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Elevated divalent cation concentration decreases potassium-induced depolarization of bullfrog primary afferent fibers
Brain Research, 240 (1982) 181-185 Elsevier Biomedical Press
181
Elevated diwalent cation concentration decreases potassium-induced depolarization of bullfrog primary afferent fibers MARK S. BRODIE, SARAH A. SHEFNER* and RICHARD A. LEVY** Department of Pharmacology, University of Illinois College of Medicine, P.O. Box 6998, Chicago, IL 60680 (U.S.A.} (Accepted February 4th, 1982) Key words: divalent cations - - potassium-induced depolarization - - primary afferent fibers
Depolarizations of bullfrog primary afferent fibers caused by increasing the K + concentration in the Ringer were extracellularly recorded from isolated, desheathed dorsal roots. In concentrations commonly used to block synaptic transmission, Mns+, Cos+, and Mgs+ decreased the amplitude and rate of rise of the K+-induceddepolarization. Cas+ had a similar action. The apparent order of effectiveness was Mn s+ m Cos+ > Cas+ > Mgs+. In a previous study on the hemisected bullfrog spinal cord Shefner and Levy~1 found that 20 mM Mg 2+ decreased K+-induced depolarizations recorded on the dorsal root, shifting the K ÷ dose-response curve to the right. This Mg ~+ effect was not due to blockade of K+-induced transmitter release onto primary afferent terminals from nearby neurons since Mg 2+ reduced K + depolarizations to a similar extent in isolated, desheathed dorsal roots where an indirect effect due to transmitter release is not possible. It was concluded that Mg 2+ can directly reduce the responsiveness of the primary afferent membrane to K +. In the present study, the effects of Mg 2+ on the K--induced depolarization of primary afferent fibers in isolated, desheathed dorsal roots was examined further, and compared to the effects of some other divalent cations. Co 2+ and Mn 2+ (refs. 1, 2, 13, 22), like Mg 2÷ (refs. 6,7), block neurotransmitter release, Co s+ and Mn z+, therefore, were tested in concentrations used to block synaptic transmission, to see if they also decrease K+-induced depolarization. Co 2+, Mn 2+ and Mg 2+ act as Ca 2+ antagonists at Ca 2+ channels (refs. 2, 14) but act in the same direction as Ca 2÷ to increase threshold 11, decrease excitability19, 2a and to shift the conductance vs voltage curves for Na + and
K + channels4,10. Therefore, we compared the effect of elevated Ca 2+ with the effects of these other divalent cations to see whether Ca 2+ exerted the same or opposite action on K+-induced depolarization. The bath for maintaining the isolated dorsal roots was made from acrylic plastic and consisted of a central superfusion chamber and adjacent oil pool. Ringer solution was circulated through the superfusion chamber with a peristaltic pump and removed with a suction pipette. A water jacket kept the bath at about 15 °C -q- 1 °C during each experiment. Rapid flow rates (6 ml/min) and a small volume superfusion chamber (0.25 ml) with minimal 'dead space' for mixing, allowed fast and complete equilibration to new steady-state concentrations following alteration in the K + concentration in the Ringer. Equilibration to new steady-state concentrations was rapid enough to permit multiple determinations of K + depolarizations in the same preparation, so that the effect of elevated divalent cation concentration on K + depolarizations could be examined using each preparation as its own control. This was desirable since the absolute size of the extracellular responses varied among preparations with such factors as the size of the root and length of root exposed to test solutions.
* To whom correspondence should be addressed. Present address: National Pharmaceutical Council, 1030 15th Street, N.W., Washington, DC 20005, U.S.A.
