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.

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.

No abstract

Adrenergic modulation of calcium channels profoundly influences cardiac function, and has served as a prime example of neurohormonal regulation of voltage-gated ion channels. Channel modulation and increased Ca influx are mediated by elevation of intracellular cyclic AMP and protein phosphorylation. The molecular mechanism of the augmented membrane Ca conductance has attracted considerable interest. An increase in the density of functional channels has often been proposed, but there has previously been no direct evidence. Single-channel recordings show that isoprenaline or 8-bromocyclic AMP increase the proportion of time individual channels spend open by prolonging openings and shortening the closed periods between openings. To look for an additional contribution of changes in the number of functional channels, we applied ensemble fluctuation analysis to whole-cell recordings of cardiac Ca channel activity. Here we present evidence that in frog ventricular heart cells beta-adrenergic stimulation increases NF, the average number of functional Ca channels per cell. We also find that isoprenaline slows the time course of both activation and inactivation, and that the enhancement of peak current decreases gradually with greater membrane depolarization.

Endocytosis following exocytosis evoked by single step depolarizations was examined in bovine adrenal chromaffin cells using high resolution capacitance measurements in perforated‐patch voltage clamp recordings. Endocytosis was detected as a smooth exponential decline in membrane capacitance to either the pre‐stimulus level (‘compensatory retrieval’) or far below the pre‐stimulus level (‘excess retrieval’). During excess retrieval, > 10 % of the cell surface could be internalized in under 5 s. Compensatory retrieval was equal in magnitude to stimulus‐evoked exocytosis for membrane additions > 100 fF (about fifty large dense‐cored vesicles). In contrast, excess retrieval surpassed both the stimulus‐evoked exocytosis, and the initial capacitance level recorded at the onset of phase‐tracking measurements. Cell capacitance was not maintained at the level achieved by excess retrieval but slowly returned to pre‐stimulus levels, even in the absence of stimulation. A large percentage of capacitance increases < 100 fF, usually evoked by 40 ms depolarizations, were not accompanied by membrane retrieval. Compensatory retrieval could occur with any amount of Ca2+ entry, but excess retrieval was never triggered below a threshold Ca2+ current integral of 70 pC. The kinetics of compensatory and excess retrieval differed by an order of magnitude. Compensatory retrieval was usually fitted with a single exponential function that had a median time constant of 5.7 s. Excess retrieval usually occurred with double exponential kinetics that had an extremely fast first time constant (median, 670 ms) and a second time constant indistinguishable from that of compensatory retrieval. The speed of compensatory retrieval was Ca2+ dependent: the largest mono‐exponential time constants occurred for the smallest amounts of Ca2+ entry and decreased with increasing Ca2+ entry. The Ca2+ dependence of mono‐exponential time constants was disrupted by cyclosporin A (CsA), an inhibitor of the Ca2+‐ and calmodulin‐dependent phosphatase calcineurin. CsA also reduced the proportion of responses with excess retrieval, but this action was caused by a shift in Ca2+ entry values below the threshold for activation. The lower total Ca2+ entry in the presence of CsA was due to an increase in the rate of Ca2+ current inactivation rather than a reduction in peak amplitude. Our data suggest that compensatory and excess retrieval represent two independent, Ca2+‐regulated mechanisms of rapid membrane internalization in bovine adrenal chromaffin cells. Alternatively, there is a single membrane internalization mechanism that can switch between two distinct modes of behaviour.

A large-conductance calcium channel in chicken dorsal root ganglion neurons was studied with patch-

Fixed and diffusible calcium (Ca) buffers shape the spatial and temporal distribution of free Ca following Ca entry through voltage-gated ion channels. This modeling study explores intracellular Ca levels achieved near the membrane and in deeper locations following typical Ca currents obtained with patch clamp experiments. Ca ion diffusion sets an upper limit on the maximal average Ca concentration achieved near the membrane. Fixed buffers restrict Ca elevation spatially to the outermost areas of the cell and slow Ca equilibration. Fixed buffer bound with Ca near the membrane can act as Ca source after termination of Ca influx. The relative contribution of fixed versus diffusible buffers to shaping the Ca transient is determined to a large extent by the binding rate of each buffer, with diffusible buffer dominating at equal binding rates. In the presence of fixed buffers, diffusible buffers speed Ca equilibration throughout the cell. The concentration profile of Ca-bound diffusible buffer differs from the concentration profile of free Ca, reflecting theoretical limits on the temporal resolution which can be achieved with commonly used diffusible Ca indicators. A Ca indicator which is fixed to an intracellular component might more accurately report local Ca concentrations.