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Inactivation of BK channels by the NH2 terminus of the beta2 auxiliary subunit: an essential role of a terminal peptide segment of three hydrophobic residues.

Xiao-Ming Xia, J P Ding, Christopher J Lingle

J. Gen. Physiol., 2003 Feb , 121, 125-48

An auxiliary beta2 subunit, when coexpressed with Slo alpha 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 beta2 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 beta2 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.