**
0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
182 Bullfrogs (Rana catesbeiana,either sex, 200-600 g) were kept in aquaria at 2-3 °C before use. The bullfrog was cooled to an anesthetic state and a laminectomy was performed. The spinal cord was removed with lumbar spinal roots attached and a lumbar dorsal root was cut off at its entry into the cord. The root was desheathed, and the proximal end (several millimeters in length) was placed in the central superfusion chamber; the remainder of the root extended into the adjacent oil pool for recording. The polarization level of the lumbar dorsal root was recorded with a pair of Ag-AgC1 hook electrodes, positioned so that the proximal hook was placed on the root close to where it exited from the oil pool into the superfusion chamber and the other was further out on the root. DC signals from these electrodes were amplied with a differential amplifier and recorded on a pen recorder. The preparation was grounded with an Ag-AgCI electrode making contact with the Ringer in the bath through a salt bridge ( 0 . 9 ~ NaC1, 5°//o Agar). The contlol Ringer contained 100 mM NaC1, 2.4
mM KCI, 9.5 mM NaHCO3, 1.9 mM CaC12, 2.8 mM D-glucose and 10 mM Tris buffer (pH 7.4). The Ringer was continuously bubbled with 95 0/o Oz/5 o/ • / 0 CO2. K+-rich Ringer solutions were made by equimolar substitution of K ~ for N a ' . For divalent cation-rich Ringer solutions MnCI2 (2 mM), COC12 (2 mM), CaC12 (2 or 10 mM) or MgSO4 (20 mM) was added to the Ringer without concomitant removal of other ions. These concentrations of Mn 2 ', Co 2+ and Mg 2+ were chosen because they are commonly used to block synaptic transmission (see for example refs. 1 and 13). Two millimolar Ca 2 ~ was used for comparison with 2 mM Mn 2~ and Co s+. Ten millimolar Ca z+ was selected because for previously studied phenomena in which Ca zt and Mg 2~ act in the same direction, Ca 2 ~ was about twice as potent as Mg 2÷ (refs. 10, 11). In previous experiments, we found no difference between the effect of MgSO4 and MgCI2 on K+-induced depolarizations (ref. 21); MgSO4 was used in this study because it is less hygroscopic and therefore is easier to weigh accurately.
A.
5.
157ram K +
9.9 rnM K "1"-
25mM K +
_ _ _ , , . - ~ ~
_
I
'9.9 rnM K÷
20raM Mg ++
_llmV Irnin
13.TrnM K+
20raM Mg *'t"
B.
_J2mV Imin
25mM K + 2 0 m M Mg t +
._] 5 mV Imin
C.
25mM K +
25rnM K +
20mMK+
2J mv
2omM.*
2 m M Mn "='+
Imin
2 m M Ca + +
/'mv Imin
Fig. 1. DC depolarization of the dorsal root when K+0 was raised from 2.4 mM to the concentration listed below each trace. The K+-rich solution was applied for the duration of the dark bars under each trace. Upper traces show control K+ depolarizations and lower traces show responses to these same K + concentrations when given in the presence of an additional 20 mM Mg2÷ (A), 2 mM Mn~+ (B), or 2 mM Ca~+ (C).
183 Control depolarizations by K+-rich Ringer solutions containing 9.9, 13.7 or 25.0 m M were determined. Each K+-rich solution was applied until the dorsal root depolarization reached a new steadystate level (6-8 min) and then was washed out for 8-10 min between K + doses allowing the D C voltage to return to baseline (see Fig. 1A). A solution rich in divalent cation was then applied for 20 min and the K+-induced depolarization repeated in the presence of the elevated divalent cation concentration (see lower traces Fig. 1A). After 20 min to wash out the high divalent cation solution, a second control determination of K+-induced depolarizations was made to make sure that changes in the K ÷ depolarizations were not due to a general deterioration of the preparation. Fig. 1A shows the effect of addition of 20 m M Mg 2+ on K--induced depolarizations. At all 3 K + concentrations, the K+-induced depolarization is reduced in amplitude by high Mg 2+. Two millimolar Mn 2+ caused a similar, though smaller magnitude change in the 25 m M K ÷ depolarization (Fig. 1B) whereas 2 m M Ca 2+ caused no detectable change in amplitude of the 25 m M K ÷ depolarization (Fig. 1C). The mean percent decreases in K ÷ depolarizations caused by the 5 high divalent cation conditions are shown in Fig. 2. The largest decreases were caused by 20 m M Mg z÷ and 10 m M Ca 2÷ (topmost curves) and there was no significant difference between these two groups when tested with a two-way analysis of variance (ANOVA) with repeated measures is (P > 0.05). Intermediate decreases in K ÷ depolarizations were seen with 2 mM CO z÷ and 2 m M Mn 2÷ while 2 m M Ca 2÷ caused smaller decreases. A similar A N O VA comparing 2 m M Co g÷, Mn 2÷ and Ca ~÷ effects showed a significant difference between treatments ( F = 9.34; df = 2,9; P < 0.01). The Dunn test for post-hoc comparisonslS showed that 2 m M Co 2÷ and 2 m M Mn 2÷ were not significantly different (P > 0.05) but Co 2÷ and 2 m M Ca z÷ (bottom two curves) did differ significantly from each other (P < 0.01). In each divalent cation treatment group, the decrease in depolarization by 9.9 m M K + was greater than the decrease in 25.0 m M K + depolarization (see Fig. 2). This observation is supported statistically, since both A N O V A s mentioned above showed a
70 E3
z
+l
~ : z 50 0 WN LU II~--I 0 ¢ WII. 1211,1 Z
m n~ w
I0 .f,r,
•
I0
,
15
,
20
~o
K ÷ CONCENTRATION (raM)
Fig. 2. The percent decrease in K+-induced depolarizations caused by the 5 divalent cation conditions: 10 mM Ca 2+ (O), 20 rnM Mg2+ (A), 2 mM Mn e+ (©), 2 mM Cos+ (A), and 2 mM Ca e+ ([]). K + concentration is shown on a logarithmic scale. Each point represents the mean 4- S.E.M. of 4 experiments.
