Single-Channel Kinetics, Inactivation, and Spatial Distribution of Inositol Trisphosphate (IP3) Receptors in Xenopus Oocyte Nucleus

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Abstract
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 μM, 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 ∼300 pS at 60 mV. The channel was frequently observed with high open probability (mean Po = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants τ < 5 ms and ∼20 ms; and at least three closed states with τ ∼1 ms, ∼10 ms, and >1 s. Higher cytoplasmic potential increased the relative frequency and τ 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 (τ ∼ 30 s) was consistently observed in the continuous presence of IP3 and the absence of change in [Ca2+]. Some (∼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 (2+-independent inactivation of IP3R and channel clustering may contribute to complex [Ca2+] signals in cells.