Depolarizing pulses

Ca2+ is necessary for transmission at the neuromuscular junction and other synapses and plays a special role in exocytosis. In most cases in the CNS and PNS, chemical transmission does not occur unless Ca2+ is present in the extracellular fluid. Katz and Miledi [16] elegantly demonstrated the critical role of Ca2+ in neurotransmitter release. The frog NMJ was perfused with salt solution containing Mg2+ but deficient in Ca2+. A twin-barrel micropipet, with each barrel filled with 1.0mmol/l of either CaCl2 or NaCl, was placed immediately adjacent to the terminal. The sodium barrel was used to depolarize the nerve terminal electrically and the calcium barrel to apply Ca2+ ionotophoretically. Depolarization without Ca2+ failed to elicit an EPP (Fig. 10-6A). If Ca2+ was applied just before the depolarization, EPPs were evoked (Fig. 10-6B). In contrast, EPPs could not be elicited if the Ca2+ pulse immediately followed the depolarization (Fig. 10-6C). EPPs occurred when a Ca2+ pulse as short as 1 ms preceded the start of the depolarizing pulse by as little as 50-100 (xs. The experiments demonstrated that Ca2+ must be present when a nerve terminal is depolarized in order for neurotransmitter to be released. 

Fig. TI.4 The exocytosis probability as it depends on the number of channels in a cluster and the duration of the depolarizing pulse. The curves are for the following duration times of the depolarizing pulse (from bottom to top) 10, 20, 30, 40 and 50 ms. The delay time is equal to 2 ms.

The simulation data were extended for other multiple channel systems, up to ten channels these data are summarized in Figs. 11.4 to 11.6. The importance of channel clustering in case of large delay times is obvious from these figures, e.g. in the case of a 10 ms latency time, depolarizing pulses shorter than 20 ms practically do not induce exocytosis, even with clusters containing ten channels. 

In synapses where the speed of exocytosis is usually most important, neutralization of the delay time in sensing the rising concentration of Ca2+ is achieved by arranging a large number of Ca2+ channels that open per individual secretory vesicle. Such an arrangement increases the probability of exocytosis practically to unity, even for short depolarizing pulses, i.e. comparable to the latency time q [14]. 

D. Skin Capacitance and the Use of Direct and Depolarizing Pulsed Currents... 

Development of the slow tail current during a depolarizing pulse was taken as a measure of the rate at which the sodium channels are modified. It had a fast and a slow phase, and the latter disappeared after removal of sodium channel inactivation with pronase. Based on these and other results, a kinetic scheme was developed (Figure 2). Tetramethrin modifies the sodium channel in both closed and open states, and the modified channel opens and is inactivated much more slowly than the normal channel (15). However, we have very recently shown that the apparent inactivation of the modified sodium channel is a result of depletion of sodium ions in the periaxonal space (20). Figure 2 incorporates the revised version of the kinetic scheme. 

Figure 1. Membrane sodium currents in a squid giant axon before (a) and during (b) internal perfusion with 1 / M (+)-trans allethrin. Sodium current associated with a step depolarization from -100 mV to -20 mV was recorded after cesium and tetramethylammonium had been substituted for internal K+ and external K+, respectively, to eliminate the potassium current. In the control record (a), the peak sodium current (Ip) is followed by a small slow sodium current (Is) during a depolarizing pulse, and the tail current (Itail) associated with step repolarization decays quickly. In the presence of allethrin (b), Ip remains unchanged while Is is greatly increased in amplitude. Itail s als0 increased in amplitude and decays very slowly.

Figure 3. Sodium currents recorded from the squid giant axons before (A) and after (B) internal application of 10 pM deltamethrin. External and internal sodium concentrations were 111 mM and 50 mM, respectively. A, a depolarizing pulse from the holding potential (V ) of -80 mV to -20 mV elicited the normal transient inward sodium current which decayed within 10 msec. Depolarization to a second depolarizing pulse (500 msec) to the sodium reversal potential (E a - +20 mV) yielded a negligible current. Repolarization to the holding potential (-80 mV) produced a very small inward sodium tail current. B, the same pulse protocol as that for A but in the presence of deltamethrin in another axon Note a large and prolonged tail current upon repolarization from +20 mV to -80 mV.

Fig. la-c. Additive after-effects of short depolarizing pulses in 15 pM veratridine (pH 8.1 21 C). a Time course of current during a 12-ms pulse (second trace) from Vh=-15 mV (holding potential) to V=48 mV, both potentials relative to the resting value (V=0 mV). Note that the pulse is followed by a fast and a much slower current tail whose start is shown, b After-effects of a 10-Hz train of seven 12-ms impulses from Vh=-6 mV to a value close to the reversal potential, c After-effects of a 4-Hz train of 18 pulses (I2-ms, Vh=-20 mV, V=128 mV). The after-effects increase as VHbecomes more negative (compare b and c). (With permission from Ulbricht 1969a)... 

Other consequences of the binding hypothesis are as follows. During trains of depolarizing pulses the peak Na current (corresponding to 0) should decrease at the rate with which the tail current (0 ) increases. This has been found in frog muscle (Sutro 1986), neuroblastoma cells (Barnes and Hille 1988), and frog nerve in which, however, the reverse process. 

A number of studies performed in voltage-clamped bovine chromaffin cells have also produced contradictory results. For instance, Artalejo et al. [82] measured KICm elicited by a train of 10 depolarizing pulses of 50 ms to -I-IO mV separated by 500 ms (5 s of stimulation) in bovine chromaffin cells 10 mM Ba (or Ca Q was used as charge carrier. They found that N or P channels contributed about 20% to exocytosis so-called facilitation Ca " channels (DHP-sensitive L-type channels), that were recruited by previous pretreatment with D1 receptor agonists or cAMP, contributed 80% of the exocytosis. The authors suggest that facilitation Ca " channels may be closer to the docking and release sites than either of the other two channels. ... 

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