significant effect of K + concentration on the magnitude of the decrease in depolarization: for the 20 m M Mg 2+ vs 10 m M Ca 2+ test ( F = 27.4; df ----2,12) and for the 2 m M Ca z+, Co 2+ and Mn 2+ test (F = 30.4; df = 2,18) P < 0.001. There was no significant interaction effect between the divalent cation condition used and K + concentration seen in either A N O V A (P > 0.05). Elevating the divalent cation concentration not only decreases the amplitude of the K+-induced depolarization but also slows the rate of rise of the depolarization (see Figs. 1A and B). This effect occurred at all 3 K + concentrations but was most apparent for 25.0 m M K + responses. The slowing of the rate of rise was quantitated by measuring the time to half amplitude for control depolarizations and in the presence of divalent cation-rich solutions, and calculating the percent of control time to half amplitude. Only depolarizations by 25 m M K + were analyzed because the accuracy of the determination of time to half amplitude for the smaller K + concentrations was questionable. The mean percent of control time to half amplitude (:k S.E.M.) after 2 m M Ca z+, 2 m M Co ~+, 2 mM Mn z+, 10 m M Ca 2+ and 20 mM Mg z+ were 128 -+- 18, 176 ± 17, 234 -+- 40, 457 zk 147 and 717 zk 166, respectively.
184 In addition to blocking synaptic transmission, 2 mM Co 2~, 2 mM Mn 2+ and 20 mM Mg 2+ can increase threshold and interfere with action potential propagationL We now report that these divalent cations, when tested in concentrations used to block synaptic transm;ssion~, 13 alsc reduce depolarizations caused by increasing the extracellular K + concentration (K+0) and decrease the rate of rise of such depolarizations. Elevated Ca 2+ also causes this effect, consistent with an early observation that Ca "~+ opposes K + depolarization of frog node of Ranvier (ref. 15). A rank order of effectiveness of the divalent cations in decreasing the K+-induced depolarization can be proposed based on the data in Fig. 2 and the statistical comparisons between groups described above. The effect of 20 m M Mg 2+ was not significantly different from the effect of 10 mM Ca 2+, therefore, Mg 2+ appears to be less effective than Ca z+ in producing the decrease. Two millimolar Ca 2+ was the only condition which caused no detectable decrease in 25 m M K ÷ depolarization and the effect of 2 m M Ca z+ was significantly less than the action of 2 mM Co 2+, the next curve above Ca 2+ in Fig. 2. There was no significant difference between 2 mM Co 2+ and Mn 2+ and therefore they appear to be equally effective. Taken together, these results suggest the following order of effectiveness in decreasing K+-induced depolarization: Mn 2+ ~ Co 2+ Ca 2÷ :> Mg 2+. Additional experiments varying the concentration of each divalent cation will be required, however, for unequivocal determination of the relative potency of these ions. The mechanism underlying the decrease in K +induced depolarization by elevated divalent cation concentration may be a reduction in the K + conductance (gK) of the membrane. A reduction in the steady-state gK would decrease the influence of K+0 in determining the resting membrane potential, and therefore should reduce the depolarization which occurs when K+0 is raised. Increasing K+0 not only causes membrane depolarization as measured in our study, but also increases the membrane conductance (ref. 17). Dubois and Bergman s have shown with voltage-clamp of frog node that either increasing K+0 or membrane depolarization can increase the steady-state gK. Furthermore, the opening of K ÷ channels by depolarization or raising K+0 depends
