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Inhibition of potassium currents by the antiarrhythmic drug E4031 in rat taste receptor cells
ELSEVIER
Neuroscience Letters 204 (1996) 149-152
NEUROSClENC[ I[IT[IIS
Inhibition of potassium currents by the antiarrhythmic drug E4031 in rat taste receptor cells Xiao-Dong Sun, M. Scott Herness* Indiana University School of Medicine, Center for Medical Education, Ball State University, Muncie, IN 47306, USA Received 26 October 1995; revised version received 28 December 1995; accepted 28 December 1995
Abstract
The effect of the class Ill antiarrhythmic agent E4031 was investigated on a non-cardiac preparation as a potential tool for studying potassium currents. Patch clamp recordings in the whole cell configuration were performed on dissociated rat taste cells. These cells possess a variety of potassium currents; they also conduct action potentials. Unlike its more specific action on a type of delayed rectifier channel in cardiac cells, three types of potassium currents were reversibly diminished in taste cells in the presence of E4031. These included transient, sustained, and inwardly-rectifying potassium currents. Activation properties were not altered but the inactivation curve was shifted to the left by approximately 10 mV. Inhibitions of whole cell currents were voltage-dependent, larger at depolarized potentials, but were never complete. E4031 significantly broadened the gustatory action potential and, at higher concentrations, inhibited spike height, suggesting an additional inhibitory effect on sodium channels that was evident in voltage-clamp records. We conclude that E4031 is an effective inhibitor of potassium currents in the micromolar range and that it likely acts at a conserved segment of the potassium channel.
Keywords: E4031 ; Taste; Gustation; Potassium channels; Transduction; Action potential; Class III antiarrthymic
The potassium channel blocker E4031 is a class III antiarrhythmic developed to prolong the cardiac action potential by inhibiting potassium currents without affecting sodium or calcium currents. In cardiac tissue, it has been demonstrated to block some but not all of the delayed rectifier potassium current [6,9,12,13]. Other cardiac potassium channels are unaffected, such as the ATPsensitive potassium current [16] or the cloned min K channel [15]. The use of E4031 has been circumscribed to cardiac preparations and little, if any, information on a non-cardiac preparation is available. Potassium currents have been characterized in taste cells from a variety of species [1-4,10,11,14]. There are multiple potassium components in taste cells that are similar in voltage-sensitivity and inactivation properties to neuronal potassium channels. These currents have multiple functions in taste cells. They are likely important in early phases of transduction schemes for sweet and bitter stimuli. Additionally, they contribute to various phases of * Corresponding author. Tel.: +1 317 2858759; fax: +1 317 2851059; e-mail: 00msherness @bsu.edu.
the resultant action potential; delayed rectifiers and transient potassium currents contribute to the repolarization phase, transient and calcium-activated currents contribute to the afterhyperpolarization (AHP); inward rectifiers contribute to the resting potential [4,11]. W e examined the effect of E4031 on potassium currents from taste cells to examine its utility as a potential probe for gustatory transduction mechanisms. Isolated taste receptor cells were obtained from male and female S p r a g u e - D a w l e y rats weighing between 2 0 0 280 g as previously described [7]. Animals were brought to a surgical level of anesthesia (ketamine 90.9 mg/ml, and acepromazine 0.909 mg/ml mixture, 0.9 ml/kg bw, Butler Laboratories, IN) prior to excision of circumvallate and foliate papillae. Individual cells were dissociated with Papain (14 U/ml, Boehringer-Mannheim, IN). Cells were voltage clamped using standard patch clamp techniques in the whole cell configuration. Electrode resistance was typically 3.5-5.5 Mr2 when filled with a pseudo-intracellular fluid (ICF) composed of (mM) 140 KCI, 2 MgC12, 1 CaC12, 11 E G T A , 10 HEPES, and 4 A T P (disodium salt), and measured in pseudo-extracell-
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 2 3 4 3 - 9
150
X.-D. Sun, M.S. Herness / Neuroscience Letters 204 (1996) 149-152
ular fluid (ECF) composed o f (mM) 126 NaCI, 5 KC1, 5 NaHEPES, 1.25 NaH2POa'H20, 10 glucose, 2 CaC12, and 2 MgCI2. Input resistance ranged from 6-15 G f l ; membrane capacitance was 3 - 6 pF; series resistance averaged 10 Mff2. Low pass filtering due to resistance-capacitance coupling was considered minimal. The average RC time constant was 37kts whereas the capacitive transient settling time averaged 131 kts [8]. Data were acquired with a high impedance amplifier (Axopatch 200A, A x o n Instruments, CA), a 486-based computer equipped with a 12 bit 330 kHz A/D converter (Digidata 1200, A x o n Instruments, CA), and commercially available software program (pClamp, v 6.0, Axon Instruments, CA). The membrane voltage was typically held at - 8 0 mV and a series o f depolarizing command potentials, in increments of 10 mV, were applied. M e m brane currents, in response to voltage perturbations, were acquired with a cut-off frequency of 5 kHz (at - 3 dB). Leak subtraction