- Email: [email protected]
A TEA-sensitive component in the conductance of a non-excitable tissue (the amphibian lens)
E&p. Eye Res. (1979) 28, 349-352
LETTER
TO THE EDITORS
A TEA-sensitive Component in the Conductance of a Non-excitable Tissue (the Amphibian Lens) Voltage-dependent, tetraethylammoniumf (TEA) sensitive membrane conductances are a well known feature of excitable tissues (Armstrong and Binstock, 1965; Hille. 1970). However, there are few reports that the conductances of non-excitable tissues have these properties. Current-voltage curves of amphibian epithelial tissues for example, are linear over a very wide range of voltage values (Lowenstein and Kanno, 1964; Smith, 1974). The lens is a good example of a “passive” tissue and even in recent literature is referred to as the crystalline lens (Eisenberg and Rae, 1976; Kinsey, 1976). Duncan (1969) and Eisenberg and Rae (1976) have shown that the lens membrane current-voltage curves are indeed linear but the voltage region over which they were able to map out the curves was relatively small and limited by the current that could be passedthrough the tip of their microelectrodes. We changed t,he lens potential through a greater range by depolarizing with high external potassium and found that the change in conductance obtained was much greater than that, predicted by conventional conductance equations (Kimuzuka and Koketsu, 1964). When the lens potential was depolarized in the presenceof TEA, the conductances a,t the different potassium concentrations were greatly reduced compared to the controls a.ndmuch closerto the predicted values. The relation between the TEA-sensitive component of the conductance and lens membrane potential could be accurately fitted using conventional Hodgkin-Huxley equations (Jack, Noble and Tsien. 1975). A two internal microelectrode technique wras used to nieasure the membrane conductance (Duncan, 1969; Delamere and Duncan, 1977). Lenseswere taken from large healthy specimensof the frog Ra,nccpipievas and placed in a 1 cm3 perfusion chamber with a flow rate of 2 cm3 min- l. Recording was made between an internal microelectrode and an agar electrode in the chamber. Current was passed through the lens via another mieroelect’rodeto an earthed silver chloride electrode in the bath. Square current pulses of O-5PA and 10 see duration were applied and the resting potential and resultant voltage deflections (of the order of 5 mV) recorded on magnet,ic tape. After changing the potassium ion concentration in the perfusing Ringer, t,he conductance was measured when the membrane potential had stabilized. The lens was perfused with normal Ringer after each increase in external potassium to check t,hat t,he resting potential and conductance had not been changed. Figure 1 showsthat the lens potential depolarizes in the presenceof potassium and the Goldman (1943) potential equation (1) can he applied to the data to obtain estimates for the relative permeabilities.
E = RT In K,K, +aNa, P +aNa,
+jIClr +@I,
(1)
where tc = P&P,, j3 = P,.JP,. The internal and external concentrations of potassium, sodium and chloride were obtained from ion analyses (Delamere and Duncan, 1977; Patmore, unpublished) and the solid line is given by CI.= 0.01, /3 = 1.4. 00144835j79/030349-to4
$01.00/0
Q I979 Academic Press Inc. (London) Limited 349
350
2.5
5
IO 15
25
[Kt]o(md
FIG. 1. Effect of increasing external potassium on lens membrane potential. (0) control values; (0) Ringer with 20 mM-TEA. The error bars indicate the meanfsn. of at least four experiments in each case. The TEA value at 15 rnM represents one experiment and the potential value for TEA at 2.5 mu (also one experiment) wa8 -76 mV, identical to the control. The solid line was computed from equation 1 with Kr = 95 m&r, Nai = 8.1, Clr = 6, Cl, = 110 and Na,+K, = 117.5 rniv; a = 0.01; j = 1.4.
I If
/
Y
I
I’
/
E ”
I
/
IO
/ / 4
:15-
i / ,*’
5-
0-
_-
2.5
--
/-
/’
5
/
/ 0
/’
15
25
FIG. 2. Relationship between conductance and external potasclium concentration. (0) control values; (0) 20 mM-TEA added. The error bars indicate meanfs.n. of at least four separate experiments. The solid line was generated from equation (2) with the concentrations and permeability ratios given in Fig. 1 and with Pg = 1.8 x 10-a m se&. The dashed line WBB generated from Equation 4 and in order to do this the relationship between lens membrane potential and external potassium was assumed to be given by the solid curve in Fig. 1.