on the extracetlular Ca 2÷ concentration, gK being lower when more Ca 2+ is present. Increases in the external Ca `'.+ concentration have been found to shift the gK vs voltage curve to the right; that is, more depolarization is required to activate gKS,9,10,12. Mg 2÷ (refs. 5, 20), Co ')T and Ni 2+ (ref. 20) exert the same effect. The shift in the steady-state gK vs voltage curve in the presence of elevated divalent cation concentration can be quantitatively accounted for by a shift in the membrane surface potential 20. Interestingly, the order of effectiveness of divalent cations in reducing K+-induced depolarization of afferent fibers that we report is the same as the order of the constants for adsorption of Co 2+, Ca 2+ and Mg 2+ to the external membrane surface calculated by Mozhayeva and Naumov 2°. The order we found: Mn 2+ ~ Co 2+ ~ Ca 2 ~ ~ Mg 2+ was also the same as the order of effectiveness found by Hille et al. 16 for causing a positive voltage shift in sodium activation, an effect which appears to be due to a change in membrane surface potential, as well. The decrease in the rate of rise of K~-induced depolarization by elevated divalent cation concentration which we report may be explained by Frankenhaueser and Hodgkin's 1° finding that increasing external Ca 2+ delayed the onset and decreased the rate of rise in gK with depolarization. These effects could be mimicked by polarizing the membrane potential, which suggests that a change in membrane surface potential may be responsible for this action of divalent cations, as well. In conclusion, changes in divalent cation concentration often used to block or enhance synaptic transmission have other actions as well. In addition to the previously documented effects on threshold and excitability, divalent cation concentration can alter very basic neuronal properties such as the influence of K+0 on the membrane potential. Our thanks to Edmund Anderson and Jeremy Shefner for their helpful comments on the manuscript.
1 Bagust, J. and Kerkut, G. A., The use of the transition elements manganese, cobalt and nickel as synaptic blocking agents on isolated, hemisected, mouse spinal cord, Brain Research, 182 (1980) 474-477. 2 Baker, P. F., Meves, H. and Ridgway, E. B., Effects of
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6
7
8
9 10
11
12
manganese and other agents on Ca z+ uptake that follows depolarization of squid axons, J. Physiol. (Lond.), 231 (1973) 511-526. Balnave, R. J. and Gage, P. W., The inhibitory effect of manganese on transmitter release at the neuromuscular junction of the toad, Brit. J. Pharmacol., 47 (1973) 339352. Blaustein, M. P. and Goldman, D. E., The action of certain polyvalent cations on the voltage-clamped lobster axon, J. gen. Physiol., 51 (1968) 279-291. Brismar, T. and Frankenhaeuser, B., The effect of calcium on the potassium permeability in the myelinated nerve fibre of Xenopus laevis, Acta physiol, scand., 85 (1972) 237-241. Del Castillo, J. and Katz, B., The effect of magnesium on the activity of motor nerve endings, J. Physiol. (Lond.), 124 (1954) 553-559. Dodge, F. A. and Rahamimoff, R., Co-operative action of calcium ions in transmitter release at the neuromuseular junction, J. Physiol. (Lond.), 193 (1967) 419-432. Dubois, J. M. and Bergman, C., The steady-state potassium conductance of the Ranvier node at various external K-concentrations, Pfliiger's Arch., 370 (1977) 185-194. Frankenhaeuser, B., The effect of calcium on the myelinated nerve fibre, J. Physiol. (Lond.), 137 (1957) 245-260. Frankenhaeuser, B. and Hodgkin, A. L., The action of calcium on the electrical properties of squid axons, J. Physiol. (Lond.), 137 (1957) 218-244. Frankenhaeuser, B. and Meves, H., The effect of magnesium and calcium on the frog myelinated nerve fibre, J. Physiol. (Lond.), 142 (1958) 360-365. Gilbert, D. L. and Ehrenstein, G., Effect of divalent cations on potassium conductance of squid axons: determination of surface charge, Biophys. J., 9 (1969) 447-463.
