Ca2+/calmodulin-dependent protein kinase regulates GABA-activated Cl- current in cockroach dorsal unpaired median neurons.
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(GABA)) when 10(-4) M of GABA was applied by pneumatic pressure-ejection pulses. The semi-logarithmic curve of I(GABA) amplitude versus the ejection time yielded a Hill coefficient of 4.0. I(GABA) 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, I(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(GABA) 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 calcium-free solution. The semi-logarithmic curve for CdCl2-resistant I(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(GABA) 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)-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(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- 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 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.
Alix
Philippe
P
Laboratoire de Neurophysiologie Unité Propre de Recherche de l'Enseignement Supérieur Equipe d'Accueil 2647, Université d'Angers, F-49045 Angers Cedex, France.
Grolleau
Francoise
F
Hue
Bernard
B
D000818:Animals; D019256:Cadmium Chloride / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D007306:Insecticides; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000201:enzymology*; D018408:Patch-Clamp Techniques; D010526:Periplaneta; D018079:Receptors, GABA / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D018118:Chloride Channels; D002712:Chlorides; D007306:Insecticides; D018079:Receptors, GABA; D005680:gamma-Aminobutyric Acid; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019256:Cadmium Chloride; D002118:Calcium