Effects of Strychnine on the Potassium Conductance of the Frog N o d e of Ranvier B. I. S H A P I R O From the Department of Biology, Harvard University, Cambridge, Massachusetts 02138, and the Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195. Dr. Shapiro's present address is the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Maryland 20014.
A B S T R A C T The nature of the block of potassium conductance by strychnine in frog node of Ranvier was investigated. The block is voltage-dependent and reaches a steady level with a relaxation time of 1 to several ms. Block is increased by depolarization or a reduction in [K+]o as well as by increasing strychnine concentration. A quaternary derivative of strychnine produces a similar block only when applied intracellularly. In general and in detail, strychnine block resembles that produced by intracellular application of the substituted tetraethylammonium compounds extensively studied by C. M. Armstrong (1969.J. Gen. Physiol. 54: 553-575. 1971. J. Gen. Physiol. 58: 413-437). The kinetics, voltage dependence, and dependence on [K+]o of strychnine block are of the same form. It is concluded that tertiary strychnine must cross the axon membrane and block from the axoplasmic side in the same fashion as these quaternary amines. INTRODUCTION
Strychnine recently has been s h o w n to affect b o t h s o d i u m a n d p o t a s s i u m currents in the squid a x o n m e m b r a n e s (Shapiro et al., 1974). Earlier e x p e r i m e n t s had s h o w n that strychnine has s o m e effects on the action potentials in toad m y e l i n a t e d fibers (Maruhashi et al., 1956). This p r e s e n t p a p e r is i n t e n d e d to e x t e n d the s t u d y to the effects o f strychnine on a v o l t a g e - c l a m p e d f r o g n o d e o f Ranvier. It is f u r t h e r d e s i g n e d to answer several questions raised by the previous work on squid axons. First is w h e t h e r strychnine p r o d u c e s its voltage- a n d timed e p e n d e n t block o f potassium channels in a m a n n e r r e s e m b l i n g that o f certain q u a t e r n a r y a m m o n i u m ions ( A r m s t r o n g , 1971). T h e s e ions act f r o m the inside o f the a x o n , m o v i n g into potassium channels a f t e r the channels are o p e n for ion flow. T h e block is c o u n t e r a c t e d by the external p o t a s s i u m ions. T h e s e comp o u n d s act similarly o n f r o g nodes o f Ranvier ( A r m s t r o n g a n d Hille, 1972). A r m s t r o n g (1971) has estimated rate constants for the blocking reaction. T h e p r e s e n t p a p e r r e p o r t s similar e x p e r i m e n t s a n d analyses to c o m p a r e the details o f strychnine blockage with that p r o d u c e d by the q u a t e r n a r y a m m o n i u m (QA) ions. MATERIALS
AND
METHODS
The methods for voltage clamping the frog (Rana pipiens) node of Ranvier are those of Hille (1971). This is a modification o f the voltage clamp apparatus o f D o d g e and THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 69, 1977 • pages 897-914
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T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y • V O L U M E 6 9 • 1 9 7 7
F r a n k e n h a e u s e r (1958) and involves the use o f four fluid pools separated by three Vaseline gaps. Two high-bandwidth, high-gain feedback amplifiers control the voltage in two o f the pools. C u r r e n t is measured by the change in voltage in one o f the end pools. T h e internode in that pool serves as a resistive lead and the conversion of measured pool voltage into current d e p e n d s on measurement o f the resistance o f that lead. Since the axon is cut in the two end pools, the internal composition at the start o f the experiment can be varied by altering the contents of these pools. As a rule, the axon is cut in isotonic (120 mM) KC1. In a few experiments N-methylstrychnine (NMS) was a d d e d to one end pool after initial voltage clamp data were obtained. In these cases the axon in the current-passing pool was recut in a new solution containing NMS. Since the resistance of the current-passing pathway was changed by this p r o c e d u r e , recalibration was necessary. Several minutes are required for the NMS to diffuse the approximately 500 ~ m from the cut end to the region inside the node. T h e standard Ringer's solution bathing the node contained 115 mM NaC1, 2 mM KCI, 2 mM CaCI2, and 2 mM Tris (hydroxymethyl) a m i n o m e t h a n e buffer, p H 7.4. In high-KC1 solutions, some o f the sodium was replaced by potassium. T h e external calcium and buffer concentrations were maintained throughout. Experiments on potassium currents were p e r f o r m e d with 200 nM tetrodotoxin (TTX) (Sigma Chemical, Corp., St. Louis, Mo.). Contamination with the sodium channel current was minimal. Leakage current, along with some capacity current, was subtracted electronically in the m a n n e r o f Armstrong and Hille (1972). T h e measured leakage value was used to obtain the resistance of the axon lead from the current pool. All measurements were at 10°C. Strychnine base and strychnine sulfate were obtained from ICN K & K Laboratories Inc., Plainview, N.Y. Strychnine contains a tertiary nitrogen with a pKa of 7.45 in 120 mM KC1. We had previously postulated (Shapiro et al., 1974) that strychnine acts in the charged form on the inside o f the m e m b r a n e . Since strychnine is effective when applied externally we proposed that it crosses the m e m b r a n e , probably largely or entirely in the uncharged form. In o r d e r to test this hypothesis I synthesized a simple quaternary derivative, N-methylstrychnine by dissolving the free base in a large excess of iodomethane (Eastman Kodak Corp., Organic Chemicals Div., Rochester, N.Y.). T h e reaction occurs at r o o m t e m p e r a t u r e and yields a fine precipitate which was filtered and washed copiously with cold methanol. T h e precipitate was the~a dissolved in water and an aliquot was titrated. No g r o u p with a pKa between p H 5 and p H 9 was found. T h e precision of the titration was such that unreacted strychnine must have been
