Assessment of Ca2+ Sensitivity of SK Channels in Rat Ventricular Cardiomyocytes using Intrinsic CA2+ Cycling Machinery

Small conductance Ca2+ activated K+ (SK) channels are thought to play significant roles in ventricular arrhythmias. It has been hypothesized that increase in Ca2+ sensitivity is the main underlying cause of functional upregulation of SKs in diseased hearts. We developed the protocol to assess Ca2+ sensitivity using simultaneous recordings of currents with intracellular Ca2+ in voltage clamped cardiomyocytes. By converting Rhod-2 fluorescence into [Ca2+]i we estimated Ca2+ sensitivity of rat SK2 channels overexpressed in cells using Adenovirus gene transfer. We obtained apparent affinity of rSK2 ∼ 500 nM which is in consistence with published values. To test whether SK channels from diseased hearts are more sensitive to Ca2+ we used rat model of cardiac hypertrophy induced by thoracic aortic banding (TAB). In contrast to Shams, myocytes from TAB hearts exhibited significant prolongation of APD upon application of SK inhibitor UCL-1684 (1 μM). Voltage clamp experiments showed SK inhibitor-sensitive current with amplitude ∼1 pA/pF. Simultaneous measurements of caffeine-induced ISK (10 mM) and [Ca]i at −30 mV before and after application of UCL-1684 demonstrated that sensitivity of SKs in TAB myocytes is low (Kd ∼900 nM). This result suggests that sensitivity to [Ca2+]i in hypertrophy is rather reduced than increased. To explore potential mechanisms responsible for changes in SK sensitivity to [Ca2+]i we treated cultured rat myocytes overexpressing SK2 with Phenylephrine (100 μM) and Propranolol (1 μM). We found that SK2 Ca2+ dependence in Phe/Pro was shifted to the right (Kd ∼850 nM), which was preventable by coexpressing CRNK, an inhibitory peptide for ROS and Ca2+-dependent tyrosine kinase Pyk2. Western blot analysis demonstrated that Pyk2 levels were upregulated in TABs. These data implicate Pyk2 as a novel negative regulator of SK Ca2+ sensitivity in diseased hearts.