Faculty Bibliography

1. Whole-cell recordings of voltage-gated Ca2+ current in single smooth muscle cells from rabbit ear artery were obtained with 110 mM-Ba2+ as charge carrier. 2. Noradrenaline (NA, 1-20 microM) produced a sustained increase in the dihydropyridine-sensitive L-type Ca2+ current, ranging up to 3-fold in some cells. The dihydropyridine-resistant T-type Ca2+ current was not affected. 3. The time and voltage dependence of activation and inactivation of the L-type current were not significantly changed during NA modulation. 4. The NA-induced increase in L-current was enhanced in magnitude and consistency by the inclusion of 200 microM-GTP in the pipette (internal) solution. 5. The effect of NA on L-current was not abolished by pre-treatment with prazosin, phentolamine or propranolol, suggesting that it is not mediated by alpha- or beta-adrenoceptors. 6. Phenylephrine (5 microM) was ineffective as an agonist, while adrenaline was approximately equipotent to NA. In these respects, the pharmacology of L-current modulation resembles that of 'gamma'-adrenergic receptors (Hirst & Nield, 1980). 7. NA modulation of L-type Ca2+ channels may be particularly important in promoting sympathetic vasoconstriction in resistance vessels where Ca2+ stores are relatively poorly developed and where NA-evoked contractions are very sensitive to organic Ca2+ channel antagonists

Changes in cytosolic free Ca2+ concentration [( Ca2+]i) due to Ca2+ entry or Ca2+ release from internal stores were spatially resolved by digital imaging with the Ca2+ indicator fura-2 in frog sympathetic neurons. Electrical stimulation evoked a rise in [Ca2+]i spreading radially from the periphery to the center of the soma. Elevated [K+]o also increased [Ca2+]i, but only in the presence of external Ca2+, indicating that Ca2+ influx through Ca2+ channels is the primary event in the depolarization response. Ca2+ release or uptake from caffeine-sensitive internal stores was able to amplify or attenuate the effects of Ca2+ influx, to generate continued oscillations in [Ca2+]i, and to persistently elevate [Ca2+]i above basal levels after the stores had been Ca2(+)-loaded

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel 'rundown' in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed 'B-type' because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms

Ca2+ imaging and single-channel recording were used to study the regulation of cytosolic free Ca2+ ([Ca2+]i) in local regions of frog sympathetic neurons. Digital imaging with the fluorescent Ca2+ indicator fura-2 demonstrated: (i) resting [Ca2+]i of 70-100 nM; (ii) significant increases in [Ca2+]i in growth cones and cell bodies following depolarization induced by extracellular electrical stimulation or increased external K+; (iii) in cell bodies, large transient increases in [Ca2+]i following exposure to caffeine and sustained oscillations in [Ca2+]i in the presence of elevated K+ and caffeine; and (iv) in growth cones, smaller and briefer changes in [Ca2+]i in response to caffeine. The nature of the depolarization-induced Ca2+ entry was studied with cell-attached patch recordings (110 mM Ba2+ in recording pipette). Ca2+ channel activity was observed in 18 of 20 patches on cell bodies, 3 of 5 patches along neurites, and 36 of 41 patch recordings from growth cones. We observed two types of Ca2+ channels: L-type channels, characterized by a 28-pS slope conductance, sensitivity to dihydropyridine Ca2+ channel agonist, and availability even with depolarizing holding potentials; and N-type channels, characterized by a 15-pS slope conductance, resistance to dihydropyridines, and inactivation with depolarized holding potentials. Both types of channels were found on growth cones and along neurites as well as on cell bodies; channels often appeared concentrated in local hot spots, sometimes dominated by one channel type

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

Properties of cardiac Ca channels have come into sharper focus with the advent of single cell preparations and suction pipette recording methods. We briefly summarize our present picture of the gating and permeation properties of the conventional, dihydropyridine-sensitive type of Ca channel (L-type). Distinctive features of a second type of voltage-gated Ca channel (T-type) are discussed

1. 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. greater than -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, tau approximately 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. greater than -60 mV and is progressively removed between -60 and -95 mV. 4. Component N activates at relatively strong depolarizations (t.p. greater than -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. 7. Cadmium ions (20-50 microM) virtually eliminate both N and L currents (greater than 90% block) but leave T relatively unaffected (less than 50% block). 200 microM-Cd2+ blocks all three components. 8. Nickel ions (100 microM) strongly reduce T current but leave N and L current little changed. 9. The dihydropyridine antagonist nifedipine (10 microM) inhibits L current (approximately 60% block) at a holding potential that inactivates half the L-type channels.(ABSTRACT TRUNCATED AT 400 WORDS)

1. 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) in the recording pipette and with isotonic potassium aspartate in the bathing solution to zero the cell membrane potential. 2. L-type channels are distinguished by a unitary slope conductance of 25 pS, activation over the range of membrane potentials between 0 and +40 mV, little inactivation over the course of a 136 ms depolarization, and availability for opening even at depolarized holding potentials (h.p. greater than -40 mV). L channels show a predominant mode of gating (mode 1) characterized by brief openings (approximately 1 ms), occasionally interspersed with another pattern of gating characterized by much longer openings (mode 2). 3. The dihydropyridine (DHP) Ca2+ agonist Bay K 8644 promotes mode 2 activity and shifts the voltage dependence of L-type channel activation towards more negative potentials. It leaves the unitary current-voltage relation unchanged. 4. Nifedipine, a DHP Ca2+ antagonist, strongly inhibits L-type channel activity through an increase in the proportion of blank sweeps. 5. T-type Ca2+ channels are distinguished by a much smaller unitary slope conductance (8 pS) and by activation and inactivation over relatively negative ranges of potential. Inactivation is complete by the end of 136 ms pulses to test potentials beyond -20 mV. 6. N-type Ca2+ channels are distinguished by an intermediate unitary slope conductance (13 pS), and by activation over a range of potentials between those of T- and L-type channels. Inactivation of N-type channels takes place over an exceptionally broad range of holding potentials (-80 to -20 mV). 7. Cell-attached patch data on the voltage dependence of activation and inactivation of T- and N-type channels are in excellent agreement with results from whole-cell recordings (Fox, Nowycky & Tsien, 1987) if allowances are made for variations in external surface potential. 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 (approximately 1 ms) openings, and a bi-exponential 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Freshly dispersed rabbit ear artery cells were studied using patch-clamp techniques to measure membrane currents in whole cells and single-channel currents in membrane patches. Whole-cell calcium currents recorded at physiologic extracellular calcium concentrations were small (approximately 10 pA). Recordings with 110 mM external barium gave much larger currents and revealed two current components with properties similar to those in other vascular smooth muscle preparations and to the calcium currents designated as T and L in heart cells and neurons. T current was activated with weak depolarizations and inactivated rapidly, while L current was activated with relatively strong depolarizations and inactivated more slowly. L current was increased by dihydropyridine agonists and decreased by dihydropyridine antagonists, while T current was unaffected. Recordings from cell-attached and outside-out membrane patches with 100 mM external barium showed unitary calcium channel currents with conductances of 8 and 25 pS. The small conductance channels had kinetic properties that accounted for T current in the whole-cell recordings, while the 25-pS channels showed the voltage dependence, the time dependence, and the dihydropyridine sensitivity expected for L-type channels. We conclude that vascular smooth muscle cells contain two types of calcium channels with properties very similar to those described for T- and L-type calcium channels in other cell types; the L current appears to be the predominant current component in whole-cell recordings