Single-channel kinetics, inactivation, and spatial distribution of inositol trisphosphate (IP3) receptors in Xenopus oocyte nucleus

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 mu M, 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 similar to 300 pS at 60 mV. The channel was frequently observed with high open probability (mean P-0=0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants tau <5 ms and similar to 20 ms; and at least three closed states with tau similar to 1 ms, similar to 10 ms, and >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: F-1 and F-2. 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 F-1 to F-2 transitions. Channel run-down or inactivation (tau similar to 30 s) was consistently observed in the continuous presence of IP3 and the absence of change in [Ca2+]. Some (similar to 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 mu m) as well as macroscopic (similar to 10 mu m) clusters. Ca2+-independent inactivation of IP3R and channel clustering may contribute to complex [Ca2+] signals in cells.