was employed except when recording inwardly rectifying currents. Solution exchange of the bathing solution was accomplished with a gravity-fed perfusion system of the recording chamber at approximately 3 ml/min. For experiments that utilized high potassium ECF, potassium was substituted for sodium on an equimolar basis. Potassium currents first activate close to - 1 0 mV, from a holding potential of - 8 0 mV, and increase in a linear fashion with more depolarized potentials reaching magnitudes o f several nanoamperes. This current is composed o f both sustained components and slowly inactivating components, with time constants in the range of 1-3 s. W h o l e cell potassium conductance becomes half maximal around 4 mV with an e-fold increase of conductance every 12 mV o f depolarization (data not shown). The addition of E4031 to the bathing medium resulted in a rapid and reversible diminution of these currents (Fig. 1A). The remaining current activated rapidly and showed no discernible inactivation, even after 1 s. Currents were inhibited at three tested concentrations of 10/~M, 100ktM, and 1 m M (Fig. 1B); IktM was without effect. Inhibition was measurable at every test potential but percent inhibition increased steadily with more depolarized potentials reaching (at +90 mV) 24.4%, 44.4%, and 69.5% inhibition for 10ktM, 100/~M, and 1 mM, respectively, suggesting a voltage-dependence of the block. Onset and offset kinetics were not noticeably altered. Inset shows dose-response curve for E4031 inhibition of outward currents produced by a test pulse of 90 mV. Half maximal inhibition was produced by approximately 200/~M. Each point is the mean (_+SE) of 4 - 6 cells. The inactivation kinetics of the remaining potassium current was altered in the presence of E4031 (data not shown). Inactivation was measured by clamping the membrane to various prepulse potentials for 10 s prior to a test pulse to 90 mV. Under control conditions half maximal inhibition occurred at - 8 . 7 _+ 3.8 mV (n = 4 )
A
....
1 m M E4031 it_ i i
! .........
~
n
1 nA
B
nA
~ 1.0[ +=
4-
~ 0.5
3.
=. Z 0.0
100 ms
ii iir
• . ]/ , 10 4
10 .4
1mME4031 ~ / / ~
~
10 .3
±
± ± ±
1-
[ E40311 (M) -100
-50
0
i
i
5O
100 mV
Fig. 1. E4031 inhibition of outward potassium currents in taste receptor cells. (A) Representative whole cell currents from a dissociated taste receptor cell are illustrated before, during, and after superfusion of the cell with a pseudoextracellular fluid containing 1 mM E4031. (B) Data from several cells are presented as mean (_+SE) in the current voltage plot for ECF (O) and for three concentrations of E4031, 10/~M (O), 100ktM (A), and 1 mM (A). E4031 did not alter the activation kinetics but did decrease the magnitude of the current in a dose-dependent manner. The concentration-response relationship is presented the inset for a test pulse to 90 mV. Half maximal inhibition occurred at approximately 200/.tM (dotted lines). with an e-fold decrease per 9.1_+0.8 mV. This agreed well with previous measurements [4]. In 1 0 0 / t M E4031, the inactivation curve was shifted to the left by approximately 10 mV without a change in slope. Half maximal inactivation occurred at - 1 8 . 7 _+ 2.4 mV (n = 4) with an efold decrease every 10.2 _ 0.5 mV. Taste receptor cells possess inwardly rectifying potassium currents that are approximately half maximal conductance at resting potentials, have conductances that vary with the square root of external potassium concentration, and display little inactivation. They are insensitive to 4-aminopyridine (4AP), mildly sensitive to tetraethylammonium (TEA), and are blocked by micromolar concentrations of cesium and barium. Sample currents recorded in 100 m M potassium ECF, in Fig. 2A, were obtained by holding the cell at its zero-current potential ( - 5 mV for this cell) and applying either depolarizing or hyperpolarizing command potentials in 10 mV increments from +30 mV to - 1 6 0 mV. These currents were inhibited
151
X.-D. Sun, M.S. Herness / Neuroscience Letters 204 (1996) 149-152
A
L
Control
---
L
I 0.5 ro~ 20 msec •
l mM E4031
Washout
B -160
-120
-8o
-40
_ _ ~
4
o
mV
~-
~Washout
_ _ l
I
I
1 m M the action potential was protracted and malformed (Fig. 3C). In voltage-clamped records (not shown) some inhibition of inward sodium currents occurred at this concentration. In contrast to its effects in cardiac tissue as a specific blocker of a component of the delayed rectifying potassium current [12,13], E4031 was an effective blocker of several types o f potassium conductance in taste receptor cells. These included sustained, transient, and inwardlyrectifying potassium currents. To our knowledge, effects of E4031 on multiple types o f potassium channels have not been previously reported. The plateau in the currentvoltage relationship of the E4031-insensitive current could be consistent with complete inhibition of a subset of potassium channels. Although the identity of this remaining current in Fig. 1 was not investigated, chloride currents likely contribute substantially (personal observation). The action of E4031 on inward rectifying current is of particular interest since these channels do not belong to the superfamily of ' s h a k e r ' - l i k e potassium channel but represent a novel gene family (e.g. [5]). They are, however, highly conserved in the P region thought to form the
-1.2.