13 HackeR, J. T., Selective antagonism of frog cerebellar synaptic transmission by manganese and cobalt ions, Brain Research, 114 (1976) 47-52. 14 Hagiwara, S. and Byerly, L., Calcium channel, Ann. Rev. Neurosci., 4 (1981) 69-125. 15 Hashimura, S. and Wright, E. B., Effect of ionic environment on excitability and electrical properties of frog single nerve fiber, J. Neurophysiol., 21 (1958) 24-44. 16 HiUe, B., Woodhull, A. M. and Shapiro, B. I., Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions and pH, Phil. Trans. B., 270 (1975) 301-318. 17 Hodgkin, A. L., The effect of potassium on the surface membrane of an isolated axon, J. Physiol. (Lond.), 106 (1947) 319-340. 18 Keppel, G., Design and Analysis: a Researcher's Handbook, Prentice-Hall, Engelwood Cliffs, NJ, 1973, 658 pp. 19 Krnjevi6, K., Lamour, Y., MacDonald, J. F. and Nistri, A., Effects of some divalent cations on motoneurones in cats, Canad. J. Physiol. Pharmacol., 57 (1979) 944-956. 20 Mozhayeva, G. N. and Naumov, A. P., Effect of surface charge on the steady-state potassium conductance of nodal membrane, Nature (Lond.), 228 (1970) 164-165. 21 Shefner, S. A. and Levy, R. A., The contribution of increases in extracellular potassium to primary afferent depolarization in the bullfrog spinal cord, Brain Research, 205 (1981) 321-335. 22 Weakly, J. N., The action of cobalt on neuromuscular transmission in the frog, J. Physiol. (Lond.), 234 (1973) 597-612. 23 Wojtowicz, J. M., Marshall, K. C. and Hendelman, W. J., Depression by magnesium ion of neuronal excitability in tissue cultures of central nervous system, Canad. J. Physiol. Pharmacol., 55 (1977) 367-372.
181
Elevated diwalent cation concentration decreases potassium-induced depolarization of bullfrog primary afferent fibers MARK S. BRODIE, SARAH A. SHEFNER* and RICHARD A. LEVY** Department of Pharmacology, University of Illinois College of Medicine, P.O. Box 6998, Chicago, IL 60680 (U.S.A.} (Accepted February 4th, 1982) Key words: divalent cations - - potassium-induced depolarization - - primary afferent fibers
Depolarizations of bullfrog primary afferent fibers caused by increasing the K + concentration in the Ringer were extracellularly recorded from isolated, desheathed dorsal roots. In concentrations commonly used to block synaptic transmission, Mns+, Cos+, and Mgs+ decreased the amplitude and rate of rise of the K+-induceddepolarization. Cas+ had a similar action. The apparent order of effectiveness was Mn s+ m Cos+ > Cas+ > Mgs+. In a previous study on the hemisected bullfrog spinal cord Shefner and Levy~1 found that 20 mM Mg 2+ decreased K+-induced depolarizations recorded on the dorsal root, shifting the K ÷ dose-response curve to the right. This Mg ~+ effect was not due to blockade of K+-induced transmitter release onto primary afferent terminals from nearby neurons since Mg 2+ reduced K + depolarizations to a similar extent in isolated, desheathed dorsal roots where an indirect effect due to transmitter release is not possible. It was concluded that Mg 2+ can directly reduce the responsiveness of the primary afferent membrane to K +. In the present study, the effects of Mg 2+ on the K--induced depolarization of primary afferent fibers in isolated, desheathed dorsal roots was examined further, and compared to the effects of some other divalent cations. Co 2+ and Mn 2+ (refs. 1, 2, 13, 22), like Mg 2÷ (refs. 6,7), block neurotransmitter release, Co s+ and Mn z+, therefore, were tested in concentrations used to block synaptic transmission, to see if they also decrease K+-induced depolarization. Co 2+, Mn 2+ and Mg 2+ act as Ca 2+ antagonists at Ca 2+ channels (refs. 2, 14) but act in the same direction as Ca 2÷ to increase threshold 11, decrease excitability19, 2a and to shift the conductance vs voltage curves for Na + and
K + channels4,10. Therefore, we compared the effect of elevated Ca 2+ with the effects of these other divalent cations to see whether Ca 2+ exerted the same or opposite action on K+-induced depolarization. The bath for maintaining the isolated dorsal roots was made from acrylic plastic and consisted of a central superfusion chamber and adjacent oil pool. Ringer solution was circulated through the superfusion chamber with a peristaltic pump and removed with a suction pipette. A water jacket kept the bath at about 15 °C -q- 1 °C during each experiment. Rapid flow rates (6 ml/min) and a small volume superfusion chamber (0.25 ml) with minimal 'dead space' for mixing, allowed fast and complete equilibration to new steady-state concentrations following alteration in the K + concentration in the Ringer. Equilibration to new steady-state concentrations was rapid enough to permit multiple determinations of K + depolarizations in the same preparation, so that the effect of elevated divalent cation concentration on K + depolarizations could be examined using each preparation as its own control. This was desirable since the absolute size of the extracellular responses varied among preparations with such factors as the size of the root and length of root exposed to test solutions.
