Two phosphatase sites on the Ca2+ channel affecting different kinetic functions

Area of Science:

• Cardiovascular Physiology
• Molecular Pharmacology
• Ion Channel Biophysics

Background:

• L-type calcium channels are crucial for cardiac function.
• Phosphorylation regulates ion channel activity.
• Okadaic acid (OA) inhibits protein phosphatases, affecting phosphorylation levels.

Purpose of the Study:

• To investigate the effects of okadaic acid (OA) on the kinetics of dihydropyridine-sensitive (L-type) Ca2+ channels.
• To determine the concentration-dependent effects of OA on channel gating properties.
• To explore the role of phosphorylation in regulating L-type Ca2+ channel activity.

Main Methods:

• Single isolated rabbit ventricular myocytes were used.
• Cell-attached patch-clamp recordings were performed with barium as the charge carrier.
• Repetitive voltage steps were applied to assess channel availability and gating kinetics.
• Okadaic acid (OA) was applied at various concentrations (0.001-750 microM).

Main Results:

• Low concentrations of OA (0.001-1 microM) decreased non-conducting sweeps and, with cAMP pretreatment, induced long-lasting openings.
• High concentrations of OA (10-750 microM) induced long-lasting openings, with open time distributions fitted by two exponentials.
• OA increased the time constant of the available state (TS) but did not affect the unavailable state (TF).
• OA exhibited concentration-dependent effects on both opening probability (mode 1) and opening duration (mode 2).

Conclusions:

• Okadaic acid (OA) alters L-type Ca2+ channel kinetics in a concentration-dependent manner.
• The findings suggest the presence of at least two distinct modulatory phosphorylation sites on the channel.
• These sites are likely dephosphorylated by different phosphatases, indicating complex regulatory mechanisms.