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

By means of the whole cell patch-clamp technique, the biophysical and pharmacological properties of voltage-dependent Ba super(2+) currents (I sub(Ba)) were characterized in embryonic cockroach brain neurons in primary culture. I sub(Ba) was characterized by a threshold of approximately -30 mV, a maximum at similar to 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 sub(0.5) of -10 plus or minus 2 (SE) mV and -30 plus or minus 1 mV, respectively. I sub(Ba) was insensitive to the dihydropyridine (DHP) agonist BayK8644 (1 mu M) and antagonist isradipine (10 mu M) but was efficiently and reversibly blocked by the phenylalkylamine verapamil in a dose-dependent manner (IC sub(50) = 170 mu M). The toxin omega -CgTxGVIA (1 mu M) had no significant effect on I sub(Ba). Micromolar doses of omega -CmTxMVIIC were needed to reduce the current amplitude significantly, and the effect was slow. At 1 mu M, 38% of the peak current was blocked after 1 h. In contrast, I sub(Ba) was potently and irreversibly blocked by nanomolar concentrations of omega -AgaTxIVA in similar to 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 mu M). The steady-state dose-response relationship was fitted with a Hill equation and yielded an IC sub(50) 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 sub(50) = 9 nM; n = 1) and nonspecific (IC sub(50) = 900 nM; n = 1) omega -AgaTxIVA-sensitive components. In the remaining 19% of the neurons, concentrations greater than or equal to 100 nM omega -AgaTxIVA had no visible effect on I sub(Ba). 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 omega -AgaTxIVA-sensitive component and DHP-, omega -CgTxGVIA-, and omega -AgaTxIVA-resistant component related respectively to the P/Q- and R-type voltage-dependent calcium channels.