LETTER
TO
THE
EDITORS
351
These permeability ratios can be used to predict the variation in conductance with external potassium by applying the Kimuzuka-Koketsu (1964) equation (2) : Gr, = (F2/RT)
+aNa, +/3Cl,)(K,
. P,[(K,
+orNa +Cl,)]”
12)
The solid line fitted through the normal bathing medium value (K, = 2.5 mM) was obtained by taking l’, = 1.8 x lOpa m set-l and LXand /? as before. This value for P, is close to that obtained from potassium flux experiments (Duncan, 1974; Duncan and Croghan, 1970). However, the conductances obtained on increasing the external potassium are much greater than the predicted values and the discrepancy increases with increasing depolarization (Fig. 2). This suggested to us that a voltagedependent conductance component was contributing to this effect and in fact TEA (20 mM) added t#o the solutions reduced the measured conductances to near the predicted values. An estimate of the contribution of a voltage-sensitive potassium channel to t)he overall conductance can be obtained from the Hodgkin-Huxley model for nerve. The conductances in the steady-state would be given by an equation of the form : G IT1 G II2
1 +exp ( -zF(E,
-E,)/RT)
1 +exp t -zWh
-E&/W
(3)
where G,,, is the voltage-sensitive component of the conductance at a membrane potential E,, GI12 the conductance at E,, z and E, are parameters defining the shape of the conductance-voltage curve. If it is assumed that the voltage-sensitive conductance Gnl lies in parallel with the other conductances in the membrane then the total measured conductance G,, at any voltage E, is given by the sum
where G,, is defined by equation (2) with K, appropriate
and
-G,>,
C’ *Hl
=
G,,
= GT2-Gp2,
G.,.,
to the voltage E,.
etc.
The dashed curve was fitted to the conductance ratios generated in this way, taking the values of z and E, as 3.25 and -42.5 mV respectively. These values are in the middle of the relatively wide range of values found in excitable tissues (Jack et al., 1975). The very great sensitivity of the conductance to TEA (Fig. 2) contrasts with the very small difference in potential between the control and TEA values (Fig. 1). In fact large changes in the potassium permeability would be expected to lead to only small changes in the potential as the resting potassium permeability is relatively high. For example a halving of P, at 25 mM K, only changes the value predicted from equation (1) by 5 m.V which is within the range observed. The role of this voltage-sensitive channel in maintaining the lens in a normal state is at present unclear but a possible function could be in restoring the normal membrane potential following depolarization due to injury. It is interesting that several drugs, which were thought formerly to interfere only with excitable processes, are known to cause cataract when applied topically to the eye (Axelsson, 1973) and in lens organ-culture conditions (Owers and Duncan, unpublished).
School of Biological X&aces, lhizlersity of East Angliu, Norwich XR4 ?TJ, E~l~ghd (Received 17 October 1978, Lonchr) REFERENCES Armst,rong, C. M. and Binstock, L. (1965). Anomalous rectification in the squid giant axon injected with tetraethylammonium chloride. J. Gen. Physiol. 48, 859-72. Axelsson, U. (1973). Miotic induced cataract. The Human Lens in Relntion to Cntnmct. Ciha symposium 19. pp. 249-63. Elsevier, Amsterdam. Delamere, N. A. and Duncan, G. (1977). A comparison of ion c,oncentrationa, potentials and conductances of amphibian, bovine and cephalopod lenses. J. Physiol., London 272,167-86. Duncan, G. (1969). The site of the ion restricting membranes in t.he toad lens. E’1~p. Eye Res. 8, 406-12. Duncan, G. (1974). Comparative physiology of the lens membranes. In The flye (Ed. Davson, K.) Vol. 5, pp. 357-97. Academic Press, New York and London. Duncan, G. and Croghan, P. C. (1970). Effect of changes of external ion concentrations and 2,4-dinitrophenol on the conductance of the toad lens membranes. F:xp. Eye Res. 10, 192-200. Eisenberg, R. S. and Rae, J. L. (1976). Current-voltage relationships in the crystalline lens. J. Physiol., London 262, 285-300. Goldman. D. E. (1943). Potential, impedance and rectification in membranes. J. Gen. Physiol. 2, 37-49. Hille, B. (197U). Ionic channels in nerve membranes. Prog. Biophys. Xolec. Biol. 21, 1-Z. Jack, J. J. B., Pioble, D. and Tsien, R. W. (1975). Nectric Current Flow in Ercitrrble Cells. Clarendon Press, Oxford. Kimuzuka, H. and Koketsu, K. (1964). Ion transport through cell membranes. J. Thor. Biol 6, 290-305. Kinsey, V. E. (1976). Studies on the crystalline lens. XXIV. Bicarbonate content and flux determinations. DOG. Ophth. Proc. Ser. 8. Lowenstein, W. R. and Kanno, Y. (1964). Studies on an epithelial gland cell membrane. J C!e(l. Biol. 22, 565-86. Smit,h
P. G. (1974).
375,124-g.
