Augmentation of Cav3.2 T-type calcium channel activity by cAMP-dependent protein kinase A.
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 Cav3.2 T-type channels reconstituted in Xenopus oocytes. Application of 10 microM forskolin, an adenylyl cyclase stimulant, increased Cav3.2 channel activity by 40+/-4% over 30 min and negatively shifted the steady-state inactivation curve (V50=-61.4+/-0.2 versus -65.5+/-0.1 mV). Forskolin did not affect other biophysical properties of Cav3.2 channels, including activation curve, current kinetics, and recovery from inactivation. Similar stimulation was achieved by applying 200 microM 8-bromo-cAMP, a membrane-permeable cAMP analog. The augmentation of Cav3.2 channel activity by forskolin was strongly inhibited by preincubation with 20 microM N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89), and reversed by subsequent application of 500 nM protein kinase A inhibitor peptide. The stimulation of Cav3.2 channel activity by PKA was mimicked by serotonin when 5HT7 receptor was coexpressed with Cav3.2 in Xenopus oocytes. Finally, using chimeric channels constructed by replacing individual cytoplasmic loops of Cav3.2 with those of the Nav1.4 channel, which is insensitive to PKA, we localized a region required for the PKA-mediated augmentation to the II-III loop of the Cav3.2.
Kim
Jin-Ah
JA
Department of Life Science, Sogang University, Mapo-Gu, Sinsu-Dong 1, Seoul 121-742, Korea.
Park
Jin-Yong
JY
Kang
Ho-Won
HW
Huh
Sung-Un
SU
Jeong
Seong-Woo
SW
Lee
Jung-Ha
JH
D000818:Animals; D020747:Calcium Channels, T-Type / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D006801:Humans; D051381:Rats; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D014982:Xenopus laevis
C509958:Cacna1h protein, rat; D020747:Calcium Channels, T-Type; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases