Ca2+/Calmodulin-Dependent Protein Kinase Regulates GABA-Activated Cl−Current in Cockroach Dorsal Unpaired Median Neurons

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Abstract
We studied γ-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 10−4M of GABA was applied by pneumatic pressure-ejection pulses. The semi-logarithmic curve of IGABAamplitude versus the ejection time yielded a Hill coefficient of 4.0. IGABAwas 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, IGABAwas 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 IGABAdisplayed 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, 10−3M) and calcium-free solution. The semi-logarithmic curve for CdCl2-resistant IGABAgave a Hill coefficient of 2.4. Hyperpolarizing voltage step from −50 to −80 mV was known to increase calcium influx through calcium-resting channels. According to this protocol, a significant increase of IGABAamplitude 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)-5-chloro-1-naphtalene-sulfonamide or the calcium-calmodulin-dependent protein kinase blocker 1-[ N,O-bis(5-isoquinolinesulfonyl)- N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) was added in the pipette solution, IGABAamplitude 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 CdCl2and 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 Ca2+-dependent mechanism via a calcium-dependent protein kinase. The consequences of the modulatory action of Ca2+in GABA receptors function and their sensitivity to insecticide are discussed.