nA
Fig. 2. E4031 produced partial inhibition of inwardly rectifying potassium currents. Representative current traces are illustrated in (A) recorded in a high potassium ECF. EA031 diminished these currents in a reversible manner though magnitude of inhibition was not uniform over all tested potentials. As evident in (B) greater inhibition was observed in the mid-region of hyperpolarizing membrane potentials. Data are mean _ SE for three cells.
20 mV
0A•E4031 5 msec
~_
~
Control
--
B by either 100/~M or 1 m M E4031; 1 0 p M E4031 was without effect. In the absence of blocker, currents increased in a relatively linear fashion at potentials that were negative to the holding potential (open circles) whereas in the presence o f drug the increase was nonlinear (solid circles) (Fig. 2B). The voltage-dependence of inhibition of E4031 displayed an inverted-U relationship when plotted as normalized inhibited current against membrane potential for both concentrations of E4031. M a x i m a l inhibition occurred at - 1 0 0 mV and at very hyperpolarized potentials inhibition was substantially reduced (data not shown). E4031 was also able to alter the gustatory action potential produced by injected current (25 p A for 0.1 ms) in current-clamp mode. At 10/zM the action potential became broader but without noticeable inhibition of the action potential height (APH; Fig. 3A). In voltage-clamp records clear inhibition of the outward currents, occurring within the first few milliseconds of activation, was evident (data not shown). As the concentration of E4031 increased its affect on the action potential became more pronounced. At 100/zM action potential duration (APD) was lengthened and A P H was reduced (Fig. 3B) and at
0 ~
I
C
l~tM
E4031
Control
~//--
":~'/"//i~/15 min Washout
o
/
*~
~
~"
Control
Fig. 3. The effect of E4031 on the action potential recorded from taste receptor cells. Action potentials were elicited by injection of 25 pA of current for 0.1 ms illustrated below each set of spikes. E4031 both broadened the width and, at higher concentration, truncated the height of the action potential. Current clamp records are presented for 10/~M (A), 100/~M (B), and 1 mM (C) concentrations from a single cell. The return of action potential shape after washout of E4031 is presented for the highest concentration (dotted line).
