ω-AgaIVA-Sensitive (P/Q-type) and -Resistant (R-type) High-Voltage-Activated Ba<SUP>2+</SUP> Currents in Embryonic Cockroach Brain Neurons

By means of the whole cell patch-clamp technique, the biophysical and pharmacological properties of voltage-dependent Ba²⁺ currents (I<INF>Ba</INF>) were characterized in embryonic cockroach brain neurons in primary culture. I<INF>Ba</INF> was 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 V<INF>0.5</INF> of −10 ± 2 (SE) mV and −30 ± 1 mV, respectively. I<INF>Ba</INF> was 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 (IC<INF>50</INF> = 170 μM). The toxin ω-CgTxGVIA (1 μM) had no significant effect on I<INF>Ba</INF>. 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, I<INF>Ba</INF> was 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 IC<INF>50</INF> of 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 (IC<INF>50</INF> = 9 nM; n = 1) and nonspecific (IC<INF>50</INF> = 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 I<INF>Ba</INF>. 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.