How many types of calcium channels exist in neurones? This question is fundamental to understanding how calcium entry contributes to diverse neuronal functions such as transmitter release, neurite extension, spike initiation and rhythmic firing. There is considerable evidence for the presence of more than one type of Ca conductance in neurones and other cells. However, little is known about single-channel properties of diverse neuronal Ca channels, or their responsiveness to dihydropyridines, compounds widely used as labels in Ca channel purification. Here we report evidence for the coexistence of three types of Ca channel in sensory neurones of the chick dorsal root ganglion. In addition to a large conductance channel that contributes long-lasting current at strong depolarizations (L), and a relatively tiny conductance that underlies a transient current activated at weak depolarizations (T), we find a third type of unitary activity (N) that is neither T nor L. N-type Ca channels require strongly negative potentials for complete removal of inactivation (unlike L) and strong depolarizations for activation (unlike T). The dihydropyridine Ca agonist Bay K 8644 strongly increases the opening probability of L-, but not T- or N-type channels.
SUMMARY1. Calcium currents in cultured dorsal root ganglion (d.r.g.) cells were studied with the whole-cell patch-clamp technique. Using experimental conditions that suppressed Na+ and K+ currents, and 3-10 mM-external Ca2+ or Ba2+, we distinguished three distinct types of calcium currents (L, T and N) on the basis of voltage-dependent kinetics and pharmacology.2. Component L activates at relatively positive test potentials (t.p. > -10 mV) and shows little inactivation during a 200 ms depolarization. It is completely reprimed at a holding potential (h.p.) of -60 mV, and can be isolated by using a more depolarized h.p. (-40 mV) to inactivate the other two types of calcium currents.3. Component T can be seen in isolation with weak test pulses. It begins activating at potentials more positive than -70 mV and inactivates quickly and completely during a maintained depolarization (time constant, r-. 20-50 ms). The current amplitude and the rate of decay increase with stronger depolarizations until both reach a maximum at approximately -40 mV. Inactivation is complete at h.p. > -60 mV and is progressively removed between -60 and -95 mV.4. Component N activates at relatively strong depolarizations (t.p. > -20 mV) and decays with time constants ranging from 50 to 110 ms. Inactivation is removed over a very broad range of holding potentials (h.p. between -40 and -110 mV).5. With 10 mM-EGTA in the pipette solution, substitution of Ba2+ for Ca2+ as the charge carrier does not alter the rates of activation or relaxation of any component. However, T-type channels are approximately equally permeable to Ca2+ and Ba2+, while L-type and N-type channels are both much more permeable to Ba2 .6. Component N cannot be explained by current-dependent inactivation of L current resulting from recruitment of extra L-type channels at negative holding potentials: raising the external Ba2+ concentration to 110 mm greatly increases the amplitude of L current evoked from h.p. = -30 mV but produces little inactivation. 9. The dihydropyridine antagonist nifedipine (10 JIM) inhibits L current (-60 % block) at a holding potential that inactivates half the L-type channels. It does not reduce T or N currents at holding potentials that produce similar degrees of inactivation.10. w-Toxin fraction GVIA (wo-CgTX VIA), a peptide from the venom of Conus geographuas, was used to produce long-lasting block of N and L currents. Properties of T currents isolated in this manner agree with those inferred from kinetic analysis.
Multiple types of calcium channels have been found in neurons, but uncertainty remains about which ones are involved in stimulus-secretion coupling. Two types of calcium channels in rat sympathetic neurons were described, and their relative importance in controlling norepinephrine release was analyzed. N-type and L-type calcium channels differed in voltage dependence, unitary barium conductance, and pharmacology. Nitrendipine inhibited activity of L-type channels but not N-type channels. Potassium-evoked norepinephrine release was markedly reduced by cadmium and the conesnail peptide toxin omega-Conus geographus toxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine at concentrations that strongly reduce calcium influx, as measured by fura-2. Thus N-type calcium channels play a dominant role in the depolarization-evoked release of norepinephrine.
Blockade of Ca2+ channels by ai-conotoxin GVIA, a 27 amino acid peptide from the venom of the marine snail Conus geographus, was investigated with patch-clamp recordings ofwhole-cell and unitary currents in a variety ofcell types. In dorsal root ganglion neurons, the toxin produces persistent block of L-and N-type Cab channels but only transiently inhibits T-type channels. Its actions appear to be neuron-specific, since it blocks high-threshold Ca2' channels in sensory, sympathetic, and hippocampal neurons of vertebrates but not in cardiac, skeletal, or smooth muscle cells. Block occurs through direct interaction of the toxin with an external site closely associated with the Ca2+ channel, without apparent involvement of a second messenger or dependence on channel gating. The tissue and channel-type specificity and the directness and slow reversibility of the block are features that favor use of co-conotoxin as a tool for purifying particular neuronal Ca2+ channels and defining their physiological function.