152
x.-D. Sun, M.S. Herness / Neuroscience Letters 204 (1996) 149-152
pore. This m a y suggest that, at least at high concentrations, E4031 acts at s o m e c o n s e r v e d site o f the pore, such as the selectivity filter, rather than at another site, such as the gate. T h o u g h r e d u c t i o n o f p o t a s s i u m currents by E4031 was e f f e c t i v e for all suprathreshold potentials voltaged e p e n d e n c e o f b l o c k was observed. This m a y h a v e s o m e i m p l i c a t i o n for its m e c h a n i s m . F o r outward currents b l o c k i n g e f f i c a c y increased with d e p o l a r i z i n g potentials and for inward currents it d e c r e a s e d with h y p e r p o l a r i z i n g potentials. T h i s suggests that the binding site o f E4031 m a y sense s o m e o f the electric field across the m e m b r a n e . Additionally, at very h y p e r p o l a r i z e d potentials inhibition o f inward currents was less effective, consistent with rel i e f o f b l o c k f r o m the internal side. E4031 had significant effects on the contour o f the gustatory action potential as has been o b s e r v e d with T E A [4] and forskolin (personal observation). Unanticipated was a truncation o f the spike height with 1 m M E4031. This s a m e c o n c e n t r a t i o n p r o d u c e d a d i m i n u t i o n o f inward s o d i u m current in v o l t a g e - c l a m p records, suggesting possible b l o c k i n g o f s o d i u m channels. A similar inhibition o f s o d i u m c h a n n e l s was o b s e r v e d for high concentrations o f forskolin (personal observation). In conclusion, unlike its effect in cardiac tissue, the antiarrhythmic drug E403 I, at m i c r o m o l a r concentrations, inhibits m o r e than one type of p o t a s s i u m c o n d u c t a n c e in taste r e c e p t o r cells. At the lowest tested c o n c e n t r a t i o n (10/.tM) outward potassium currents w e r e substantially inhibited without effect on inward p o t a s s i u m currents. This w o r k was supported by grant D C 0 0 4 0 1 f r o m the National Institute o f C o m m u n i c a t i v e and Sensory Disorders o f the N a t i o n a l Institutes o f Health. W e thank Eisai Laboratories Co., Ltd., T s u k u b a , Japan for their generous donation of E4031. [1] Avenet, P. and Lindemann, B., Patch-clamp study of isolated taste receptor cells of the frog, J. Membr. Biol., 97 (1987) 223-240.
[2] B6h6, P., DeSimone, J.A., Avenet, P. and Lindemann, B., Membrane currents in taste cells of the rat fungiform papilla: evidence for two types of Ca currents and inhibition of K currents by saccharin, J. Gen. Physiol., 96 (1990) 1061-1084. [3] Bigiani, A. and Roper, S., Identification of electrophysiologically distinct cell subpopulations in Necturus taste buds, J. Gen. Physiol., 102 (1993) 143-170. [4] Chen, Y. Sun, X.-D. and Herness, M.S., Characteristics of action potentials and their underlying outward currents in rat taste cells, J. Neurophysiol., 75 (2) (1996) 820-831. [5] Doupnik, C.A., Davidson, N. and Lester, H.A., The inward rectifier potassium channel family, Curr. Biol., 5 (1995) 268-277. [6] Follmer, C.H. and Colatsky, T.J., Block of delayed rectifier potassium current, IK, by flecainide and E4031 in cat ventricular myocytes, Circulation, 82 (1990) 289-293. [7] Herness, M.S., A dissociation procedure for mammalian taste cells, Neurosci. Lett., 106 (1989) 60-64. [8] Hemess, M.S. and Sun, X-.D., Voltage-dependent sodium currents recorded from dissociated rat taste cells, J. Membr. Biol., 146 (1995) 73-84. [9] Liu, Y., Taffet, S.M., Anumonwe, J.M. and Delmar, M., Characterization of an E4031-sensitive potassium current in quiescent AT-I cells, J. Cardiovasc. Electrophysiol., 5 (1990) 1017-1030. [10] Kinnamon, S.C., and Roper, S.D., Membrane properties of isolated mudpuppy taste cells, J. Gen. Physiol., 91 (1988) 351-371. [11] Roper, S.D. and McBride, D.W., Distribution of ion channels on taste cells and its relationship to chemosensory transduction, J. Membr. Biol., 109 (1989) 29-39. [12] Sanguinetti, M.C. and Jurkiewicz, N.K., Two components of cardiac delayed rectifier K+ current: differential sensitivity to block by class III antiarrhythmic agents, J. Gen. Physiol., 96 (1990) 195-215. [13] Sanguinetti, M.C. and Jurkiewicz, N.K., Delayed rectifier outward K+ current is composed of two currents in guinea pig atrial cells, Am. J. Physiol., 260 (1991) H393-H399. [14] Sugimoto, K. and Teeter, J.H., Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander, J. Gen. Physiol., 96 (1990) 809-834. [15] Varnum, M.D., Busch, A.E., Bond, C.T., Maylie, J. and Adelman, J.P., The min K channel underlies the cardiac potassium current /Ks and mediates species-specific responses to protein kinase C, Proc. Natl. Acad. Sci. USA, 90 (1993) 11528-11532. [16] Wu, B., Sato, T., Kiyoshe, T. and Arita, M., Blockade of 2,4nitrophenol-induced ATP-sensitive potassium current in guinea pig ventricular myocytes by class I antiarrhythmic drugs, Cardiovasc. Res., 26 (1992) 1095-1101.