A B S T R A C T The nature of the block of potassium conductance by strychnine in frog node of Ranvier was investigated. The block is voltage-dependent and reaches a steady level with a relaxation time of 1 to several ms. Block is increased by depolarization or a reduction in [K+]o as well as by increasing strychnine concentration. A quaternary derivative of strychnine produces a similar block only when applied intracellularly. In general and in detail, strychnine block resembles that produced by intracellular application of the substituted tetraethylammonium compounds extensively studied by C. M. Armstrong (1969.J. Gen. Physiol. 54: 553-575. 1971. J. Gen. Physiol. 58: 413-437). The kinetics, voltage dependence, and dependence on [K+]o of strychnine block are of the same form. It is concluded that tertiary strychnine must cross the axon membrane and block from the axoplasmic side in the same fashion as these quaternary amines. INTRODUCTION
Strychnine recently has been s h o w n to affect b o t h s o d i u m a n d p o t a s s i u m currents in the squid a x o n m e m b r a n e s (Shapiro et al., 1974). Earlier e x p e r i m e n t s had s h o w n that strychnine has s o m e effects on the action potentials in toad m y e l i n a t e d fibers (Maruhashi et al., 1956). This p r e s e n t p a p e r is i n t e n d e d to e x t e n d the s t u d y to the effects o f strychnine on a v o l t a g e - c l a m p e d f r o g n o d e o f Ranvier. It is f u r t h e r d e s i g n e d to answer several questions raised by the previous work on squid axons. First is w h e t h e r strychnine p r o d u c e s its voltage- a n d timed e p e n d e n t block o f potassium channels in a m a n n e r r e s e m b l i n g that o f certain q u a t e r n a r y a m m o n i u m ions ( A r m s t r o n g , 1971). T h e s e ions act f r o m the inside o f the a x o n , m o v i n g into potassium channels a f t e r the channels are o p e n for ion flow. T h e block is c o u n t e r a c t e d by the external p o t a s s i u m ions. T h e s e comp o u n d s act similarly o n f r o g nodes o f Ranvier ( A r m s t r o n g a n d Hille, 1972). A r m s t r o n g (1971) has estimated rate constants for the blocking reaction. T h e p r e s e n t p a p e r r e p o r t s similar e x p e r i m e n t s a n d analyses to c o m p a r e the details o f strychnine blockage with that p r o d u c e d by the q u a t e r n a r y a m m o n i u m (QA) ions. MATERIALS
AND
METHODS
The methods for voltage clamping the frog (Rana pipiens) node of Ranvier are those of Hille (1971). This is a modification o f the voltage clamp apparatus o f D o d g e and THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 69, 1977 • pages 897-914
897
898
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y • V O L U M E 6 9 • 1 9 7 7
F r a n k e n h a e u s e r (1958) and involves the use o f four fluid pools separated by three Vaseline gaps. Two high-bandwidth, high-gain feedback amplifiers control the voltage in two o f the pools. C u r r e n t is measured by the change in voltage in one o f the end pools. T h e internode in that pool serves as a resistive lead and the conversion of measured pool voltage into current d e p e n d s on measurement o f the resistance o f that lead. Since the axon is cut in the two end pools, the internal composition at the start o f the experiment can be varied by altering the contents of these pools. As a rule, the axon is cut in isotonic (120 mM) KC1. In a few experiments N-methylstrychnine (NMS) was a d d e d to one end pool after initial voltage clamp data were obtained. In these cases the axon in the current-passing pool was recut in a new solution containing NMS. Since the resistance of the current-passing pathway was changed by this p r o c e d u r e , recalibration was necessary. Several minutes are required for the NMS to diffuse the approximately 500 ~ m from the cut end to the region inside the node. T h e standard Ringer's solution bathing the node contained 115 mM NaC1, 2 mM KCI, 2 mM CaCI2, and 2 mM Tris (hydroxymethyl) a m i n o m e t h a n e buffer, p H 7.4. In high-KC1 solutions, some o f the sodium was replaced by potassium. T h e external calcium and buffer concentrations were maintained throughout. Experiments on potassium currents were p e r f o r m e d with 200 nM tetrodotoxin (TTX) (Sigma Chemical, Corp., St. Louis, Mo.). Contamination with the sodium channel current was minimal. Leakage current, along with some capacity current, was subtracted electronically in the m a n n e r o f Armstrong and Hille (1972). T h e measured leakage value was used to obtain the resistance of the axon lead from the current pool. All measurements were at 10°C. Strychnine base and strychnine sulfate were obtained from ICN K & K Laboratories Inc., Plainview, N.Y. Strychnine contains a tertiary nitrogen with a pKa of 7.45 in 120 mM KC1. We had previously postulated (Shapiro et al., 1974) that strychnine acts in the charged form on the inside o f the m e m b r a n e . Since strychnine is effective when applied externally we proposed that it crosses the m e m b r a n e , probably largely or entirely in the uncharged form. In o r d e r to test this hypothesis I synthesized a simple quaternary derivative, N-methylstrychnine by dissolving the free base in a large excess of iodomethane (Eastman Kodak Corp., Organic Chemicals Div., Rochester, N.Y.). T h e reaction occurs at r o o m t e m p e r a t u r e and yields a fine precipitate which was filtered and washed copiously with cold methanol. T h e precipitate was the~a dissolved in water and an aliquot was titrated. No g r o u p with a pKa between p H 5 and p H 9 was found. T h e precision of the titration was such that unreacted strychnine must have been
