Abstract
Low voltage-activated (LVA), T-type, calcium channels mediate diverse biological functions and are inhibited by Gbetagamma dimers, yet the molecular events required for channel inhibition remain unknown. Here, we identify protein kinase A (PKA) as a molecular switch that allows Gbeta(2)gammax dimers to effect voltage-independent inhibition of Ca(v)3.2 channels. Inhibition requires phosphorylation of Ser(1107), a critical serine residue on the II-III loop of the channel pore protein. S1107A prevents inhibition of unitary currents by recombinant Gbeta(2)gamma(2) dimers but does not disrupt dimer binding nor change its specificity. Gbetagamma dimers released upon receptor activation also require PKA activity for their inhibitory actions. Hence, dopamine inhibition of Ca(v)3.2 whole cell current is precluded by Gbetagamma-scavenger proteins or a peptide that blocks PKA catalytic activity. Fittingly, when used alone at receptor-selective concentrations, D(1) or D(2) agonists do not elicit channel inhibition yet together synergize to inhibit Ca(v)3.2 channel currents. We propose that a dual-receptor regulatory mechanism is used by dopamine to control Ca(v)3.2 channel activity. This mechanism, for example, would be important in aldosterone producing adrenal glomerulosa cells where channel dysregulation would lead to overproduction of aldosterone and consequent cardiac, renal, and brain target organ damage.
Highlights
By contrast, Low voltage-activated (LVA) channels of Cav3 family have a less restricted distribution [9] and are expressed in peripheral tissues [10]
protein kinase A (PKA) Activity Enables the Inhibition of Cav3.2 Unitary Currents by G2␥2—In patches excised from HEK293 cells stably expressing Cav3.2 channels, recombinant G2␥2 subunits reduce the frequency of Cav3.2 unitary openings [23]
To test the importance of the II-III loop in the regulation of LVA channels by dopamine, we evaluated the regulation of a chimeric Cav3.2 channel (Cav3.2 (Cav3.1 II-III) that contains the II-III loop from Cav3.1 channels
Summary
Cell Culture and Transfection—H295R cells (human adrenocortical carcinoma cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 10% cosmic calf serum, 1 g/ml gentamicin. PKA and G␥ Dimer Regulation of Cav3.2 Channels embryonic kidney cells) were cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 1% pen/strep. Currents were sampled at 100 kHz and filtered at 2 kHz. The pipette solution (in mM): 75 CsCl, 60 CaCl2, and 10 HEPES, pH 7.4 (adjusted with CsOH). CHAPS and dithiothreitol were added to the bath solution to maintain the stability and solubility of G␥ recombinant proteins. Immunoprecipitation and Binding—HEK293 cells transfected with FLAG-tagged Cav3.2 and Cav3.2 (S1107A) channels were stimulated with 10 M 8-bromo-cAMP (10 min at 37 °C). Immunoprecipitated channel and bound recombinant proteins were eluted with 2ϫ SDS sample buffer at 80 °C. Each phosphorylation reaction (30 l) contained (in mM): 50 Tris-HCl pH 7.4, 11 MgCl2, 0.2 ATP, 0.2 M recombinant protein, 0.5 l PKA (Calbiochem, CA), 10 Ci [␥-32P]ATP. Phosphorylation levels were assessed by the Storm 820 Phosphorimager (Amersham Biosciences)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