Neuroscience Letters 204 (1996) 149-152
NEUROSClENC[ I[IT[IIS
Inhibition of potassium currents by the antiarrhythmic drug E4031 in rat taste receptor cells Xiao-Dong Sun, M. Scott Herness* Indiana University School of Medicine, Center for Medical Education, Ball State University, Muncie, IN 47306, USA Received 26 October 1995; revised version received 28 December 1995; accepted 28 December 1995
Abstract
The effect of the class Ill antiarrhythmic agent E4031 was investigated on a non-cardiac preparation as a potential tool for studying potassium currents. Patch clamp recordings in the whole cell configuration were performed on dissociated rat taste cells. These cells possess a variety of potassium currents; they also conduct action potentials. Unlike its more specific action on a type of delayed rectifier channel in cardiac cells, three types of potassium currents were reversibly diminished in taste cells in the presence of E4031. These included transient, sustained, and inwardly-rectifying potassium currents. Activation properties were not altered but the inactivation curve was shifted to the left by approximately 10 mV. Inhibitions of whole cell currents were voltage-dependent, larger at depolarized potentials, but were never complete. E4031 significantly broadened the gustatory action potential and, at higher concentrations, inhibited spike height, suggesting an additional inhibitory effect on sodium channels that was evident in voltage-clamp records. We conclude that E4031 is an effective inhibitor of potassium currents in the micromolar range and that it likely acts at a conserved segment of the potassium channel.
Keywords: E4031 ; Taste; Gustation; Potassium channels; Transduction; Action potential; Class III antiarrthymic
The potassium channel blocker E4031 is a class III antiarrhythmic developed to prolong the cardiac action potential by inhibiting potassium currents without affecting sodium or calcium currents. In cardiac tissue, it has been demonstrated to block some but not all of the delayed rectifier potassium current [6,9,12,13]. Other cardiac potassium channels are unaffected, such as the ATPsensitive potassium current [16] or the cloned min K channel [15]. The use of E4031 has been circumscribed to cardiac preparations and little, if any, information on a non-cardiac preparation is available. Potassium currents have been characterized in taste cells from a variety of species [1-4,10,11,14]. There are multiple potassium components in taste cells that are similar in voltage-sensitivity and inactivation properties to neuronal potassium channels. These currents have multiple functions in taste cells. They are likely important in early phases of transduction schemes for sweet and bitter stimuli. Additionally, they contribute to various phases of * Corresponding author. Tel.: +1 317 2858759; fax: +1 317 2851059; e-mail: 00msherness @bsu.edu.
the resultant action potential; delayed rectifiers and transient potassium currents contribute to the repolarization phase, transient and calcium-activated currents contribute to the afterhyperpolarization (AHP); inward rectifiers contribute to the resting potential [4,11]. W e examined the effect of E4031 on potassium currents from taste cells to examine its utility as a potential probe for gustatory transduction mechanisms. Isolated taste receptor cells were obtained from male and female S p r a g u e - D a w l e y rats weighing between 2 0 0 280 g as previously described [7]. Animals were brought to a surgical level of anesthesia (ketamine 90.9 mg/ml, and acepromazine 0.909 mg/ml mixture, 0.9 ml/kg bw, Butler Laboratories, IN) prior to excision of circumvallate and foliate papillae. Individual cells were dissociated with Papain (14 U/ml, Boehringer-Mannheim, IN). Cells were voltage clamped using standard patch clamp techniques in the whole cell configuration. Electrode resistance was typically 3.5-5.5 Mr2 when filled with a pseudo-intracellular fluid (ICF) composed of (mM) 140 KCI, 2 MgC12, 1 CaC12, 11 E G T A , 10 HEPES, and 4 A T P (disodium salt), and measured in pseudo-extracell-
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 2 3 4 3 - 9
150
X.-D. Sun, M.S. Herness / Neuroscience Letters 204 (1996) 149-152
ular fluid (ECF) composed o f (mM) 126 NaCI, 5 KC1, 5 NaHEPES, 1.25 NaH2POa'H20, 10 glucose, 2 CaC12, and 2 MgCI2. Input resistance ranged from 6-15 G f l ; membrane capacitance was 3 - 6 pF; series resistance averaged 10 Mff2. Low pass filtering due to resistance-capacitance coupling was considered minimal. The average RC time constant was 37kts whereas the capacitive transient settling time averaged 131 kts [8]. Data were acquired with a high impedance amplifier (Axopatch 200A, A x o n Instruments, CA), a 486-based computer equipped with a 12 bit 330 kHz A/D converter (Digidata 1200, A x o n Instruments, CA), and commercially available software program (pClamp, v 6.0, Axon Instruments, CA). The membrane voltage was typically held at - 8 0 mV and a series o f depolarizing command potentials, in increments of 10 mV, were applied. M e m brane currents, in response to voltage perturbations, were acquired with a cut-off frequency of 5 kHz (at - 3 dB). Leak subtraction was employed except when recording inwardly rectifying currents. Solution