Tonic Protein Kinase A-Dependent Effects on K V Channels in Mesenteric Smooth Muscle Cells

Abstract

We examined the regulatory role of tonic cAMP-dependent protein kinase (PKA) activity on Kv current of smooth muscle cells isolated from 3rd/4th-order rat mesenteric arteries using whole-cell recording. Applying KT5720 (1 µM), a PKA inhibitor, attenuated the Kv current by 40.2 ± 7.4% (n = 4) at +50 mV; a similar inhibition was seen with Rp-cAMPS (100 µM). Since PKA activity is dependent on adenylyl cyclase (AC)-mediated generation of cAMP, inhibiting AC ought to mimic PKA inhibition. Application of the AC inhibitor 2’,5’-dideoxyadenosine inhibited the Kv current by 43.7 ± 7.1% (n=6). To assess whether these inhibitory effects involve a reduction in channel phosphorylation we examined whether inhibiting dephosphorylation maintains the activity of Kv channels even if PKA is inhibited. We found that cyclosporine A (4 µM), a protein phosphatase 2B inhibitor, abolished the inhibitory effect of KT5720, consistent with KT5720 reducing PKA-dependent phosphorylation of Kv channels. The targeting of PKA to phosphorylate K+ channels has been shown previously to involve A-kinase anchoring proteins (AKAP). Application of Ht-31 (20 µM ), an AKAP-mimetic peptide, did not inhibit Kv current, suggesting that PKA modulation of Kv channels in mesenteric smooth muscle may occur independently of AKAP. Attempts to enhance Kv current by activating PKA directly with dibutryl cyclic AMP (100 µM) were unsuccessful. Furthermore, β-adrenergic receptor activation with isoprenaline (100 nM), which increases cAMP levels substantially, was also ineffective in increasing Kv current amplitude, suggesting that under our experimental conditions the tonic effect of PKA on Kv channels is already maximal. [Funding acknowledgement: British Heart Foundation, UK (RG/06/008/22062)].