open state 
transition rate is strongly dependent on the membrane 
potential, but in the presence of 0.5 mM halothane, this 
transition rate is not affected by the membrane poten- 
tial. The interaction of the voltage sensor domain of the 
channel with halothane may block its voltage-sensing 
action. On the other hand, the sensitivity of the open to 
closed state transition rate constant to the membrane 
potential is not altered by halothane suggesting the 
closed to open state transition and the open to closed 
state transition may not share a common voltage sen- 
sor. 
The role of halothane mediated inhibition of the 
Ca2+-activated K+ channel in the physiological response 
to halothane is not known. Inhibition of a K’ channel 
would seem to increase the excitability of membranes by 
decreasing the hyperpolarization which accompanies 
the opening of the channel. However increasing the ex- 
citability of some cells may lead to the decrease activity 
of other cells that are linked through a negative feed- 
back. 
Acknowledgements: This work was supported by Grants GM 29300 
and GM 46495 from the National Institutes of Health and Grant 
CO7lBK the Uniformed University of the Health Sciences. The opin- 
ions or assertions contained herein are private ones of the authors and 
are not to be construed as official or reflecting the views of the Depart- 
ment of Defense or the Uniformed Services University of the Health 
Sciences. 
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Effect of the general anesthetic halothane on the activity of the transverse tubule Ca\u3csup\u3e2+\u3c/sup\u3e-activated K\u3csup\u3e+\u3c/sup\u3e channel

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