Received for publication January 17, 2006.
Revised March 24, 2006.
Accepted for publication March 27, 2006.

Augmentation of Ca_v3.2 T-type Calcium Channel Activity by cAMP-dependent Protein Kinase A

Abstract

Ca²⁺ influx through T-type Ca²⁺ channels is crucial for important physiological activities such as hormone secretion and neuronal excitability. However, it is not clear whether these channels are regulated by cAMP-dependent protein kinase A (PKA). In the present study, we examined whether PKA modulates Ca_v3.2 T-type channels reconstituted in Xenopus oocytes. Application of 10 µM forskolin, an adenylyl cyclase stimulant, increased Ca_v3.2 channel activity by 40 ± 4% over 30 min, and negatively shifted the steady state inactivation curve (V₅₀ = -61.4 ± 0.2 mV vs -65.5 ± 0.1 mV). Forskolin did not affect other biophysical properties of Ca_v3.2 channels including activation curve, current kinetics, and recovery from inactivation. Similar stimulation was achieved by applying 200 µM 8-Br-cAMP, a membrane permeable cAMP analog. The augmentation of Ca_v3.2 channel activity by forskolin was strongly inhibited by preincubation with 20 µM H-89, and reversed by subsequent application of 500 nM of the inhibitor peptide (PKI) of PKA. The stimulation of Ca_v3.2 channel activity by PKA was mimicked by serotonin when 5HT₇ receptor was coexpressed with Ca_v3.2 in Xenopus oocytes. Finally, using chimeric channels constructed by replacing individual cytoplasmic loops of Ca_v3.2 with those of the Na_v1.4 channel, which is insensitive to PKA, we localized a region required for the PKA mediated augmentation to the II-III loop of the Ca_v3.2.

Key words: Cav3.2 T-type calcium channel, H-89, PKI, forskolin, protein kinase A, two-electrode voltage clamping

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