Studies of single calcium channel currents in rat clonal pituitary cells.

142

SUMMARY1. Single and multiple Ca2+ channel currents were recorded from outside-out and cell-attached patches of cultured chick and rat dorsal root ganglion cells, using the patch-clamp technique.2. Outside-out patches containing a large number of Ca2+ channels revealed Ca2+ currents resembling those from the whole cell. A low-voltage-activated (l.v.a.) and a high-voltage-activated (h.v.a.) Ca2+ current similar to those described in the accompanying paper (Carbone & Lux, 1987b) could be distinguished. The h.v.a. current component subsided within 10 min following the formation of the patch, while the l.v.a. component lasted much longer.3. Unitary events related to the l.v.a. Ca2+ channel could be clearly resolved in outside-out patches formed in Na+-and K+-free media containing 5-50 mM-CaCl2.4. The amplitudes of l.v.a. channel openings were bimodally distributed, indicating the presence of two conductive states. At -40 mV, mean amplitudes of the two events were -0-29 + 0-07 pA and -0 47 + 0-085 pA in 50 mM-CaCl2, with apparent slope conductances of about 3-6 and 5-2 pS, respectively. In 5 mM-CaCl2 both slope conductances were about 3 times smaller. The mean open times were similar for both states and were fitted by a simple exponential with a time constant of about 2-5 ms at -40 mV. The time constant decreased with more-negative membrane potentials and was 0 9 ms at -100 mV. Openings frequently occurred in bursts separated by longer-lasting closures. The mean closed time during bursts was 1-33 ms at -40 mV.5. Time and amplitude distributions of elementary events were similar for chick and rat sensory neurones and with Ba2+ and Sr2+ replacing external Ca2+. 6. In the potential range examined (from -60 to -30 mV), the first-latency distribution function revealed a distinct rise to peak which occurred at considerably earlier times than peaks of macroscopic currents. The time course of macroscopic l.v.a. Ca2+ currents could be simulated in two ways: (a) by using a five-state Markov-chain model with rate constants estimated from the transition probabilities and dwell times of the channel states, and (b) by evaluating the convolution integral of the first-latency function and the burst open probability of the channel. Both approaches suggest that activation and inactivation are weakly coupled and that the l.v.a. channel of sensory neurones reopens several times before inactivating. during bursts were between 0-2 and 0 3 ms at -50 mV. These currents disappeared in the absence of external Na+. They could be blocked by 5 mM-Ni2+ or 1 mM-Cd2+ and had activation and inactivation properties similar to those of l.v.a. currents.These observations suggest that at a low [Ca2+]0, Na+ ions flow through l.v.a. channels. Increases of external, but not internal, Ca2+ concentration, from 1 x 10-9 to 1 x 10-4 M blocked the Na+ currents through l.v.a. channels with the half-block occurring at a [Ca2+]0 of 2-3 /tM.8. Several types of unitary events were resolved in cell-attached patches formed with pipettes containing 95 mM-BaCl2. At pote...

Section: Resultsmentioning

confidence: 99%

Section: Inactivation In Single-channel Recordingsmentioning

confidence: 99%

See 1 more Smart Citation

Single low‐voltage‐activated calcium channels in chick and rat sensory neurones.

Carbone

Lux

1987

142

“…If this hypothesis is valid, reduction of the surface potential by lowering pHi would decrease i without changing the reversal potential or the maximum slope conductance at negative membrane potentials (Adrian, 1969). Another possible explanation is that H+ causes a fast block of the Ca2+ channel, as suggested for the H+ block of Na+ channels (Hille, 1968) and the block of unitary Ba2+ current through the Caa2+ channel by external Caa2+ (Cavalie et al 1983;Cachelin et al 1983;Brum et al 1984;Hess et al 1984;Ochi et al 1984b;Trautwein & Pelzer, 1985;Cavalie et al 1986) and in other tissues (Fenwick et al 1982;Brown, Camerer, Kunze & Lux, 1982;Hagiwara & Ohmori, 1983;Brown et al 1984 The most pronounced effect of Hi on the Ba2+ current through the Ca2+ channel has been detected in the slow gating behaviour. In a number of consecutive sweeps, runs of non-blank and blank sweeps alternate slowly, representing a slow transition between the available (A) and unavailable (B) states of the channel.…”

Section: Reduction Of the Unitary Currentmentioning

confidence: 96%

Inhibition of the calcium channel by intracellular protons in single ventricular myocytes of the guinea‐pig.

Kaibara

Kameyama

1988

SUMMARY1. The inhibitory effects of intracellular protons (Hi) on the L-type Ca2l channel activity were investigated in single ventricular myocytes of guinea-pigs by using the patch-clamp method in the open-cell-attached patch configuration, where 'run down' of the channel was partially prevented.2. Hi reduced the unitary Ba2+ current of the Ca21 channel by 10-20 % without changing the maximum slope conductance.3. Hi did not alter the number of channels in patches containing one or two channels.4. Hi markedly reduced the mean current normalized by the unitary current, which gave the open-state probability multiplied by the number of channels in the patch. The dose-response curve between Ht and the open-state probability indicated half-maximum inhibition at pHi 6-6 and an apparent Hill coefficient of 1.5. H+ shifted both the steady-state activation and inactivation curves in a negative direction by 10-15 mV, and the effects were reversible.6. H+ did not affect the fast open-closed kinetics represented by the C-C-O scheme, apart from increasing the slow time constant of the closed time.7. Hi increased the percentage of blank sweeps and reduced that of non-blank sweeps resulting in a decreased probability of channel opening.8. Photo-oxidation with Rose Bengal abolished the reducing effect of Hi on the open-state probability (P.) in two out of ten experiments, suggesting the possible involvement of histidine residues in the Ht effect. 9. The above results indicate that Ht inhibits the Ba2+ current mainly by affecting the slow gating mechanism of the channel.

“…Alternatively, the discrepancy could be explained by a model with four states as suggested by Hagiwara & Ohmori (1983) which, with a third closed state connected to the open state with voltage-independent rates, predicts the turn-on to be faster than the turn-off.…”

Section: Activation Of the Ca2+ Currentmentioning

confidence: 99%

Activation kinetics of calcium currents in bull‐frog sympathetic neurones.

Sala

1991

SUMMARY1. Calcium currents were recorded in dissociated bull-frog sympathetic neurones (BSNs) through patch pipettes using discontinuous voltage clamp. Activation kinetics were examined by analysing turn-on and turn-off currents.2. After short depolarizing pulses turn-off tail currents were fitted with the sum of two exponentials. The fast component (time constant, Tr t 240 jts at -40 mV) was undoubtedly due to the closure of calcium channels. The significance of a small and slower component is discussed.3. Neither activation nor deactivation time courses changed as channels inactivated during progressively longer pulses or when the holding potential was less negative. No specific component was selectively suppressed by these manipulations.4. Steady-state activation of the Ca2+ current was described by the Boltzmann distribution raised to the second power. Currents had an apparent threshold at -30 mV and were half-activated at + 5 mV.5. Calcium current turned on following m2 kinetics throughout the range of activation. The slowest time constant was around 1-2 ms between 0 and + 10 mV. Turn-on was faster at negative or more positive potentials.6. The time course of decay of tail currents became progressively faster at more negative potentials.7. The instantaneous current-voltages (I-V) curve was obtained from tail current measurements and fitted by a modified constant-field equation.8. The measured peak I-V curve could be reconstructed from the activation curve and the instantaneous I-V curve.9. The activation kinetics of the calcium current in BSNs were consistent with the existence of a single kinetic class of channels and can be described with a simple m2 Hodgkin-Huxley model.