omega-AgaIVA-sensitive (P/Q-type) and -resistant (R-type) high-voltage-activated Ba2+ currents in embryonic cockroach brain neurons

By means of the whole cell patch-clamp technique, the biophysical and pharm acological properties of voltage-dependent Ba2+ currents (I-Ba) were charac terized in embryonic cockroach brain neurons in primary culture. I-Ba was c haracterized by a threshold of approximately -30 mV, a maximum at similar t o 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, vol tage-dependent activation and inactivation properties of the current were d etermined by fitting the corresponding curves with the Boltzmann equation a nd yielded V-0.5 of -10 +/- 2 (SE) mV and -30 +/- 1 mV, respectively. I-Ba was insensitive to the dihydropyridine (DHP) agonist BayK8644 (I mu M) and antagonist isradipine (10 mu M) but was efficiently and reversibly blocked by the phenylalkylamine verapamil in a dose-dependent manner (IC50 = 170 mu M) The toxin omega-CgTxGVIA (1 mu M) had no significant effect on I-Ba. Mi cromolar doses of omega-CmTxMIIC were needed to reduce the current amplitud e significantly, and the effect was slow. At I mu M, 38% of the peak curren t was blacked after 1 h. In contrast, I-Ba was potently and irreversibly bl ocked by nanomolar concentrations of omega-AgaTxIVA in similar to 81% of th e neurons. Approximately 20% of the current was unaffected after treatment of the neurons with high concentrations of the toxin (0.4-1 mu M). The stea dy-state dose-response relationship was fitted with a Hill equation and yie lded an IC50 of 17 nM and a Hill coefficient (n) of 0.6. A better fit was o btained with a combination of two Hill equations corresponding to specific (IC50 = 9 nM; n = 1) and nonspecific (IC50 = 900 nM; n = 1) omega-AgaTxIVA- sensitive components. In the remaining 19% of the neurons, concentrations g reater than or equal to 100 nM omega-AgaTxIVA had no visible effect on I-Ba . On the basis of these results, it is concluded that embryonic cockroach b rain neurons in primary culture express at least two types of voltage-depen dent high-voltage-activated (HVA) calcium channels: a specific omega-AgaTxI VA-sensitive component and DHP-, omega-CgTxGVIA-, and omega-AgaTxIVA-resist ant component related respectively to the P/Q- and R-type voltage-dependent calcium channels.