ω-AgaIVA–Sensitive (P/Q-type) and –Resistant (R-type) High-Voltage–Activated Ba2+Currents in Embryonic Cockroach Brain Neurons

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Abstract
By means of the whole cell patch-clamp technique, the biophysical and pharmacological properties of voltage-dependent Ba2+currents ( IBa) were characterized in embryonic cockroach brain neurons in primary culture. IBawas characterized by a threshold of approximately −30 mV, a maximum at ∼0 mV, and a reversal potential near +40 mV. Varying the holding potential from −100 to −40 mV did not modify these properties. The steady-state, voltage-dependent activation and inactivation properties of the current were determined by fitting the corresponding curves with the Boltzmann equation and yielded V0.5of −10 ± 2 (SE) mV and −30 ± 1 mV, respectively. IBawas insensitive to the dihydropyridine (DHP) agonist BayK8644 (1 μM) and antagonist isradipine (10 μM) but was efficiently and reversibly blocked by the phenylalkylamine verapamil in a dose-dependent manner ( IC50= 170 μM). The toxin ω-CgTxGVIA (1 μM) had no significant effect on IBa. Micromolar doses of ω-CmTxMVIIC were needed to reduce the current amplitude significantly, and the effect was slow. At 1 μM, 38% of the peak current was blocked after 1 h. In contrast, IBawas potently and irreversibly blocked by nanomolar concentrations of ω-AgaTxIVA in ∼81% of the neurons. Approximately 20% of the current was unaffected after treatment of the neurons with high concentrations of the toxin (0.4–1 μM). The steady-state dose-response relationship was fitted with a Hill equation and yielded an IC50of 17 nM and a Hill coefficient ( n) of 0.6. A better fit was obtained with a combination of two Hill equations corresponding to specific ( IC50= 9 nM; n = 1) and nonspecific ( IC50= 900 nM; n = 1) ω-AgaTxIVA–sensitive components. In the remaining 19% of the neurons, concentrations ≥100 nM ω-AgaTxIVA had no visible effect on IBa. On the basis of these results, it is concluded that embryonic cockroach brain neurons in primary culture express at least two types of voltage-dependent, high-voltage–activated (HVA) calcium channels: a specific ω-AgaTxIVA–sensitive component and DHP-, ω-CgTxGVIA–, and ω-AgaTxIVA–resistant component related respectively to the P/Q- and R-type voltage-dependent calcium channels.