Frequency
dependance
of the frog
skin
impedance.
&o&m.
et Biophy
.rlrtcc
LETTER
TO THE EDITORS
A TEA-sensitive Component in the Conductance of a Non-excitable Tissue (the Amphibian Lens) Voltage-dependent, tetraethylammoniumf (TEA) sensitive membrane conductances are a well known feature of excitable tissues (Armstrong and Binstock, 1965; Hille. 1970). However, there are few reports that the conductances of non-excitable tissues have these properties. Current-voltage curves of amphibian epithelial tissues for example, are linear over a very wide range of voltage values (Lowenstein and Kanno, 1964; Smith, 1974). The lens is a good example of a “passive” tissue and even in recent literature is referred to as the crystalline lens (Eisenberg and Rae, 1976; Kinsey, 1976). Duncan (1969) and Eisenberg and Rae (1976) have shown that the lens membrane current-voltage curves are indeed linear but the voltage region over which they were able to map out the curves was relatively small and limited by the current that could be passedthrough the tip of their microelectrodes. We changed t,he lens potential through a greater range by depolarizing with high external potassium and found that the change in conductance obtained was much greater than that, predicted by conventional conductance equations (Kimuzuka and Koketsu, 1964). When the lens potential was depolarized in the presenceof TEA, the conductances a,t the different potassium concentrations were greatly reduced compared to the controls a.ndmuch closerto the predicted values. The relation between the TEA-sensitive component of the conductance and lens membrane potential could be accurately fitted using conventional Hodgkin-Huxley equations (Jack, Noble and Tsien. 1975). A two internal microelectrode technique wras used to nieasure the membrane conductance (Duncan, 1969; Delamere and Duncan, 1977). Lenseswere taken from large healthy specimensof the frog Ra,nccpipievas and placed in a 1 cm3 perfusion chamber with a flow rate of 2 cm3 min- l. Recording was made between an internal microelectrode and an agar electrode in the chamber. Current was passed through the lens via another mieroelect’rodeto an earthed silver chloride electrode in the bath. Square current pulses of O-5PA and 10 see duration were applied and the resting potential and resultant voltage deflections (of the order of 5 mV) recorded on magnet,ic tape. After changing the potassium ion concentration in the perfusing Ringer, t,he conductance was measured when the membrane potential had stabilized. The lens was perfused with normal Ringer after each increase in external potassium to check t,hat t,he resting potential and conductance had not been changed. Figure 1 showsthat the lens potential depolarizes in the presenceof potassium and the Goldman (1943) potential equation (1) can he applied to the data to obtain estimates for the relative permeabilities.
E = RT In K,K, +aNa, P +aNa,
+jIClr +@I,
(1)
where tc = P&P,, j3 = P,.JP,. The internal and external concentrations of potassium, sodium and chloride were obtained from ion analyses (Delamere and Duncan, 1977; Patmore, unpublished) and the solid line is given by CI.= 0.01, /3 = 1.4. 00144835j79/030349-to4
$01.00/0
Q I979 Academic Press Inc. (London) Limited 349
350
2.5
5
IO 15
25
[Kt]o(md
FIG. 1. Effect of increasing external potassium on lens membrane potential. (0) control values; (0) Ringer with 20 mM-TEA. The error bars indicate the meanfsn. of at least four experiments in each case. The TEA value at 15 rnM represents one experiment and the potential value for TEA at 2.5 mu (also one experiment) wa8 -76 mV, identical to the control. The solid line was computed from equation 1 with Kr = 95 m&r, Nai = 8.1, Clr = 6, Cl, = 110 and Na,+K, = 117.5 rniv; a = 0.01; j = 1.4.
I If
/
Y
I
I’
/
E ”
I
/
IO
/ / 4
:15-
i / ,*’
5-
0-
_-
2.5
--
/-
/’
5
/
/ 0
/’
15
25
FIG. 2. Relationship between conductance and external potasclium concentration. (0) control values; (0) 20 mM-TEA added. The error bars indicate meanfs.n. of at least four separate experiments. The solid line was generated from equation (2) with the concentrations and permeability ratios given in Fig. 1 and with Pg = 1.8 x 10-a m se&. The dashed line WBB generated from Equation 4 and in order to do this the relationship between lens membrane potential and external potassium was assumed to be given by the solid curve in Fig. 1.