exchange of the bathing solution was accomplished with a gravity-fed perfusion system of the recording chamber at approximately 3 ml/min. For experiments that utilized high potassium ECF, potassium was substituted for sodium on an equimolar basis. Potassium currents first activate close to - 1 0 mV, from a holding potential of - 8 0 mV, and increase in a linear fashion with more depolarized potentials reaching magnitudes o f several nanoamperes. This current is composed o f both sustained components and slowly inactivating components, with time constants in the range of 1-3 s. W h o l e cell potassium conductance becomes half maximal around 4 mV with an e-fold increase of conductance every 12 mV o f depolarization (data not shown). The addition of E4031 to the bathing medium resulted in a rapid and reversible diminution of these currents (Fig. 1A). The remaining current activated rapidly and showed no discernible inactivation, even after 1 s. Currents were inhibited at three tested concentrations of 10/~M, 100ktM, and 1 m M (Fig. 1B); IktM was without effect. Inhibition was measurable at every test potential but percent inhibition increased steadily with more depolarized potentials reaching (at +90 mV) 24.4%, 44.4%, and 69.5% inhibition for 10ktM, 100/~M, and 1 mM, respectively, suggesting a voltage-dependence of the block. Onset and offset kinetics were not noticeably altered. Inset shows dose-response curve for E4031 inhibition of outward currents produced by a test pulse of 90 mV. Half maximal inhibition was produced by approximately 200/~M. Each point is the mean (_+SE) of 4 - 6 cells. The inactivation kinetics of the remaining potassium current was altered in the presence of E4031 (data not shown). Inactivation was measured by clamping the membrane to various prepulse potentials for 10 s prior to a test pulse to 90 mV. Under control conditions half maximal inhibition occurred at - 8 . 7 _+ 3.8 mV (n = 4 )
A
....
1 m M E4031 it_ i i
! .........
~
n
1 nA
B
nA
~ 1.0[ +=
4-
~ 0.5
3.
=. Z 0.0
100 ms
ii iir
• . ]/ , 10 4
10 .4
1mME4031 ~ / / ~
~
10 .3
±
± ± ±
1-
[ E40311 (M) -100
-50
0
i
i
5O
100 mV
Fig. 1. E4031 inhibition of outward potassium currents in taste receptor cells. (A) Representative whole cell currents from a dissociated taste receptor cell are illustrated before, during, and after superfusion of the cell with a pseudoextracellular fluid containing 1 mM E4031. (B) Data from several cells are presented as mean (_+SE) in the current voltage plot for ECF (O) and for three concentrations of E4031, 10/~M (O), 100ktM (A), and 1 mM (A). E4031 did not alter the activation kinetics but did decrease the magnitude of the current in a dose-dependent manner. The concentration-response relationship is presented the inset for a test pulse to 90 mV. Half maximal inhibition occurred at approximately 200/.tM (dotted lines). with an e-fold decrease per 9.1_+0.8 mV. This agreed well with previous measurements [4]. In 1 0 0 / t M E4031, the inactivation curve was shifted to the left by approximately 10 mV without a change in slope. Half maximal inactivation occurred at - 1 8 . 7 _+ 2.4 mV (n = 4) with an efold decrease every 10.2 _ 0.5 mV. Taste receptor cells possess inwardly rectifying potassium currents that are approximately half maximal conductance at resting potentials, have conductances that vary with the square root of external potassium concentration, and display little inactivation. They are insensitive to 4-aminopyridine (4AP), mildly sensitive to tetraethylammonium (TEA), and are blocked by micromolar concentrations of cesium and barium. Sample currents recorded in 100 m M potassium ECF, in Fig. 2A, were obtained by holding the cell at its zero-current potential ( - 5 mV for this cell) and applying either depolarizing or hyperpolarizing command potentials in 10 mV increments from +30 mV to - 1 6 0 mV. These currents were inhibited
151
X.-D. Sun, M.S. Herness / Neuroscience Letters 204 (1996) 149-152
A
L
Control
---
L
I 0.5 ro~ 20 msec •
l mM E4031
Washout
B -160
-120
-8o
-40
_ _ ~
4
o
mV
~-
~Washout
_ _ l
I
I
1 m M the action potential was protracted and malformed (Fig. 3C). In voltage-clamped records (not shown) some inhibition of inward sodium currents occurred at this concentration. In contrast to its effects in cardiac tissue as a specific blocker of a component of the delayed rectifying potassium current [12,13], E4031 was an effective blocker of several types o f potassium conductance in taste receptor cells. These included sustained, transient, and inwardlyrectifying potassium currents. To our knowledge, effects of E4031 on multiple types o f potassium channels have not been previously reported. The plateau in the currentvoltage relationship of the E4031-insensitive current could be consistent with complete inhibition of a subset of potassium channels. Although the identity of this remaining current in Fig. 1 was not investigated, chloride currents likely contribute substantially (personal observation). The action of E4031 on inward rectifying current is of particular interest since these channels do not belong to the superfamily of ' s h a k e r ' - l i k e potassium channel but represent a novel gene family (e.g. [5]). They are, however, highly conserved in the P region thought to form the
-1.2.
