Ca super(2+)/Calmodulin-Dependent Protein Kinase Regulates GABA-Activated Cl super(-) Current in Cockroach Dorsal Unpaired Median Neurons

• Published in: Journal of neurophysiology Vol. 87; no. 6; pp. 2972 - 2982
• Main Authors: Alix, P, Grolleau, F, Hue, B
• Format: Journal Article
• Language: English
• Published: 01.06.2002
• ISSN: 0022-3077

We studied gamma -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 (I sub(GABA)) when 10 super(-4) M of GABA was applied by pneumatic pressure-ejection pluses. The semi-logarithmic curve of I sub(GABA) amplitude versus the ejection time yielded a Hill coefficient of 4.0. I sub(GABA) was chloride (Cl super(-)) because the reversal potential given by the current-voltage (I-V) curve varied according to the value predicted by the Nernst equation for Cl super(-) dependence. In addition, I sub(GABA) 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 I sub(GABA) displayed a unexpected biphasic aspect and was best fitted by two linear regressions giving two slope conductances of 35.6 plus or minus 2.1 and 80.9 plus or minus 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 (CdCl sub(2), 10 super(-3) M) and calcium-free solution. The semi-logarithmic curve for CdCl sub(2)-resistant I sub(GABA) gave 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 I sub(GABA) amplitude was observed. However, this effect was never obtained when the same protocol was applied on cell body pretreated with CdCl sub(2). 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, I sub(GABA) 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 super(-) inward current which was insensitive to CdCl sub(2). 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 plus or minus 1.1 nS. These results, similar to those obtained with GABA in the presence of CdCl sub(2) and KN-62, indicated that CACA activated one subtype of GABA receptor. Our study demonstrated that at least two distinct subtypes of Cl super(-)-dependent GABA receptors were expressed in DUM neurons, one of which is regulated by an intracellular Ca super(2+)-dependent mechanism via a calcium-dependent protein kinase. The consequences of the modulatory action of Ca super(2+) in GABA receptors function and their sensitivity to insecticide are discussed.