Kinetics of calcium inward current activation

Dear Sir: Recently Lee et al. (1978) and Akaike et al. (1978) have published in this Journa l two very interest ing papers , repor t ing elegant exper iments with the internally per fused nerve cell bodies. We are especially happy with the success of these authors: the idea o f how simple it is to substitute the cytoplasm of the cell body by artificial saline has once more appeared to be frui tful since our first repor ts on the approach (Kostyuk et al., 1975; Krishtal and Pidoplichko, 1975) and on the separat ion o f Na and Ca currents (Kostyuk et al., 1977). Akaike et al. (1978) have presented a descript ion o f the activation o f Ca inward cu r r en t using first o rd e r kinetics (the Hodgk in -Huxley variable m raised to the first power). T h e authors note that the time resolution of their modification o f the cell body perfus ion me thod could be insufficient. As the question o f how Ca channels funct ion seems now to be essential (because of the powerful new methods o f their investigation), we want to present some evidence that the kinetic scheme of Ca cu r ren t activation is m raised to the second power, as suggested in o u r earl ier pape r (Kostyuk et al., 1977). In this s tudy we used a fu r the r modification of the me thod o f intracellular dialysis (Krishtal, 1978) which achieves quite short resolut ion times near 70-150 /xs. In such exper iments the isolated cell is located between the two pores. One of them is used for the measu remen t of potential, and the feedback cur ren t is injected th rough the other . This technique eliminates about 95% of the series resistance and simultaneously makes the substitution of the internal medium very rapid and effective. We have p e r f o r m e d precise measurements of Ca inward currents and co r respond ing gating currents in neurones enzymatically isolated f rom the ganglia of the snail Helix pomatia. T e m p e r a t u r e s f rom 2 to 22~ were used. Gating and ionic cur rents were digitally separated as shown in Fig. 1 A to obtain reliable informat ion on the initial per iod o f the Ca cu r ren t development . A defini te delay in the deve lopment of the Ca cu r ren t is seen af ter the subtraction o f the gating cur ren t . T h e residual net ionic cu r ren t is perfectly app rox ima ted by m 2 model as shown in Fig. 1 B. It is very impor tan t that the time constants ~'m for ionic cur ren t and ton for cor responding gating cur ren t are practically the same. A good fit o f the Ca cu r ren t to m 2 kinetics is well suppor ted by the voltage d e p e n d e n c e of the gat ing-charge distribution curve which coincides with the m voltage d e p e n d e n c e (Fig. 2). Such cor respohdence seems to be peculiar since