Simulation of Single Channel Kinetics Based on Ligand Binding and Electrophysiological Analysis

Cytosolic calcium acts as both a coagonist and an inhibitor of the type 1 inositol 1,4,5-trisphosphate (InsP 3 )–gated Ca channel, resulting in a bell-shaped Ca dependence of channel activity (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature. 351:751–754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science. 252: 443–446; Iino, M. 1990. J. Gen. Physiol. 95:1103–1122). The ability of Ca to inhibit channel activity, however, varies dramatically depending on InsP 3 concentration (Combettes, L., Z. Hannaert-Merah, J.F. Coquil, C. Rousseau, M. Claret, S. Swillens, and P. Champeil. 1994. J. Biol. Chem. 269:17561–17571; Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529–538). In the present report, we have extended the characterization of the effect of cytosolic Ca on both InsP 3 binding and InsP 3 -gated channel kinetics, and incorporated these data into a mathematical model capable of simulating channel kinetics. We found that cytosolic Ca increased the K d of InsP 3 binding z 3.5-fold, but did not influence the maximal number of binding sites. The ability of Ca to decrease InsP 3 binding is consistent with the rightward shift in the bell-shaped Ca dependence of InsP 3 -gated Ca channel activity. High InsP 3 concentrations are able to overcome the Ca-dependent inhibition of channel activity, apparently due to a low affinity InsP 3 binding site (Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529–538). Constants from binding analyses and channel activity determinations were used to develop a mathematical model that fits the complex Ca-dependent regulation of the type 1 InsP 3 -gated Ca channel. This model accurately simulated both steady state data (channel open probability and InsP 3 binding) and kinetic data (channel activity and open time distributions), and yielded testable predictions with regard to the regulation of this intracellular Ca channel. Information gained from these analyses, and our current molecular model of this Ca channel, will be important for understanding the basis and regulation of intracellular Ca waves and oscillations in intact cells. key words: cerebellum • inositol 1,4,5-trisphosphate receptor • intracellular calcium channel • calcium signaling • kinetic model i n t r o d u c t i o n The inositol 1,4,5-trisphosphate (InsP 3 ) 1 –gated Ca channel plays a critical role in the regulation of intracellular Ca concentrations after hormonal activation of the phosphoinositide cascade in a wide variety of cell types (Abdel-Latif, 1986; Berridge and Irvine, 1989; Clapham, 1995). The mechanisms underlying regulation of the InsP 3 -gated channel are poorly understood; however, the involvement of both cytosolic Ca and InsP 3 in the regulation of this channel is well established (Iino, 1990; Payne et al., 1990; Bezprozvanny et al., 1991; Finch et al., 1991; Bootman et al., 1995; Kaftan et al., 1997). That is, InsP 3 is absolutely required for the opening of the channel, though channel activity can be modified by cytosolic Ca. Elevating the cytosolic Ca concentration from 0.01 to 0.3 m M in the presence of 2 m M InsP 3 , for example, resulted in a dramatic increase in channel activity (Bezprozvanny et al., 1991). Further elevation of cytosolic Ca, however, decreased channel activity, with near complete inhibition of the channel in the presence of 5 m M Ca (Bezprozvanny et al., 1991). Three basic models have been proposed to explain this bell-shaped Ca dependence of the channel differing in terms of the effects of InsP 3 on the bellshaped Ca dependence. In one model (Bezprozvanny, 1994), the peak of the bell-shaped Ca dependence is unaffected by InsP 3 concentration. The other two models predict either a shift to the left (Tang et al., 1996) or the right (De Young and Keizer, 1992) in the peak of the bell-shaped Ca dependence of channel activity as InsP 3 concentrations increase. Address correspondence to Dr. J. Watras, Department of Physiology, 263 Farmington Avenue, University of Connecticut, Farmington, CT 06030. Fax: 860-679-1269 E-mail: [email protected] 1 Abbreviation used in this paper: InsP 3 , inositol 1,4,5-trisphosphate. on A uust 0, 2013 jgp.rress.org D ow nladed fom Published June 1, 1999