ENERGETICS OF SHAKER K-CHANNELS BLOCK BY INACTIVATION PEPTIDES

A synthetic peptide of the NH2-terminal inactivation domain of the ShB channel blocks Shaker channels which have an NH2-terminal deletion an d mimics many of the characteristics of the intramolecular inactivatio n reaction. To investigate the role of electrostatic interactions in b oth peptide block and the inactivation process we measured the kinetic s of block of macroscopic currents recorded from the intact ShB channe l, and from ShBDELTA6-46 channels in the presence of peptides, at diff erent ionic strengths. The rate of inactivation and the association ra te constants (k(on)) for the ShB peptides decreased with increasing io nic strength. k(on) for a more positively charged peptide was more ste eply dependent on ionic strength consistent with a simple electrostati c mechanism of enhanced diffusion. This suggests that a rate limiting step in the inactivation process is the diffusion of the NH2-terminal domain towards the pore. The dissociation rates (k(off)) were insensit ive to ionic strength. The temperature dependence of k(on) for the ShB peptide was very high, (Q10 = 5.0 +/- 0.58), whereas k(off) was relat ively temperature insensitive (Q10 almost-equal-to 1.1). The results s uggest that at higher temperatures the proportion of time either the p eptide or channel spends in the correct conformation for binding is in creased. There were two components to the time course of recovery from block by the ShB peptide, indicating two distinct blocked states, one of which has similar kinetics and dependence on external K+ concentra tion as the inactivated state of ShB. The other is voltage-dependent a nd at -120 mV is very unstable. Increasing the net charge on the pepti de did not increase sensitivity to knockoff by external K+. We propose that the free peptide, having fewer constraints than the tethered NH2 -terminal domain binds to a similar site on the channel in at least tw o different conformations.