Abstract 2330: A Key Role of Ca2+-Activated Adenylyl Cyclase I in Ca2+-Dependence of β-Adrenergic Modulation of Pacemaker Current
Previous observations show that β-adrenergic modulation of pacemaker current (If) and pacing rate in sinoatrial node (SAN) cells is impaired by disruption of normal Ca2+-homeostasis with ryanodine or Bapta. Recently, the presence of neuronal Ca2+-activated adenylyl cyclase (AC) I and VIII was reported in SAN, and was proposed as a possible mechanism of Ca2+-dependence of β-adrenergic modulation. However, direct evidence that pacemaker (HCN) channels can be regulated by Ca2+-activated AC and that such regulation introduces Ca dependence, is lacking. Here we report the effect of AC1 or AC6 over-expression on expressed HCN2 current in neonatal rat ventricular myocytes (NRVM). Co-expression of HCN2 with FLAG-tagged ACI or AC6 (provided by Dr. R. Feldman, U W Ontario) were confirmed by Western Blot analysis. AC1 and AC6 were equivalently expressed. Moreover, immunoprecipitation experiments of membrane fractions from over-expressing NRVM revealed that both AC1 and AC6 interact with HCN2. Co-expression of HCN2 with AC1 resulted in a faster beating rate of NRVM monolayer compared to GFP co-expression (201±5 bpm vs 155±6 bpm, p<.05), while AC6 expression did not (159±7 bpm). Measured HCN2 current in the AC1 group activated ~10mV more positive than in GFP group (V50: −58±1.8 mV vs −69±1.8 mV in GFP, n=8, p<.05). 1μM of βAR agonist isoproterenol (Iso) induced a further positive shift (−53±1.2 mV, n=5, p<.05). Significantly, the Iso shift was lost when cells were pre-incubated in 5μM Bapta-AM (−58±2.0 mV, n=8 in Bapta-AM plus Iso vs −59±1.4 mV, n=9 in Bapta-AM alone). In contrast to the effect of AC1 over-expression, over-expression of the ventricular AC6 isoform did not induce a statistically significant positive shift of HCN2 current (−65±1.2 mV, n=9, p>.05 vs GFP). Importantly, Iso shifted the HCN2 activation relation to a similar extent in the absence and presence of Bapta-AM (−59±1.5 mV and −58±1.3 mV respectively). Thus, AC1 but not AC6 over-expression introduces Ca-sensitivity to the βAR 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 βAR-response of heart rate.