Differential effect of PKA on the Ca2+ release kinetics of the type I and III InsP3 receptors.

The effects of protein kinase A (PKA) on the inositol 1,4,5-trisphosphate (InsP(3)) receptor isoforms type I and type III were studied. The effects of PKA on the extent and rate constants for InsP(3)-induced Ca(2+) release (IICR) were different for the two isoforms. The effects of PKA on the type I isoform showed a biphasic relationship dependent upon the concentration of PKA used. At low concentrations of PKA (<50U/ml), both the extent and rate constants for IICR increased, while at higher concentrations (>200U/ml) the extent and rate constants decreased. The type III isoform showed only an increase in the extent of IICR and not in the rate constants. The effects of PKA on the type I InsP(3) receptor using single channel electrophysiological studies were also investigated. The stimulatory effect of PKA is due to an increase in conductance levels and not to a change in the mean open time of the channel.

Kinetic model of the inositol trisphosphate receptor that shows both steady-state and quantal patterns of Ca2+ release from intracellular stores.

The release of Ca(2+) from intracellular stores via InsP(3) receptors shows anomalous kinetics. Successive additions of low concentrations of InsP(3) cause successive rapid transients of Ca(2+) release. These quantal responses have been ascribed to all-or-none release from stores with differing sensitivities to InsP(3) or, alternatively, to a steady-state mechanism where complex kinetic properties of the InsP(3) receptor allow partial emptying of all the stores. We present here an adaptive model of the InsP(3) receptor that can show either pattern, depending on the imposed experimental conditions. The model proposes two interconvertible conformational states of the receptor: one state binds InsP(3) rapidly, but with low affinity, whereas the other state binds slowly, but with high affinity. The model shows repetitive increments of Ca(2+) release in the absence of a Ca(2+) gradient, but more pronounced incremental behaviour when released Ca(2+) builds up at the mouth of the channel. The sensitivity to Ins P (3) is critically dependent on the density of InsP(3) receptors, so that different stores can respond to different concentration ranges of Ins P (3). Since the model generates very high Hill coefficients (h approximately 7), it allows all-or-none release of Ca(2+) from stores of differing receptor density, but questions the validity of the use of h values as a guide to the number of InsP(3) molecules needed to open the channel. The model presents a mechanism for terminating Ca(2+) release in the presence of positive feedback from released Ca(2+), thereby providing an explanation of why elementary Ca(2+) signals ('blips' and 'puffs') do not inevitably turn into regenerative waves.

Biophysics of gating phenomena in voltage-dependent OmpC mutant porin channels (R74C and R37C) of Escherichia coli outer membranes.

The mechanism by which the membrane potential closes and opens voltage-dependent beta-barrel membrane channels is not fully understood. OmpC porins form trimeric water-filled channels when incorporated into artificial bilayers, each monomer having a conductance of approximately 510 pS in 1 M KCl. These channels are relatively insensitive to membrane potential difference (pd) and close only when the pd exceeds +/-250 mV. Another well-known trimer, OmpF, has a monomer conductance of approximately 780 pS in 1 M NaCl, is more sensitive to pd, and can be closed reversibly when a pd of more than +/-150 mV is applied to the channel-containing membranes. With the aid of the 3D atomic structure of these channels determined by X-ray crystallography, and using site-directed mutagenesis, specific amino acids can be substituted in desired locations in the channel lumen. In this study we have used mutants 37C and 74C and attached fluorescence probes to them to monitor polarity changes in the channel lumen during gating. From the observed changes in polarity, we conclude that conformational changes occur in the channel which interrupt the electrolyte conducting pathway.

The cytotoxic domain of colicin E9 is a channel-forming endonuclease.

Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. Here we demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltage-gated, channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein.

Modulation of type-1 Ins(1,4,5)P3 receptor channels by the FK506-binding protein, FKBP12.

FK506-binding protein (FKBP12) is highly expressed in neuronal tissue, where it is proposed to localize calcineurin to intracellular calcium-release channels, ryanodine receptors and Ins(1,4,5)P(3) receptors (InsP(3)Rs). The effects of FKBP12 on ryanodine receptors have been well characterized but the nature and function of binding of FKBP12 to InsP(3)R is more controversial, with evidence for and against a tight interaction between these two proteins. To investigate this, we incorporated purified type-1 InsP(3)R from rat cerebellum into planar lipid bilayers to monitor the effects of exogenous recombinant FKBP12 on single-channel activity, using K(+) as the current carrier. Here we report for the first time that FKBP12 causes a substantial change in single-channel properties of the type-1 InsP(3)R, specifically to increase the amount of time the channel spends in a fully open state. In the presence of ATP, FKBP12 can also induce co-ordinated gating with neighbouring receptors. The effects of FKBP12 were reversed by FK506. We also present data showing that rapamycin, at sub-optimal concentrations of Ins(2,4,5)P(3), decreases the rate of calcium release from cerebellar microsomes. These results provide evidence for a direct functional interaction between FKBP12 and the type-1 InsP(3)R.