Functional role of the NH2-terminal cytoplasmic domain of a mammalian A-type K channel.
It has been shown for a Shaker channel (H-4) that its NH2-terminal cytoplasmic domain may form a "ball and chain" structure, with the "chain" tethering the "ball" to the channel while the "ball" capable of binding to the channel in its open state and causing inactivation. Equivalent structures have not been identified in mammalian Shaker homologues. We studied the functional role of the NH2-terminal region of a fast-inactivating mammalian K channel, RHK1 (Kv1.4), by deleting different domains in this region and examining the resultant changes in channel properties at whole cell and single channel levels. Deleting the NH2-terminal hydrophobic domain (domain A) or the subsequent positive charges (domain I) from RHK1 greatly slowed the decay of whole cell currents, suggesting the existence of a ball-like structure in RHK1 similar to that in the Shaker channel. The function of the ball appeared to be abolished by deleting domain A, while modified but maintained by deleting domain I. In the latter case, the data suggest that the positive charges needed for the function of the ball can be replaced by amino acids from a following region (domain III) that has a high positive charge density. Deleting multiple domains from the NH2 terminus of RHK1 corresponding to the chain in Shaker H-4 did not induce expected changes in channel properties that might result from a shortening of a chain. A comparison of single channel kinetics of selected mutant channels with those of the wild-type channel indicated that these deletion mutations slowed whole cell currents by prolonging burst durations and by increasing the probability of reopening during depolarization. There were no changes in single channel current amplitude or latency to first opening. In conclusion, our observations indicate that the inactivation mechanism of RHK1 is similar to that of Shaker H-4 in that a positively charged cytoplasmic domain is important for such a process. The NH2-terminal domain is not involved in channel activation or ion permeation process.
Tseng-Crank
J
J
Molecular Biology Department, Glaxo Incorporated Research Institute, Research Triangle Park, North Carolina 27709.
Yao
J A
JA
Berman
M F
MF
Tseng
G N
GN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D015640:Ion Channel Gating / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000138:chemical synthesis; D012150:Polymorphism, Restriction Fragment Length; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D018076:DNA, Complementary; D009841:Oligonucleotides; D015221:Potassium Channels