Structure-activity relationships of the Kvbeta1 inactivation domain and its putative receptor probed using peptide analogs of voltage-gated potassium channel alpha- and beta-subunits.
Certain beta-subunits exert profound effects on the kinetics of voltage-gated (Kv) potassium channel inactivation through an interaction between the amino-terminal "inactivation domain" of the beta-subunit and a "receptor" located at or near the cytoplasmic mouth of the channel pore. Here we used a bacterial random peptide library to examine the structural requirements for this interaction. To identify peptides that bind Kv1.1 we screened the library against a synthetic peptide corresponding to the predicted S4-S5 cytoplasmic loop of the Kv1.1 alpha-subunit (residues 313-328). Among the highest affinity interactors were peptides with significant homology to the amino terminus of Kvbeta1. We performed a second screen using a peptide from the amino terminus of Kvbeta1 (residues 2-31) as "bait" and identified peptide sequences with significant homology to the S4-S5 loop of Kv1.1. A series of synthetic peptides containing mutations of the wild-type Kvbeta1 and Kv1.1 sequences were examined for their ability to inhibit Kvbeta1/Kv1.1 binding. Amino acids Arg20 and Leu21 in Kvbeta1 and residues Arg324 and Leu328 in Kv1.1 were found to be important for the interaction. Taken together, these data provide support for the contention that the S4-S5 loop of the Kv1.1 alpha subunit is the likely acceptor for the Kvbeta1 inactivation domain and provide information about residues that may underlie the protein-protein interactions responsible for beta-subunit mediated Kv channel inactivation.
Lombardi
S J
SJ
Wyeth-Ayerst Research, CNS-Disorders, Princeton, New Jersey 08543-8000, USA. lombars@war.wyeth.com
Truong
A
A
Spence
P
P
Rhodes
K J
KJ
Jones
P G
PG
D000595:Amino Acid Sequence; D016384:Consensus Sequence; D006801:Humans; D051662:Kv1.1 Potassium Channel; D051663:Kv1.2 Potassium Channel; D008969:Molecular Sequence Data; D019151:Peptide Library; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
C496946:KCNA1 protein, human; C496969:KCNA2 protein, human; D051663:Kv1.2 Potassium Channel; D019151:Peptide Library; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D051662:Kv1.1 Potassium Channel