Ca /Calmodulin-Dependent Protein Kinase Regulates GABA-Activated Cl Current in Cockroach Dorsal Unpaired Median Neurons

Alix, Philippe, Francoise Grolleau, and Bernard Hue. Ca /Calmodulin-dependent protein kinase regulates GABA-activated chloride current in cockroach dorsal unpaired median (DUM) neurons. J Neurophysiol 87: 2972–2982, 2002; 10.1152/jn.00631.2001. We studied -aminobutyric acid (GABA)-mediated currents in short-term cultured dorsal unpaired median (DUM) neurons of cockroach Periplaneta americana using the whole cell patch-clamp technique in symmetrical chloride solutions. All DUM neurons voltage-clamped at 50 mV displayed inward currents (IGABA) when 10 4 M of GABA was applied by pneumatic pressure-ejection pulses. The semi-logarithmic curve of IGABA amplitude versus the ejection time yielded a Hill coefficient of 4.0. IGABA was chloride (Cl ) because the reversal potential given by the current-voltage (I-V) curve varied according to the value predicted by the Nernst equation for Cl dependence. In addition, IGABA was almost completely blocked by bath application of the chloride channel blockers picrotoxin (PTX) or 3,3-bis(trifluoromethyl)bicyclo-[2,2,1]heptane-2,2-diacarbonitrile (BIDN). The I-V curve for IGABA displayed a unexpected biphasic aspect and was best fitted by two linear regressions giving two slope conductances of 35.6 2.1 and 80.9 4.1 nS for potentials ranging from 0 to 30 and 30 to 70 mV, respectively. At 50 mV, the current amplitude was decreased by cadmium chloride (CdCl2, 10 3 M) and calciumfree solution. The semi-logarithmic curve for CdCl2-resistant IGABA gave a Hill coefficient of 2.4. Hyperpolarizing voltage step from 50 to 80 mV was known to increase calcium influx through calciumresting channels. According to this protocol, a significant increase of IGABA amplitude was observed. However, this effect was never obtained when the same protocol was applied on cell body pretreated with CdCl2. When the calmodulin blocker N-(6-aminohexyl)-5chloro-1-naphtalene-sulfonamide or the calcium-calmodulin-dependent protein kinase blocker 1-[N,O-bis(5-isoquinolinesulfonyl)-Nmethyl-L-tyrosyl]-4-phenylpiperazine (KN-62) was added in the pipette solution, IGABA amplitude was decreased. Pressure ejection application of the cis-4-aminocrotonic acid (CACA) on DUM neuron cell body held at 50 mV, evoked a Cl inward current which was insensitive to CdCl2. The Hill plot yielded a Hill coefficient of 2.3, and the I-V curve was always linear in the negative potential range with a slope conductance of 32.4 1.1 nS. These results, similar to those obtained with GABA in the presence of CdCl2 and KN-62, indicated that CACA activated one subtype of GABA receptor. Our study demonstrated that at least two distinct subtypes of Cl -dependent GABA receptors were expressed in DUM neurons, one of which is regulated by an intracellular Ca -dependent mechanism via a calcium-dependent protein kinase. The consequences of the modulatory action of Ca in GABA receptors function and their sensitivity to insecticide are discussed.