* To whom correspondence should be addressed. Present address: National Pharmaceutical Council, 1030 15th Street, N.W., Washington, DC 20005, U.S.A.
**
0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
182 Bullfrogs (Rana catesbeiana,either sex, 200-600 g) were kept in aquaria at 2-3 °C before use. The bullfrog was cooled to an anesthetic state and a laminectomy was performed. The spinal cord was removed with lumbar spinal roots attached and a lumbar dorsal root was cut off at its entry into the cord. The root was desheathed, and the proximal end (several millimeters in length) was placed in the central superfusion chamber; the remainder of the root extended into the adjacent oil pool for recording. The polarization level of the lumbar dorsal root was recorded with a pair of Ag-AgC1 hook electrodes, positioned so that the proximal hook was placed on the root close to where it exited from the oil pool into the superfusion chamber and the other was further out on the root. DC signals from these electrodes were amplied with a differential amplifier and recorded on a pen recorder. The preparation was grounded with an Ag-AgCI electrode making contact with the Ringer in the bath through a salt bridge ( 0 . 9 ~ NaC1, 5°//o Agar). The contlol Ringer contained 100 mM NaC1, 2.4
mM KCI, 9.5 mM NaHCO3, 1.9 mM CaC12, 2.8 mM D-glucose and 10 mM Tris buffer (pH 7.4). The Ringer was continuously bubbled with 95 0/o Oz/5 o/ • / 0 CO2. K+-rich Ringer solutions were made by equimolar substitution of K ~ for N a ' . For divalent cation-rich Ringer solutions MnCI2 (2 mM), COC12 (2 mM), CaC12 (2 or 10 mM) or MgSO4 (20 mM) was added to the Ringer without concomitant removal of other ions. These concentrations of Mn 2 ', Co 2+ and Mg 2+ were chosen because they are commonly used to block synaptic transmission (see for example refs. 1 and 13). Two millimolar Ca 2 ~ was used for comparison with 2 mM Mn 2~ and Co s+. Ten millimolar Ca z+ was selected because for previously studied phenomena in which Ca zt and Mg 2~ act in the same direction, Ca 2 ~ was about twice as potent as Mg 2÷ (refs. 10, 11). In previous experiments, we found no difference between the effect of MgSO4 and MgCI2 on K+-induced depolarizations (ref. 21); MgSO4 was used in this study because it is less hygroscopic and therefore is easier to weigh accurately.
A.
5.
157ram K +
9.9 rnM K "1"-
25mM K +
_ _ _ , , . - ~ ~
_
I
'9.9 rnM K÷
20raM Mg ++
_llmV Irnin
13.TrnM K+
20raM Mg *'t"
B.
_J2mV Imin
25mM K + 2 0 m M Mg t +
._] 5 mV Imin
C.
25mM K +
25rnM K +
20mMK+
2J mv
2omM.*
2 m M Mn "='+
Imin
2 m M Ca + +
/'mv Imin
Fig. 1. DC depolarization of the dorsal root when K+0 was raised from 2.4 mM to the concentration listed below each trace. The K+-rich solution was applied for the duration of the dark bars under each trace. Upper traces show control K+ depolarizations and lower traces show responses to these same K + concentrations when given in the presence of an additional 20 mM Mg2÷ (A), 2 mM Mn~+ (B), or 2 mM Ca~+ (C).
