Inactivation of BK channels by the NH 2 terminus of the beta 2 auxiliary subunit : An essential role of a terminal peptide segment of three hydrophobic residues

An auxiliary 2 subunit, when coexpressed with Slo subunits, produces inactivation of the resulting large-conductance, Ca 2 and voltage-dependent K (BK-type) channels. Inactivation is mediated by the cytosolic NH 2 terminus of the 2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH 2 terminus in the inactivation process. The 2 NH 2 terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2–4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH 2 -terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process. key words: inactivation mechanisms • inactivation domains • K channels • BK channels • Ca 2 and voltagegated K channels I N T R O D U C T I O N Rapid inactivation of Ca 2 and voltage-gated BK-type K channels arises from coexpression of the slo1 pore– forming subunits with particular auxiliary subunits (Wallner et al., 1999; Xia et al., 1999, 2000; Uebele et al., 2000; Lingle et al., 2001). Of the four members of the BK subunit family, inactivation arises from the short cytosolic NH 2 terminus of either the 2 subunit (Wallner et al., 1999; Xia et al., 1999) or of particular splice variants of the 3 subunit (Uebele et al., 2000; Xia et al., 2000; Lingle et al., 2001). Since slo1 and subunits assemble in a 1:1 stoichiometry (Knaus et al., 1994b; Wang et al., 2002), up to four inactivation-competent NH 2 termini can be present in any inactivating BK channel (Wang et al., 2002). Similar to inactivation of voltage-dependent K (Kv) channels mediated by NH 2 -terminal domains of subunits (MacKinnon et al., 1993; Gomez-Lagunas and Armstrong, 1995), inactivation arises from the independent action of each NH 2 terminus (Xia et al., 1999; Wang et al., 2002). Thus, at least superficially similar elements would appear to contribute both to inactivation of Kv channels and BK channels. Of the kinetic behaviors exhibited by voltage-gated ion channels, the phenomenon of rapid inactivation of Kv channels has perhaps been most amenable to a correlation of the structural elements of the channel with an actual mechanism of gating. For Kv channels, to produce inactivation, the cytosolic NH 2 terminus, either of the pore-forming subunits (Hoshi et al., 1990; Ruppersberg et al., 1991) or of cytosolic auxiliary subunits (Rettig et al., 1994), appears to move into a position that closely abuts the mouth of the ion permeation pathway. The close association of the Kv blocking domain and the ion permeation pathway is supported by the fact that cytosolic channel blockers compete with the blocking domain for occupancy of the channel (Choi et al., 1991; Demo and Yellen, 1991). Furthermore, once the inactivation domain occupies its blocking position, it impedes closure of the channels (Demo and Yellen, 1991; RupXiao-Ming Xia and J.P. Ding contributed equally to this work. Address correspondence to C. Lingle, Department of Anesthesiology, Washington University School of Medicine, 600 S. Euclid Ave., Box 8054, St. Louis, MO 63110. Fax: (314) 362-8571; E-mail: clingle@morpheus.wustl. edu on Jauary 6, 2012 jgp.rress. org D ow nladed fom Published February 3, 2003