The effects of internal Ca2+ and Mg2+ on ion channels in the squid giant axon
The effects of internal Ca2+ and Mg2+ on ion channels in the squid giant axon
Abstract and Keywords
This chapter deals with the regulatory effects of internally added Ca2+ and Mg2+ on the Na+ and K+ channel activities of an excitable membrane. The regulatory effects of internally applied Ca2+ and Mg2+ ions, using intracellularly perfused squid axons, is discussed. It has previously beeen shown using squid giant axons that the action of internal Ca2+ on Na+ channel activity causes deterioration, or little effect. The internally added Ca2+ produces a Na+ action potential without any electrical stimulation in squid axons. Low concentrations of Ca2+ and Mg2+ exist in the cytoplasm of animal cells. Changes in concentration of Ca2+ are closely related to several functions, including membrane processes such as excitability and synaptic transmission. The internally applied Ca2+ and Mg2+ commonly have two types of effect on ion channel activities. First, they cause a lowering of the threshold voltage level for Na+ and K+ channel activation. The lowering of threshold potential caused by Ca2+ and Mg2+ is a notable and important effect which suggests that the membrane becomes more excitable in the presence of increased levels of intracellular divalent cations. Second, the internally applied Ca2+ and Mg2+ reduce Na+ and K+ currents by a concentration-dependent amount that differs for internally applied Ca2+ and Mg2+ respectively. The similarities and differences between Ca2+ and Mg2+ effects on ion channel activities are also described.
During intracellular perfusion with a 25 mM K+ solution containing Ca2+ in the range 0.1 to 10 mM, the action potential of the squid giant axon gradually decreased in amplitude, while the duration was transiently prolonged. The threshold potential for the initiation of the action potential was markedly lowered towards the resting potential level. A standard voltage clamp method was applied for the measurement of Na+ (I Na) and K+ currents (I K). The amplitude of I Na and I K were reduced by internal addition of 1–30 mM Ca2+ or Mg2+ to a 100 mM K+ solution. At the same time, a shift of the I–V curve in the negative voltage direction was observed. Up to 4 mM, the values for the voltage shift of I Na for an e-fold change in Ca2+ and Mg2+ concentration were 7 mV and 8 mV respectively. I Na and I K were gradually reduced by the increase in internal Ca2+ or Mg2+ concentration. The time constants for I Na reduction by Ca2+ and Mg2+ were 2.0 min and 1.8 min, respectively. The apparent dissociation constants (K D) of I Na reduction by Ca2+ and Mg2+ were 2.3 mM and 7.0 mM, respectively. I K was reduced more quickly but less effectively than I Na by Ca2+ and Mg2+ application.
Low concentrations of Ca2+ and Mg2+ exist in the cytoplasm of animal cells. Changes in concentration of Ca2+ are closely related to several functions, including membrane processes such as excitability and synaptic transmission, but less is known about the role of Mg2+. In the case of squid axons, the free Ca2+ concentration in the axoplasm has been estimated to be in the range 50–150 nM (Dipolo et al. 1976; Requena et al. 1986; Baker and Umbach 1987), while the free Mg concentration has been reported as 2–4 mM (Baker and Crawford 1972; Brinley and Scarpa 1975; DeWeer 1976).
This chapter deals with the regulatory effects of internally added Ca2+ and Mg2+ on the Na+ and K+ channel activities of an excitable membrane. Using squid giant axons, the action of internal Ca2+ on Na+ channel activity was reported as causing deterioration (Tasaki et al. 1965), or little effect (Begenisich and Lynch, 1974). We have previously shown that internally added Ca2+ produced a Na+ action potential without any electrical stimulation in squid axons (Yamagishi and Furuya 1981).
(p.154) Concerning the action of internal Ca2+ on K+ channel activity, the existence of Ca2+ activated K+ channels has been well established, starting with studies on Aplysia nerve cells (Meech and Strumwasser 1970) and cat motor neurones (Krnjevic and Lisiewicz 1972). Concerning Mg2+ effects on K+ channels, Matsuda et al. (1987) reported the blockage of outward K+ current in heart muscle cells. However, our understanding of the effect of divalent cations on the kinetics of Na+ and K+ channels is still very incomplete.
In this report, we show that internally applied Ca2+ and Mg2+ commonly have two types of effect on ion channel activities. First, they cause a lowering of the threshold voltage level for Na+ and K+ channel activation. Second, they reduce Na+ and K+ currents by a concentration-dependent amount that differs for internally applied Ca2+ and Mg2+, respectively. The similarities and differences between Ca2+ and Mg2+ effects on ion channel activities are described.
