Differential control of the hyperpolarization‐activated current (i(f)) by cAMP gating and phosphatase inhibition in rabbit sino‐atrial node myocytes.

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Abstract
1. The actions of the phosphatase inhibitor calyculin A on the hyperpolarization‐activated cardiac ‘pacemaker’ current (i(f)) were determined in single cells isolated from the sino‐atrial (SA) node of the rabbit. 2. Cells were incubated for 8 min in Tyrode solution containing calyculin A (0.5 microM) and then superfused with normal Tyrode solution. The mean normalized i(f) measured in eight cells at mid‐activation voltages during and after exposure to calyculin A increased maximally by 47% with a time constant of 466 s, a time much longer than that required for cAMP‐mediated i(f) stimulation (about 8 s). 3. In two‐pulse protocols, calyculin A treatment increased i(f) at full as well as at mid‐activation voltages, indicating a higher i(f) conductance. 4. Measurement of the conductance‐voltage (gf(V)) relation by voltage ramp protocols confirmed a conductance increase by calyculin A, with no significant change in the position of the activation curve on the voltage axis. Data pooled together from ramp and two‐pulse protocols yielded a calyculin A‐induced increase in fully activated i(f) conductance of 39.6 +/‐ 6.4% (n = 16 cells). 5. The positive and negative shift of i(f) voltage dependence in response to beta‐adrenergic (1 microM isoprenaline) and muscarinic stimulation (1 microM acetylcholine), respectively, was preserved after the calyculin A‐induced increase in conductance. The shift of the i(f) activation curve induced by 1 microM isoprenaline was significantly larger in calyculin A‐treated cells (8.8 vs. 5.8 mV). 6. These data indicate that phosphatase inhibition increases i(f) in a manner distinct from the direct cAMP pathway and potentiates the beta‐adrenergic‐mediated i(f) modulation.