nA
Fig. 2. E4031 produced partial inhibition of inwardly rectifying potassium currents. Representative current traces are illustrated in (A) recorded in a high potassium ECF. EA031 diminished these currents in a reversible manner though magnitude of inhibition was not uniform over all tested potentials. As evident in (B) greater inhibition was observed in the mid-region of hyperpolarizing membrane potentials. Data are mean _ SE for three cells.
20 mV
0A•E4031 5 msec
~_
~
Control
--
B by either 100/~M or 1 m M E4031; 1 0 p M E4031 was without effect. In the absence of blocker, currents increased in a relatively linear fashion at potentials that were negative to the holding potential (open circles) whereas in the presence o f drug the increase was nonlinear (solid circles) (Fig. 2B). The voltage-dependence of inhibition of E4031 displayed an inverted-U relationship when plotted as normalized inhibited current against membrane potential for both concentrations of E4031. M a x i m a l inhibition occurred at - 1 0 0 mV and at very hyperpolarized potentials inhibition was substantially reduced (data not shown). E4031 was also able to alter the gustatory action potential produced by injected current (25 p A for 0.1 ms) in current-clamp mode. At 10/zM the action potential became broader but without noticeable inhibition of the action potential height (APH; Fig. 3A). In voltage-clamp records clear inhibition of the outward currents, occurring within the first few milliseconds of activation, was evident (data not shown). As the concentration of E4031 increased its affect on the action potential became more pronounced. At 100/zM action potential duration (APD) was lengthened and A P H was reduced (Fig. 3B) and at
0 ~
I
C
l~tM
E4031
Control
~//--
":~'/"//i~/15 min Washout
o
/
*~
~
~"
Control
Fig. 3. The effect of E4031 on the action potential recorded from taste receptor cells. Action potentials were elicited by injection of 25 pA of current for 0.1 ms illustrated below each set of spikes. E4031 both broadened the width and, at higher concentration, truncated the height of the action potential. Current clamp records are presented for 10/~M (A), 100/~M (B), and 1 mM (C) concentrations from a single cell. The return of action potential shape after washout of E4031 is presented for the highest concentration (dotted line).