183 Control depolarizations by K+-rich Ringer solutions containing 9.9, 13.7 or 25.0 m M were determined. Each K+-rich solution was applied until the dorsal root depolarization reached a new steadystate level (6-8 min) and then was washed out for 8-10 min between K + doses allowing the D C voltage to return to baseline (see Fig. 1A). A solution rich in divalent cation was then applied for 20 min and the K+-induced depolarization repeated in the presence of the elevated divalent cation concentration (see lower traces Fig. 1A). After 20 min to wash out the high divalent cation solution, a second control determination of K+-induced depolarizations was made to make sure that changes in the K ÷ depolarizations were not due to a general deterioration of the preparation. Fig. 1A shows the effect of addition of 20 m M Mg 2+ on K--induced depolarizations. At all 3 K + concentrations, the K+-induced depolarization is reduced in amplitude by high Mg 2+. Two millimolar Mn 2+ caused a similar, though smaller magnitude change in the 25 m M K ÷ depolarization (Fig. 1B) whereas 2 m M Ca 2+ caused no detectable change in amplitude of the 25 m M K ÷ depolarization (Fig. 1C). The mean percent decreases in K ÷ depolarizations caused by the 5 high divalent cation conditions are shown in Fig. 2. The largest decreases were caused by 20 m M Mg z÷ and 10 m M Ca 2÷ (topmost curves) and there was no significant difference between these two groups when tested with a two-way analysis of variance (ANOVA) with repeated measures is (P > 0.05). Intermediate decreases in K ÷ depolarizations were seen with 2 mM CO z÷ and 2 m M Mn 2÷ while 2 m M Ca 2÷ caused smaller decreases. A similar A N O VA comparing 2 m M Co g÷, Mn 2÷ and Ca ~÷ effects showed a significant difference between treatments ( F = 9.34; df = 2,9; P < 0.01). The Dunn test for post-hoc comparisonslS showed that 2 m M Co 2÷ and 2 m M Mn 2÷ were not significantly different (P > 0.05) but Co 2÷ and 2 m M Ca z÷ (bottom two curves) did differ significantly from each other (P < 0.01). In each divalent cation treatment group, the decrease in depolarization by 9.9 m M K + was greater than the decrease in 25.0 m M K + depolarization (see Fig. 2). This observation is supported statistically, since both A N O V A s mentioned above showed a
70 E3
z
+l
~ : z 50 0 WN LU II~--I 0 ¢ WII. 1211,1 Z
m n~ w
I0 .f,r,
•
I0
,
15
,
20
~o
K ÷ CONCENTRATION (raM)
Fig. 2. The percent decrease in K+-induced depolarizations caused by the 5 divalent cation conditions: 10 mM Ca 2+ (O), 20 rnM Mg2+ (A), 2 mM Mn e+ (©), 2 mM Cos+ (A), and 2 mM Ca e+ ([]). K + concentration is shown on a logarithmic scale. Each point represents the mean 4- S.E.M. of 4 experiments.
significant effect of K + concentration on the magnitude of the decrease in depolarization: for the 20 m M Mg 2+ vs 10 m M Ca 2+ test ( F = 27.4; df ----2,12) and for the 2 m M Ca z+, Co 2+ and Mn 2+ test (F = 30.4; df = 2,18) P < 0.001. There was no significant interaction effect between the divalent cation condition used and K + concentration seen in either A N O V A (P > 0.05). Elevating the divalent cation concentration not only decreases the amplitude of the K+-induced depolarization but also slows the rate of rise of the depolarization (see Figs. 1A and B). This effect occurred at all 3 K + concentrations but was most apparent for 25.0 m M K + responses. The slowing of the rate of rise was quantitated by measuring the time to half amplitude for control depolarizations and in the presence of divalent cation-rich solutions, and calculating the percent of control time to half amplitude. Only depolarizations by 25 m M K + were analyzed because the accuracy of the determination of time to half amplitude for the smaller K + concentrations was questionable. The mean percent of control time to half amplitude (:k S.E.M.) after 2 m M Ca z+, 2 m M Co ~+, 2 mM Mn z+, 10 m M Ca 2+ and 20 mM Mg z+ were 128 -+- 18, 176 ± 17, 234 -+- 40, 457 zk 147 and 717 zk 166, respectively.