Effects of internal Ca2+ and Mg2+ on the action potential
The first intracellular recording of the action potential was made by A. L. Hodgkin (1939), using a giant axon of squid Loligo forbesi. He showed a beautiful action potential of an amplitude of about 90 mV, including a 40-mV overshoot. It was a great discovery and a starting point for the elucidation of the membrane phenomena of excitable cells. In the early 1960s, the groups of Hodgkin (Baker et al. 1961, 1962) and Tasaki (Oikawa et al. 1961) independently introduced successful intracellular perfusion of the squid giant axon.
This study discusses the regulatory effects of internally applied Ca2+ and Mg2+ ions, using intracellularly perfused squid axons. A giant axon of the squid Doryteuthis bleekeri was mounted in a Lucite chamber filled with artificial sea water (ASW; 450 mM NaCl, 10 mM KC1, 10 mM CaCl2, 20 mM MgCl2, 10 mM Tris–Cl, pH 8.0). As a standard internal solution, 25–100 mM KC1, K-glutamate, or KF solution was used, and the pH was adjusted to 7.4 with 4 mM Tris–HEPES. The tonicity was adjusted to that of the external solution by adding glycerol or glucose.
Figure 11.1 illustrates the typical change in action potential caused by the addition of 1 mM CaCl2 to the internal 25 mM KC1 solution. The resting potential and action potential amplitudes were −35 and 140 mV, respectively. On adding Ca2+ internally, the resting potential shifted slightly to the negative direction (hyperpolarization), while the action potential amplitude decreased to 110 mV over 2.2 min. At that time, the duration of the action potential at the level of half amplitude was prolonged to 12 ms, which was four times longer than the control. The most prominent feature was a lowering of the threshold potential at which the action potential initiates. The threshold value during resting conditions was −19 mV, which was 16 mV depolarized from the resting potential. On adding Ca2+, the threshold quickly dropped to (p.155)
This large lowering of threshold potential caused by Ca2+ and Mg2+ is a notable and important effect, and suggests that the membrane becomes more excitable in the presence of increased levels of intracellular divalent cations.
Shift of Na+ channel activation in a negative voltage direction
A standard voltage-clamp method was applied for the measurement of membrane currents. The axon immersed in ASW was perfused internally with a 100-mM K-glutamate and 10-mM tetraethylammonium-Cl solution for the measurement of Na+ current (I Na). Tetrodotoxin at a concentration of 0.3 μM was added to the ASW to block Na+ current and to allow measurement of K+ current (I k).
Figure 11.2 shows the effect of internally applied Ca2+. As shown in record 2 of the figure, the amplitude of the Na+ current was reduced by a 4-mM (p.156)
Figure 11.3 shows the values of the voltage shift caused by a range of concentrations of Ca2+ and Mg2+ (0.3–30 mM). The voltage shifts were plotted as the voltage difference at the level of 1/2 peak I Na before and after the Ca2+ or Mg2+ application. For an e-fold increase of Ca2+ and Mg2+ concentration, the voltage shifted by 7 and 8 mV, respectively, within the concentration range 0.3–4 mM. Compared with the effect of externally applied Ca2+ (Frankenhaeuser and Hodgkin 1957), the direction of the voltage shift of the Na+ activation curve was opposite, but the value of the shift was similar.
In the present experiments, it is proposed that negatively charged sites at the inner mouth of the Na+ channel are neutralized by the divalent cations, whereas the experiment of Frankenhaeuser and Hodgkin neutralized charged sites at the outer mouth of the Na+ channel. It would be predicted that charge neutralization at the two sites would shift the I–V curve in opposite directions, as observed. Shift of the I–V curve to the right (in the positive voltage direction) is associated with membrane stabilization, while shift to the left (negative voltage direction) increases excitability. The effects of elevated (p.157)
Reduction of Na+ and K+ currents
Figure 11.4 shows the time course of reduction of I Na and I K by different concentrations of internal Ca2+ and Mg2+. I Na and I K were reduced more strongly at higher Ca2+ or Mg2+ concentrations. The degrees and time course of I Na and I K reduction were different. I Na was reduced more slowly but more strongly compared with I K. The time constants of I Na and I K reduction were 2.0 and 0.5 min, respectively, while the apparent dissociation constants (K D) with 100-mM internal K+ were 2.3 and 5.8 mM for I Na and I K reduction, respectively.
