Summary. The slow inward current carried by Na + through potential-dependent calcium channels in conditions when divalent cations were removed from the extracellular solution by EDTA has been investigated on isolated internally perfused neurons of the snail Helix pomatia. The calcium channels also acquire the capability to pass monovalent cations if other caIcium-binding substances are added to the extracellular solution. Based on these facts the conclusion is made that the immediate reason for the modification of the channel selectivity is the absence of divalent cations in the extracellular medium. All potential-dependent characteristics of the modified calcium channel are shifted by 60 to 70 mV in the hyperpolarizing direction compared with those of the original calcium channel. The series of relative permeabilities for modified calcium channels The induced sodium current decreases immediately when the concentration of divalent cations in the extracellular solution is elevated. This decrease is not potential dependent and can be approximated by Langmuir~ isotherm with dissociation constants pKc~ :PKsr :pKB,:pK~g = 6.6:5.5:4.8:4.2. The conclusion is drawn that the calcium channels in the somatic membrane have two ion-selecting filters with different functionsan external one consisting, probably, of several carboxylic groups which bind divalent cations in a highly specific manner and determine the impermeability of the channel to monovalent cations in physiological conditions, and the channel ion-selecting filter including a single carboxylic group normally determining the channel selectivity for different divalent cations.

Three types of electrically-operated calcium channels have been identified in the membrane of cultured dorsal root ganglion neurons from mouse embryos using patch clamp technique. Low-threshold inactivating (LTI) channels with the lowest unitary conductance (5.7 pS with 60 mM Sr2+ or 7.2 pS with 60 mM Ba2+) preserved their activity for a long time on excised membrane patches and were insensitive to dihydropyridine Ca channel agonist Bay K8644. Corresponding whole-cell current could be decreased by 40% with 25 microM D-600. High-threshold inactivating (HTI) channels had somewhat higher unitary conductance (7 pS and 11.4 pS for Sr2+ or Ba2+ at 60 mM concentration) and were much more dependent upon intracellular metabolic support. D-600 inhibited the corresponding HTI whole-cell current by about 10%. High-threshold non-inactivating (HTN) channels were the most conducting ones (9 pS and 18.4 pS for 60 mM Sr2+ or 60 mM Ba2+) and their functioning was strongly metabolic dependent. Whole-cell HTN current could be slightly enhanced by 10 microM Bay K8644 due to some prolongation of channel mean open time. Effect of Bay K8644 was much less pronounced than that reported for cardiac cells. HTN whole-cell current could be almost completely blocked by 25 microM D-600. The described three types of calcium channels revealed different potential dependence and absolute values of mean channel open time.

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Activation kinetics of single high-threshold inactivating (HTI or N-type) calcium channels of cultured dorsal root ganglion cells from mouse embryos was studied using a patch-clamp method. Calcium channels displayed bursting activity. The open-time histogram was single exponential with an almost potential-independent mean open time tau op = 1.2 msec. The closed-time histogram was multicomponent; at least three of the components were associated with the activation process. The "fast" exponential component with the potential-independent time constant tau fcl = 0.9 msec included all intraburst gaps, while two "slower" ones with potential-dependent time constants tau scl and tau vscl described shut times between bursts and between clusters of bursts. The burst length histogram was biexponential. The "fast" component with a relatively potential-independent time constant tau fbur = 0.6 msec described short, isolated channel openings while the "slow" component characterized real bursts with a potential-dependent mean life time. The waiting-time histogram could be fitted by a difference of two exponentials with time constants being the same as tau scl and tau vscl. The data obtained were described in the frame of a 4-state sequential model of calcium channel activation, in which the first two stages are formally attributed to potential-dependent transmembrane transfer of two charged gating particles accompanying the channel transitions between three closed states, and the third one to fast conformational changes in channel protein leading to the opening of the channel. The rate constants for all transitions were defined. The validity of the proposed model for both low-threshold inactivating (LTI or T-type) and high-threshold noninactivating (HTN or L-type) calcium channels is discussed.