Calcium currents in the A7r5 smooth muscle-derived cell line. An allosteric model for calcium channel activation and dihydropyridine agonist action.

We have investigated the gating kinetics of calcium channels in the A7r5 cell line at the level of single channels and whole cell currents, in the absence and presence of dihydropyridine (DHP) calcium channel agonists. Although latencies to first opening and macroscopic currents are strongly voltage dependent, analysis of amplitude histograms indicates that the primary open-closed transition is voltage independent. This suggests that the molecular mechanisms for voltage sensing and channel opening are distinct, but coupled. We propose a modified Monod-Wyman-Changeux (MWC) model for channel activation, where movement of a voltage sensor is analogous to ligand binding, and the closed and open channels correspond to inactive (T) and active (R) states. This model can account for the activation kinetics of the calcium channel, and is consistent with the existence of four homologous domains in the main subunit of the calcium channel protein. DHP agonists slow deactivation kinetics, shift the activation curve to more negative potentials with an increase in slope, induce intermingled fast and slow channel openings, and reduce the latency to first opening. These effects are predicted by the MWC model if we make the simple assumption that DHP agonists act as allosteric effectors to stabilize the open states of the channel.

Calcium currents in bullfrog sympathetic neurons. II. Inactivation.

Calcium currents in bullfrog sympathetic neurons inactivate slowly and partially during depolarizations lasting 0.5-1 s. There is also a slower (minutes) inactivation process with a broad voltage dependence. An irreversible loss of current (rundown) is prominent with low concentrations of intracellular Ca2+ buffers, with either Ca2+ or Ba2+ as the charge carrier. The extent and rate of the more rapid inactivation process are maximal near the voltage at which the peak inward current is generated, suggesting that inactivation might be Ca2+ dependent. However, inactivation occurs with either Ca2+ or Ba2+ as the charge carrier, is not prevented by strong buffering of intracellular Ca2+ with 10 mM BAPTA, and varies little as the peak current is changed 10-fold by changing the divalent ion concentration. That is, rapid inactivation is not explained by simple versions of voltage, Ca2+- or current-dependent inactivation models. A model in which ion binding within the channel allows a slower, rate-limiting inactivation process fits some but not all of the observed features of inactivation. A purely voltage-dependent three-state cyclic model fits the data if microscopic inactivation is favored by hyperpolarization.

Calcium currents in bullfrog sympathetic neurons. I. Activation kinetics and pharmacology.

The calcium current of bullfrog sympathetic neurons activates and deactivates rapidly (tau less than 3 ms). For brief depolarizations, the current can be fit reasonably well by a Hodgkin-Huxley-type model with a single gating particle of charge +3. With 2 mM Ca2+ as the charge carrier, half-maximal activation occurs at approximately -5 mV, near the voltage where activation and deactivation are slowest. When extracellular divalent ion concentrations are reduced, monovalent ions (e.g., Na+ and methylammonium) produce kinetically similar inward currents. Current carried by Ba2+ is blocked by Cd2+ at micromolar concentrations, and by 100 nM omega-conotoxin. Commercially available saxitoxin blocks the current, but different batches have quantitatively different potency. The dihydropyridine agonist Bay K 8644 induces a slight shift in activation kinetics to more negative voltages, with little effect on the peak current. Nifedipine at least partially reverses the effect of Bay K 8644, but has little effect on its own. Muscarinic agonists and other ligands that inhibit the M-type potassium current of frog sympathetic neurons have weak inhibitory effects on the calcium current as well. One interpretation of these results is that the N-type calcium current predominates in these cells, with a minor contribution of L-type current.

Calcium currents in the A7r5 smooth muscle-derived cell line.

We have studied voltage-dependent calcium channels in the A7r5 smooth muscle cell line by measuring the high-affinity binding of radiolabelled dihydropyridines (DHPs), whole-cell and single-channel currents in patch-clamped cells, as well as cytosolic calcium ([Ca2+]i) in fura-2-loaded cell suspensions and monolayers. Intact A7r5 cells express saturable, high-affinity, voltage-sensitive DHP binding sites with pharmacological properties characteristic of L-type calcium channels. When cells were voltage clamped in the whole-cell configuration with near normal intra- and extracellular solutions, a DHP-sensitive inward current resembling the L-type calcium current was dominant. With barium (10 mM) as the charge carrier, peak inward currents were typically recorded at test potentials between 0 and +20 mV. Currents were blocked by extracellular cadmium with a half-maximal inhibitory concentration of approximately 1 microM. Isoproterenol (1 microM) or forskolin (10 microM) increased currents in approximately half of the cells tested. Forskolin (10 microM) increased single-channel activity in five of eight cell-attached patches. After cells had been quiescent for several weeks, cell suspensions showed changes in resting [Ca2+]i in response to DHPs and increased potassium. Most confluent monolayers of cells showed spontaneous transient elevations in [Ca2+]i. Bath application of Bay K 8644 increased the frequency and magnitude of these [Ca2+]i transients, whereas nifedipine abolished the transients. These data suggest that the [Ca2+]i transients were due to synchronous action potentials in electrically coupled cell monolayers.