SUMMARY1. The distribution of pharmacologically and/or biophysically unique Ca2+ current subtypes was studied in different diameter rat dorsal root ganglion (DRG) neuron cell bodies. DRG cells which fell into three diameter ranges, small (20-27 ,tm), medium (33-38 ,um) and large (45-51 ,um), were studied. T-type Ca2+ current was defined as low-threshold, rapidly inactivating current evoked by a weak test depolarization (-50 mV) from negative holding potentials (-80 to -100 mV), and which was sensitive to changes in holding potential. L-type Ca2+ current was defined as peak high-threshold Ca2+ current evoked from a holding potential of -60 mV and sensitive to blockade by 2 ,aM-nimodipine. N-type Ca2+ current was defined as peak high-threshold Ca2+ current evoked from a holding potential of -60 mV and sensitive to blockade by 0 9 JIM-cw-conotoxin GVIA.2. T-type Ca2+ currents were observed in small and medium diameter, but not in large diameter, DRG cell bodies. Large diameter DRG cell bodies had a small amount of low-threshold Ca2+ current but this current did not inactivate and was insensitive to a change in holding potential from -80 to -90 mV, and thus did not appear to be conducted through T-type Ca2+ channels. The T-type Ca2+ currents observed in medium diameter DRG cell bodies were considerably larger in amplitude (1-6 nA) than those observed in small diameter DRG cell bodies (100 pA-1 nA). This difference could not be accounted for by the difference in membrane surface area of small versus medium diameter DRG cell bodies. 3. The T-type Ca2+ currents observed in medium diameter DRG cells were sensitive to blockade by amiloride. Amiloride (500 /tM) blocked 79-4 + 0-9 % (mean+ S.E.M.) of T-type Ca2+ current amplitude in six medium diameter DRG cell bodies which were held at -80 mV and depolarized to -50 or -40 mV. Amiloride (500 1tM) failed to block high-threshold current in five medium diameter DRG cell bodies, indicating that it was specific for T-type Ca2+ current in these cells. 4. The percentage of peak whole-cell L-type Ca2+ current was significantly larger in small diameter DRG cell bodies (52-9 + 4-7 % of total whole-cell Ca2+ current) than in medium diameter DRG cell bodies (6-6 + 3-9 % of total whole-cell Ca2+ current) or large diameter DRG cell bodies (19-4+5-7% of total whole-cell Ca2+ current). The percentage of N-type Ca2+ current was similar in small, medium and large diameter M'S 9176 R. S. SCROGGS AND A. P. FOX DRG cell bodies, averaging 29-5 + 3-8, 35 9 + 4-5 and 24-7 + 2-5 % of total whole-cell Ca2+ current, respectively. There also was not a significant difference in N-type Ca2+ current density between the three size ranges.5. In the DRG neurons treated with a combination of 2 ,M-nimodipine and 0 9 ,mM-w-conotoxin, significantly more current was left unblocked in medium diameter DRG cell bodies (57-5 + 4-3 % of total whole-cell Ca2+ current) and large diameter cell bodies (55X8 +5*7 % of total whole-cell Ca2+ current) than in small diameter DRG cell bodies (17-6+2'7% of total whole-cell Ca...
SUMMARY1. T-, and L-type Ca2+ channels were studied in cell-attached patch recordings from the cell bodies of chick dorsal root ganglion neurones. All experiments were performed with isotonic BaCl2 (110 mM) A. P. FOX, M. C. NOWYCKY AND R. W. TSIEN 8. Patches containing one or two channels of a single type were used for analysis of gating kinetics. The predominant pattern of activity for each of the channel types is an exponential distribution of relatively brief (-1 ms) openings, and a biexponential distribution of short and long closings. 9. Patches containing all possible combinations of channel types were observed. However, preliminary evidence suggests that channels are distributed unevenly over the cell body; clustering of N-type channels is particularly prominent.10. In summary, each of the channel types displays a unique pattern of conductance, kinetic and pharmacological properties. The functional roles of the channel types and their distribution among cells are discussed.
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