184 In addition to blocking synaptic transmission, 2 mM Co 2~, 2 mM Mn 2+ and 20 mM Mg 2+ can increase threshold and interfere with action potential propagationL We now report that these divalent cations, when tested in concentrations used to block synaptic transm;ssion~, 13 alsc reduce depolarizations caused by increasing the extracellular K + concentration (K+0) and decrease the rate of rise of such depolarizations. Elevated Ca 2+ also causes this effect, consistent with an early observation that Ca "~+ opposes K + depolarization of frog node of Ranvier (ref. 15). A rank order of effectiveness of the divalent cations in decreasing the K+-induced depolarization can be proposed based on the data in Fig. 2 and the statistical comparisons between groups described above. The effect of 20 m M Mg 2+ was not significantly different from the effect of 10 mM Ca 2+, therefore, Mg 2+ appears to be less effective than Ca z+ in producing the decrease. Two millimolar Ca 2+ was the only condition which caused no detectable decrease in 25 m M K ÷ depolarization and the effect of 2 m M Ca z+ was significantly less than the action of 2 mM Co 2+, the next curve above Ca 2+ in Fig. 2. There was no significant difference between 2 mM Co 2+ and Mn 2+ and therefore they appear to be equally effective. Taken together, these results suggest the following order of effectiveness in decreasing K+-induced depolarization: Mn 2+ ~ Co 2+ Ca 2÷ :> Mg 2+. Additional experiments varying the concentration of each divalent cation will be required, however, for unequivocal determination of the relative potency of these ions. The mechanism underlying the decrease in K +induced depolarization by elevated divalent cation concentration may be a reduction in the K + conductance (gK) of the membrane. A reduction in the steady-state gK would decrease the influence of K+0 in determining the resting membrane potential, and therefore should reduce the depolarization which occurs when K+0 is raised. Increasing K+0 not only causes membrane depolarization as measured in our study, but also increases the membrane conductance (ref. 17). Dubois and Bergman s have shown with voltage-clamp of frog node that either increasing K+0 or membrane depolarization can increase the steady-state gK. Furthermore, the opening of K ÷ channels by depolarization or raising K+0 depends
on the extracetlular Ca 2÷ concentration, gK being lower when more Ca 2+ is present. Increases in the external Ca `'.+ concentration have been found to shift the gK vs voltage curve to the right; that is, more depolarization is required to activate gKS,9,10,12. Mg 2÷ (refs. 5, 20), Co ')T and Ni 2+ (ref. 20) exert the same effect. The shift in the steady-state gK vs voltage curve in the presence of elevated divalent cation concentration can be quantitatively accounted for by a shift in the membrane surface potential 20. Interestingly, the order of effectiveness of divalent cations in reducing K+-induced depolarization of afferent fibers that we report is the same as the order of the constants for adsorption of Co 2+, Ca 2+ and Mg 2+ to the external membrane surface calculated by Mozhayeva and Naumov 2°. The order we found: Mn 2+ ~ Co 2+ ~ Ca 2 ~ ~ Mg 2+ was also the same as the order of effectiveness found by Hille et al. 16 for causing a positive voltage shift in sodium activation, an effect which appears to be due to a change in membrane surface potential, as well. The decrease in the rate of rise of K~-induced depolarization by elevated divalent cation concentration which we report may be explained by Frankenhaueser and Hodgkin's 1° finding that increasing external Ca 2+ delayed the onset and decreased the rate of rise in gK with depolarization. These effects could be mimicked by polarizing the membrane potential, which suggests that a change in membrane surface potential may be responsible for this action of divalent cations, as well. In conclusion, changes in divalent cation concentration often used to block or enhance synaptic transmission have other actions as well. In addition to the previously documented effects on threshold and excitability, divalent cation concentration can alter very basic neuronal properties such as the influence of K+0 on the membrane potential. Our thanks to Edmund Anderson and Jeremy Shefner for their helpful comments on the manuscript.
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