Patch clamp analysis of Xenopus IP3R revealed multiple conductance states, a novel flicker kinetic mode, voltage-dependent gating, and Ca2+-independent inactivation with spatial clustering.
The study characterizes the complex single-channel kinetics, voltage-dependent gating, and spatial clustering of IP3 receptors in Xenopus oocytes, providing insights into intracellular calcium signaling mechanisms.
Single-channel properties of the Xenopus inositol trisphosphate receptor (IP3R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K+ as the charge carrier (cytoplasmic IP3 = 10 microM, free Ca2+ = 200 nM), the IP3R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and approximately 300 pS at 60 mV. The channel was frequently observed with high open probability (mean P(o) = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants tau 1 s. Higher cytoplasmic potential increased the relative frequency and tau of the longest closed state. A novel "flicker" kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F1 and F2. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F1 to F2 transitions. Channel run-down or inactivation (tau approximately 30 s) was consistently observed in the continuous presence of IP3 and the absence of change in Ca2+. Some (approximately 10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage-reversible inactivation. Mapping of functional channels indicates that the IP3R tends to aggregate into microscopic (<1 microm) as well as macroscopic (approximately 10 microm) clusters. Ca2+-independent inactivation of IP3R and channel clustering may contribute to complex Ca2+ signals in cells.
Mak et al. (Thu,) reported a other. Patch clamp electrophysiology was evaluated on Single-channel properties (conductance, kinetics, inactivation, spatial distribution). Patch clamp analysis of Xenopus IP3R revealed multiple conductance states, a novel flicker kinetic mode, voltage-dependent gating, and Ca2+-independent inactivation with spatial clustering.