Dual modulation of unitary L-type Ca2+ channel currents by [Ca2+]i in fura-2-loaded guinea-pig ventricular myocytes

Abstract

1. Single-channel studies were performed to clarify how tonic changes in intracellular Ca2+ concentrations ([Ca2+]i) modulate cardiac L-type Ca2+ channels. Currents were recorded from fura-2-loaded guinea-pig ventricular myocytes in the cell-attached configuration. Fura-2 fluorescence signals were recorded simultaneously during pulses to elicit channel activity. 2. The myocyte [Ca2+]i was altered through changes in bath Ca2+ concentration during K+ depolarization. When [Ca2+]i exceeded approximately 2 times the resting level (estimated [Ca2+]i around 180-400 nM), the activity of Ca2+ channels was reversibly potentiated without changes in unitary current amplitudes. 3. Increased channel open probability during Ca(2+)-dependent potentiation resulted from increased availability and increased open probability during non-blank sweeps. Closed time analysis revealed a distribution best fitted with two exponentials. Increased [Ca2+]i reduced the longer time constant, but had no effect on the shorter time constant. The open time constant was unchanged in most cases. Current records occasionally included sweeps with long openings (approximately 10 ms or more), whose appearance increased during potentiation. 4. When [Ca2+]i was increased after cAMP-dependent upregulation of Ca2+ channels, the change in channel activity was diminished. Similar results were observed when Ca(2+)-dependent potentiation was examined in myocytes exposed to a membrane-permeant protein kinase inhibitor, H-89. This suggests that channel phosphorylation may be responsible for Ca(2+)-dependent potentiation. 5. When [Ca2+]i was further increased, but remained below the threshold for contraction (estimated [Ca2+]i above 600 nM), Ca2+ channel activity was suppressed. 6. Our results demonstrate directly at the single-channel level that [Ca2+]i modulates the activity of cardiac L-type Ca2+ channels, enhancing it with modest [Ca2+]i increases and decreasing it with greater [Ca2+]i increases.