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

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 ( 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.

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Mechanism of activation by cGMP-dependent protein kinase of large Ca(2+)-activated K+ channels in mesangial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.5.c1669 ◽

1996 ◽

Vol 271 (5) ◽

pp. C1669-C1677 ◽

Cited By ~ 36

Author(s):

J. D. Stockand ◽

S. C. Sansom

Keyword(s):

Protein Kinase ◽

Mesangial Cells ◽

Hill Coefficient ◽

Voltage Sensitivity ◽

Open Probability ◽

Dependent Protein Kinase ◽

K Channels ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein ◽

Excised Patches

The patch clamp method was employed to establish the mechanism of regulation by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) of large Ca(2+)-activated K+ channels (BKCa) in human mesangial cells. Dibutyryl cGMP (DBcGMP) significantly increased open probability (Po) of BKCa in the absence but not in the presence of staurosporine in cell-attached patches. In excised patches, BKCa was activated by simultaneous addition of MgATP plus cGMP but not cAMP plus MgATP. Activation by cGMP plus MgATP was blocked by KT-5823, an inhibitor of PKG, but not by KT-5720, an inhibitor of cAMP-dependent protein kinase (PKA). Thus a cGMP-specific endogenous kinase is associated with mesangial BKCa. In excised patches, exogenous PKG but not PKA or protein kinase C activated BKCa. The half-activation potential (V1/2), defined as the potential at which the Po = 0.5 with 1 microM Ca2+, was -34 and 42 mV for activated and inactivated BKCa, respectively; however, the gating charge (Zg), a measure of voltage sensitivity, was not affected by PKG. Similarly, the Ca1/2 (free Ca2+ concentration required to activate to Po = 0.5 at 40 mV) decreased from 1.74 to 0.1 microM on addition of PKG, but the Hill coefficient, a measure of Ca2+ sensitivity, was not affected. Activation of BKCa by PKG was heterogeneous with two populations: the majority (67%) activated by PKG and the minority unaffected. It is concluded that an endogenous PKG activates BKCa by decreasing the Ca2+ and voltage activation thresholds independently of sensitivities.

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