Ca2+-activated adenylyl cyclase 1 introduces Ca2+-dependence to beta-adrenergic stimulation of HCN2 current

Previous observations show that beta-adrenergic modulation of pacemaker current (I-f) in sinoatrial node (SAN) cells is impaired by disruption of normal Ca2+-homeostasis with ryanodine or BAFTA. Recently, the presence of Ca2+-activated adenylyl cyclase (AC) 1 was reported in SAN, and was proposed as a possible mechanism of Ca2+-dependence of beta-adrenergic modulation. However, direct evidence that pacemaker (HCN) channels can be regulated by Ca2+-activated AC and that such regulation introduces Ca2+ dependence, is lacking. Here we co-expressed AC1 or AC6 with HCN2 in neonatal rat ventricular myocytes, which lack AC1. Although both isoforms have equivalent expression level and ability to interact with HCN2, only AC1 increases intracellular cAMP content, accelerates spontaneous beating rate and modifies HCN2 biophysics. Measured HCN2 current in the AC1 group activated similar to 10 mV more positive than in GFP or AC6. The beta-adrenergic agonist isoproterenol induced a further positive shift under control conditions, but failed to do so after pretreatment with the Ca2+ chelator BAPTA. In the AC6 group, isoproterenol shifted the HCN2 activation relation to a similar extent in the absence and presence of BAPTA. Thus, AC1 but not AC6 over-expression introduces Ca2+-sensitivity to the beta-adrenergic response of HCN2. These results demonstrate physical and functional interaction between AC isoforms and the HCN2 pacemaker channel and support a key role of Ca2+ activated AC1 as a molecular mechanism in Ca2+-dependent modulation of beta-adrenergic response of heart rate. (C) 2012 Elsevier Ltd. All rights reserved.