• K+ channels and mechanisms of inactivation
  • Structure and function of BK-type Ca2+ and voltage-dependent K+ channels
  • Cloning and functional characterization of accessory subunits of BK-type channels.
  • Coupling of BK channels to specific Ca2+ channel subtypes

Inactivation mechanisms. Inactivation is a general property of a wide variety of ion channels. Specifically, under conditions which initially result in opening of the ion channels, channels eventually enter closed states from which opening of the channels is unlikely. Understanding the structural components of ion channels that contribute to inactivation has been very informative about the molecular architecture of ion channels and about the conformational changes that occur in ion channel proteins during channel opening and inactivation. One of the best understood inactivation mechanisms is that involving the N-terminus of the a subunits of particular voltage-dependent K+ channels, including the ShakerB K+ channel. In this case, inactivation involves movement of residues on the N-terminus into a position along the S6 transmembrane helices that define the ion permeation pathway. In fact, when the N-terminus is in its blocking position, the channel is prevented from closing.

Inactivation of BK channels shares many features with voltage-dependent K+ channel rapid inactivation, but key differences suggest that the mechanism and structural basis underlying BK channel inactivation are different (Solaro et al., 1997). Inactivation of BK channels is also mediated by cytosolic N-terminal segments of channel subunits, in this case  by auxiliary b subunits (Xia et al., 1999; Xia et al., 2000). However, many features of the inactivation are unique.We have shown that inactivation mediated by the b3b subunit involves two kinetic steps, involving formation of a novel preinactivated open state (Lingle et al., 2001).  Two critical questions are being addressed in future work. First, what is the physical basis for each of the two kinetic steps in the blocking process? Second, where does blockade of the channel occur?

One approach we are taking to address these issues has utilized a mutational analysis of inactivation mediated by the b2 auxiliary subunit.  Single point mutations in the b2 N-terminus allow the two step inactivation characteristic of the b3b subunit (Lingle et al., 2001) to be revealed as shown below for mutation of the tryptophan residue in position 4 to a glutamate (b2-W4E). Single channel studies of such mutants are providing insight into the physical basis of each of the kinetic steps.

Although both the b2 and b3b auxiliary subunits produce inactivation, the temporal properties of that inactivation are quite different: b2 currents inactivate with a time constant of 25 ms and b3b currents inactivate with a time constant of less than 1 ms. Based on the two-step blocking model, the simulation below shows that a change in a single kinetic rate constant can account qualitatively for the differences between the two types of behavior, yielding either a complete but slower (25 ms) inactivation, and a rapid (1 ms), but incomplete inactivation.

Structure and function of BK-type Ca2+ and voltage-dependent K+ channels. One of the more interesting properties of BK channels is their regulation by two distinct physiological stimuli, membrane voltage and cytosolic Ca2+. This ability for two independent signals to regulate BK channels allows them to play quite distinct functional roles dependent on the concentrations and time course of changes in Ca2+ within a cell.  Although the voltage-sensitivity of BK channels is thought to arise by mechanisms similar to other voltage-dependent channels, the sites and mechanisms by which Ca2+ regulates BK channels is less well understood. We are pursuing several strategies to try to identify structural components of BK channels that are important in Ca2+-dependent regulation.