Regulation of the frequency-dependent facilitation of L-type Ca2+ currents in rat ventricular myocytes.

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1. An increase in the rate of stimulation induces an augmentation of L-type Ca2+ currents (ICa) and concomitant slowing of current decay in rat ventricular cells. This facilitation is quasi immediate (1-3 s), graded with the rate of stimulation, and occurs only from negative holding potentials. We investigated this effect using trains of stimulation at 1 Hz and the whole-cell patch-clamp technique (18-22 degrees C). 2. The decay of ICa is normally bi-exponential and comprises fast and slow current components (ICa,fc and ICa,sc, respectively). Facilitation of ICa was observed only when ICa,fc was predominant. 3. Facilitation developed during the run-up of ICa with the interconversion of ICa,sc into ICa,fc, and vanished during the run-down of ICa with the loss of ICa,fc.Ni2+ (300 microM) and nifedipine (1 microM) suppressed facilitation owing to the preferential inhibition of ICa,fc. 4. Facilitation of ICa was not altered (when present) or favoured (when absent) by the cAMP-dependent phosphorylation of Ca2+ channels promoted by isoprenaline or by intracellular application of cAMP or of the catalytic subunit of protein kinase A (C-sub). A similar effect was observed when the dihydropyridine agonist Bay K 8644 was applied. In both cases, facilitation was linked to a preferential increase of ICa,fc. 5. Following intracellular application of inhibitors of protein kinase A in combination with a non-hydrolysable ATP analogue, ICa consisted predominantly of ICa,sc and no facilitation was observed. The calmodulin antagonist naphthalenesulphonamide had no effect on facilitation. 6. When Bay K 8644 was applied in combination with isoprenaline, cAMP or C-sub, the decay of ICa was slowed with the predominant development of ICa,sc, and facilitation of ICa was nearly abolished. Facilitation also depended on extracellular Ca2+, and was suppressed when Ba2+ replaced Ca2+ as the permeating ion. 7. When no EGTA was included in the patch pipette, facilitation was not further enhanced but a use-dependent decrease of ICa frequently occurred. When BAPTA was used in place of EGTA, the rate of inactivation of ICa was reduced and facilitation was abolished. 8. In conclusion, the facilitation of ICa that reflects a voltage-driven interconversion of ICa,fc into ICa,sc is also regulated by Ca2+ and by cAMP-dependent phosphorylation. The presence of the gating pattern typified by ICa,fc is required. Ca2+ may exert its effect near the inner pore of the Ca2+ channel protein and control the distribution between the closed states of the two gating pathways.

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