On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

Abstract

The Ca2+ channel subunits alpha 1C-a and alpha 1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba2+ current (IBa) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 microM cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 microM PKA and 1 microM okadaic acid. The activity of the alpha 1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 microM H 89 and was not increased by superfusion with 5 microM forskolin plus 20 microM isobutyl-methylxanthine (IBMX). The alpha 1C-a.beta 2.alpha 2/delta complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 microM H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the alpha 1C-a.beta 2.alpha 2/delta channel with 10 microM PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca2+ current (ICa) of cardiac myocytes increased threefold during internal dialysis with 5 microM PKA or 25 microM microcystin and during external superfusion with 0.1 microM isoproterenol or 5 microM forskolin plus 50 microM IBMX. These results indicate that the L-type Ca2+ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.

The β1‐subunit is essential for modulation by protein kinase C of an human and a non‐human L‐type Ca2+ channel

“…This agrees with recent results by others [22] who showed that expression of L-type Ca 2+ channels in CHO or HEK cells were not sensitive to PKA. Again in agreement with our results, but in contrast to others [16,17], these authors could not observe double-pulse facilitation in artificially expressed L-type Ca 2+ channels.…”

β-Adrenergic regulation requires direct anchoring of PKA to cardiac Ca V 1.2 channels via a leucine zipper interaction with A kinase-anchoring protein 15

“…Despite much effort, it has not been possible to fully reconstitute ␤-adrenergic regulation or PKA regulation of holomeric Ca V 1.2 channels in nonmuscle cells (e.g., refs. [54][55][56]. Small increments in Ca V 1.2 channel activity have been observed, but typically Ͻ10% of the increases in I Ca that were observed in cardiac myocytes.…”

A potential site of functional modulation by protein kinase A in the cardiac Ca2+ channel α1C subunit

“…The presence of 13 may be necessary for a voltage-dependent facilitation of Ca 2+ channel current which, in turn, may be modulated by PKA [22]. However, most studies in expression systems do not support a role for the I] subunit, since coexpression of 13 with alc does not restore the positive modulation of the channel by PKA in Xenopus oocytes and in CHO cells and does not significantly alter the reducing effects of PKA inhibitors [20][21][22][23]. Our finding that the removal of a single PKA site in ~lC completely eliminates the effects of PKA inhibitors, and that the presence of the ~2A subunit does not restore these effects, also argues against the involvement of the 13 subunit in PKA modulation in the expression systems.…”

“…This assumption is supported by the finding that PKA inhibitors reduce the Ca 2+ current via channels formed by C~lc alone or with some or all of the auxiliary subunits in CHO and HEK293 cells and in Xenopus oocytes [17,[20][21][22], suggesting that Cqc is the main target for this modulation. There is little doubt that the observed decrease in Ca 2--channel current is caused by dephosphorylation of PKA sites due to the activity of endogenous protein phosphatases, because the peptide and proteins used to inhibit PKA in these experiments are highly specific toward PKA [21].…”

“…However, others [19,20] could not reproduce these results. When expressed in Xenopus oocytes, channels composed of cardiac or neuronal ale or cqc + (x2/~ are insensitive to PKA or PKA-activating treatments [21][22][23].…”