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x.-D. Sun, M.S. Herness / Neuroscience Letters 204 (1996) 149-152
pore. This m a y suggest that, at least at high concentrations, E4031 acts at s o m e c o n s e r v e d site o f the pore, such as the selectivity filter, rather than at another site, such as the gate. T h o u g h r e d u c t i o n o f p o t a s s i u m currents by E4031 was e f f e c t i v e for all suprathreshold potentials voltaged e p e n d e n c e o f b l o c k was observed. This m a y h a v e s o m e i m p l i c a t i o n for its m e c h a n i s m . F o r outward currents b l o c k i n g e f f i c a c y increased with d e p o l a r i z i n g potentials and for inward currents it d e c r e a s e d with h y p e r p o l a r i z i n g potentials. T h i s suggests that the binding site o f E4031 m a y sense s o m e o f the electric field across the m e m b r a n e . Additionally, at very h y p e r p o l a r i z e d potentials inhibition o f inward currents was less effective, consistent with rel i e f o f b l o c k f r o m the internal side. E4031 had significant effects on the contour o f the gustatory action potential as has been o b s e r v e d with T E A [4] and forskolin (personal observation). Unanticipated was a truncation o f the spike height with 1 m M E4031. This s a m e c o n c e n t r a t i o n p r o d u c e d a d i m i n u t i o n o f inward s o d i u m current in v o l t a g e - c l a m p records, suggesting possible b l o c k i n g o f s o d i u m channels. A similar inhibition o f s o d i u m c h a n n e l s was o b s e r v e d for high concentrations o f forskolin (personal observation). In conclusion, unlike its effect in cardiac tissue, the antiarrhythmic drug E403 I, at m i c r o m o l a r concentrations, inhibits m o r e than one type of p o t a s s i u m c o n d u c t a n c e in taste r e c e p t o r cells. At the lowest tested c o n c e n t r a t i o n (10/.tM) outward potassium currents w e r e substantially inhibited without effect on inward p o t a s s i u m currents. This w o r k was supported by grant D C 0 0 4 0 1 f r o m the National Institute o f C o m m u n i c a t i v e and Sensory Disorders o f the N a t i o n a l Institutes o f Health. W e thank Eisai Laboratories Co., Ltd., T s u k u b a , Japan for their generous donation of E4031. [1] Avenet, P. and Lindemann, B., Patch-clamp study of isolated taste receptor cells of the frog, J. Membr. Biol., 97 (1987) 223-240.
[2] B6h6, P., DeSimone, J.A., Avenet, P. and Lindemann, B., Membrane currents in taste cells of the rat fungiform papilla: evidence for two types of Ca currents and inhibition of K currents by saccharin, J. Gen. Physiol., 96 (1990) 1061-1084. [3] Bigiani, A. and Roper, S., Identification of electrophysiologically distinct cell subpopulations in Necturus taste buds, J. Gen. Physiol., 102 (1993) 143-170. [4] Chen, Y. Sun, X.-D. and Herness, M.S., Characteristics of action potentials and their underlying outward currents in rat taste cells, J. Neurophysiol., 75 (2) (1996) 820-831. [5] Doupnik, C.A., Davidson, N. and Lester, H.A., The inward rectifier potassium channel family, Curr. Biol., 5 (1995) 268-277. [6] Follmer, C.H. and Colatsky, T.J., Block of delayed rectifier potassium current, IK, by flecainide and E4031 in cat ventricular myocytes, Circulation, 82 (1990) 289-293. [7] Herness, M.S., A dissociation procedure for mammalian taste cells, Neurosci. Lett., 106 (1989) 60-64. [8] Hemess, M.S. and Sun, X-.D., Voltage-dependent sodium currents recorded from dissociated rat taste cells, J. Membr. Biol., 146 (1995) 73-84. [9] Liu, Y., Taffet, S.M., Anumonwe, J.M. and Delmar, M., Characterization of an E4031-sensitive potassium current in quiescent AT-I cells, J. Cardiovasc. Electrophysiol., 5 (1990) 1017-1030. [10] Kinnamon, S.C., and Roper, S.D., Membrane properties of isolated mudpuppy taste cells, J. Gen. Physiol., 91 (1988) 351-371. [11] Roper, S.D. and McBride, D.W., Distribution of ion channels on taste cells and its relationship to chemosensory transduction, J. Membr. Biol., 109 (1989) 29-39. [12] Sanguinetti, M.C. and Jurkiewicz, N.K., Two components of cardiac delayed rectifier K+ current: differential sensitivity to block by class III antiarrhythmic agents, J. Gen. Physiol., 96 (1990) 195-215. [13] Sanguinetti, M.C. and Jurkiewicz, N.K., Delayed rectifier outward K+ current is composed of two currents in guinea pig atrial cells, Am. J. Physiol., 260 (1991) H393-H399. [14] Sugimoto, K. and Teeter, J.H., Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander, J. Gen. Physiol., 96 (1990) 809-834. [15] Varnum, M.D., Busch, A.E., Bond, C.T., Maylie, J. and Adelman, J.P., The min K channel underlies the cardiac potassium current /Ks and mediates species-specific responses to protein kinase C, Proc. Natl. Acad. Sci. USA, 90 (1993) 11528-11532. [16] Wu, B., Sato, T., Kiyoshe, T. and Arita, M., Blockade of 2,4nitrophenol-induced ATP-sensitive potassium current in guinea pig ventricular myocytes by class I antiarrhythmic drugs, Cardiovasc. Res., 26 (1992) 1095-1101.