LETTER
TO
THE
EDITORS
351
These permeability ratios can be used to predict the variation in conductance with external potassium by applying the Kimuzuka-Koketsu (1964) equation (2) : Gr, = (F2/RT)
+aNa, +/3Cl,)(K,
. P,[(K,
+orNa +Cl,)]”
12)
The solid line fitted through the normal bathing medium value (K, = 2.5 mM) was obtained by taking l’, = 1.8 x lOpa m set-l and LXand /? as before. This value for P, is close to that obtained from potassium flux experiments (Duncan, 1974; Duncan and Croghan, 1970). However, the conductances obtained on increasing the external potassium are much greater than the predicted values and the discrepancy increases with increasing depolarization (Fig. 2). This suggested to us that a voltagedependent conductance component was contributing to this effect and in fact TEA (20 mM) added t#o the solutions reduced the measured conductances to near the predicted values. An estimate of the contribution of a voltage-sensitive potassium channel to t)he overall conductance can be obtained from the Hodgkin-Huxley model for nerve. The conductances in the steady-state would be given by an equation of the form : G IT1 G II2
1 +exp ( -zF(E,
-E,)/RT)
1 +exp t -zWh
-E&/W
(3)
where G,,, is the voltage-sensitive component of the conductance at a membrane potential E,, GI12 the conductance at E,, z and E, are parameters defining the shape of the conductance-voltage curve. If it is assumed that the voltage-sensitive conductance Gnl lies in parallel with the other conductances in the membrane then the total measured conductance G,, at any voltage E, is given by the sum
where G,, is defined by equation (2) with K, appropriate
and
-G,>,
C’ *Hl
=
G,,
= GT2-Gp2,
G.,.,
to the voltage E,.
etc.
The dashed curve was fitted to the conductance ratios generated in this way, taking the values of z and E, as 3.25 and -42.5 mV respectively. These values are in the middle of the relatively wide range of values found in excitable tissues (Jack et al., 1975). The very great sensitivity of the conductance to TEA (Fig. 2) contrasts with the very small difference in potential between the control and TEA values (Fig. 1). In fact large changes in the potassium permeability would be expected to lead to only small changes in the potential as the resting potassium permeability is relatively high. For example a halving of P, at 25 mM K, only changes the value predicted from equation (1) by 5 m.V which is within the range observed. The role of this voltage-sensitive channel in maintaining the lens in a normal state is at present unclear but a possible function could be in restoring the normal membrane potential following depolarization due to injury. It is interesting that several drugs, which were thought formerly to interfere only with excitable processes, are known to cause cataract when applied topically to the eye (Axelsson, 1973) and in lens organ-culture conditions (Owers and Duncan, unpublished).
School of Biological X&aces, lhizlersity of East Angliu, Norwich XR4 ?TJ, E~l~ghd (Received 17 October 1978, Lonchr) REFERENCES Armst,rong, C. M. and Binstock, L. (1965). Anomalous rectification in the squid giant axon injected with tetraethylammonium chloride. J. Gen. Physiol. 48, 859-72. Axelsson, U. (1973). Miotic induced cataract. The Human Lens in Relntion to Cntnmct. Ciha symposium 19. pp. 249-63. Elsevier, Amsterdam. Delamere, N. A. and Duncan, G. (1977). A comparison of ion c,oncentrationa, potentials and conductances of amphibian, bovine and cephalopod lenses. J. Physiol., London 272,167-86. Duncan, G. (1969). The site of the ion restricting membranes in t.he toad lens. E’1~p. Eye Res. 8, 406-12. Duncan, G. (1974). Comparative physiology of the lens membranes. In The flye (Ed. Davson, K.) Vol. 5, pp. 357-97. Academic Press, New York and London. Duncan, G. and Croghan, P. C. (1970). Effect of changes of external ion concentrations and 2,4-dinitrophenol on the conductance of the toad lens membranes. F:xp. Eye Res. 10, 192-200. Eisenberg, R. S. and Rae, J. L. (1976). Current-voltage relationships in the crystalline lens. J. Physiol., London 262, 285-300. Goldman. D. E. (1943). Potential, impedance and rectification in membranes. J. Gen. Physiol. 2, 37-49. Hille, B. (197U). Ionic channels in nerve membranes. Prog. Biophys. Xolec. Biol. 21, 1-Z. Jack, J. J. B., Pioble, D. and Tsien, R. W. (1975). Nectric Current Flow in Ercitrrble Cells. Clarendon Press, Oxford. Kimuzuka, H. and Koketsu, K. (1964). Ion transport through cell membranes. J. Thor. Biol 6, 290-305. Kinsey, V. E. (1976). Studies on the crystalline lens. XXIV. Bicarbonate content and flux determinations. DOG. Ophth. Proc. Ser. 8. Lowenstein, W. R. and Kanno, Y. (1964). Studies on an epithelial gland cell membrane. J C!e(l. Biol. 22, 565-86. Smit,h
P. G. (1974).
375,124-g.
Frequency
dependance
of the frog
skin
impedance.
&o&m.
et Biophy
.rlrtcc