Figure 11.5 shows the dose-response curves for the effect of Ca2+ and Mg2+ on I Na and I K with 100 mM internal K+. The K D for I Na reduction was 2.3 mM for Ca2+ and 7 mM for Mg2+. The Ca2+ effect was three times greater than the Mg2+ effect. The K D for I K reduction was 5.8 mM for Ca2+ and 10.2 mM for Mg2+.
We have mainly used an internal K+ concentration in the range up to 100 mM, in order to avoid the reduction of membrane excitability caused by high KC1 or K+-glutamate solution. In the axoplasm, the normal internal K+ concentration is about 400 mM (Hodgkin 1964). In the present experiments, the Ca2+ and Mg2+ effects were dependent on the internal K+ concentration [K+]i, and were roughly proportional to the [Ca2+]i/[K+]i or [Mg2+]i/[K+]i (p.159)
Intracellularly applied Ca2+ and Mg2+ exhibited dual effects on ion channel activities of squid axons. First, increased internal Ca2+ and Mg2+ caused the membrane to become more easily excitable, by charge neutralization at the inner mouth of the Na+ channels. Second, they reduced the I Na and I K amplitudes in a concentration-dependent manner. The Ca2+ concentration used was higher than that present in axoplasm, whereas the applied Mg2+ concentration was of the same order as that in axoplasm (2–4 mM). Thus, internal Mg2+ may play a role in physiological regulation of membrane excitability.
The authors wish to thank Drs J. N. Abbott, I. Inoue, and I. Tsutsui for their helpful suggestions and comments on the manuscript.
Baker, P. F., Hodgkin, A. L., and Shaw, T. I. (1961). Replacement of the protoplasm of a giant nerve fibre with artificial solutions. Nature, 190, 885−7.
Baker, P. F. and Crawford, A. C. (1972). Mobility and transport of magnesium in squid giant axons. Journal of Physiology, 227, 855−74.
Baker, P. F. and Umbach, J. A. (1987). Calcium buffering in axons and axoplasm of Loligo. Journal of Physiology, 383, 369−94.
Baker, P. F., Hodgkin, A. L., and Shaw, T. I. (1962). Replacement of axoplasm of giant nerve fibres with artificial solutions. Journal of Physiology, 164, 330−54.
Begenisich, T. and Lynch, C. (1974). Effects of divalent cations on voltage clamped squid axons. Journal of General Physiology, 63, 675−89.
Brinley, F. J. and Scarpa, A. (1975). Ionized magnesium concentration in axoplasm of dialysed squid axons. Federation of European Biochemical Societies Letters, 50, 82−5.
DeWeer, P. (1976). Axoplasmic free magnesium levels and magnesium extraction from squid giant axons. Journal of General Physiology, 68, 159−78.
Dipolo, R., Requena, J., Brinley, Jr, F. J., Mullins, L. J., Scarpa, A., and Tiffert, Y. (1976). Ionized Ca concentration in squid axons. Journal of General Physiology, 67, 433−67.
Frankenhaeuser, B. and Hodgkin, A. L. (1957). The action of calcium on the electrical properties of squid axons. Journal of Physiology, 137, 218−44.
Hodgkin, A. L. (1939). Action potentials recorded from inside a nerve fibre. Nature, 144, 710−11.
Hodgkin, A. L. (1964). The conduction of nerve impulses. The Sherrington Lecture VIII. Liverpool University Press.
Krnjevic, K. and Lisiewicz, A. (1972). Injection of calcium ions into spinal motoneurones. Journal of Physiology, 225, 363−90.
Matsuda, H., Saigusa, A., and Irisawa, H. (1987). Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+. Nature, 325, 156−9.
Meech, R. W. and Strumwasser, F. (1970). Intracellular calcium injection activates potassium conductance in Aplysia nerve cells. Federation Proceedings, 29, 834.
Oikawa, T., Spyropoulos, C. S., Tasaki, I., and Teorell, T. (1961). Methods for perfusing the giant axon of Loligo pealii. Acta Physiologica Scandinavica, 52, 195−6.
Requena, J., Mullins, L. J., Whittembury, J., and Brinley, Jr, F. J. (1986). Dependence of ionized and total Ca in squid axons on Nao-free or high Ko conditions. Journal of General Physiology, 87, 143−59.
Tasaki, I., Singer, L., and Takenaka, T. (1965). Effects of internal and external ionic environment on excitability of squid giant axon: A macromolecular approach. Journal of General Physiology, 48, 1095−123.
Yamagishi, S. and Furuya, K. (1981). Initiation of the spike by intracellularly perfused calcium in squid giant axons. Proceedings of the Japan Academy, 57B, 54−8.