Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca(2+)-sensitive K+ channels: an additional transmembrane region at the N terminus.
The pore-forming alpha subunit of large conductance voltage- and Ca(2+)-sensitive K (MaxiK) channels is regulated by a beta subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming alpha subunit necessary for beta-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to beta-subunit modulation, and analyzed the topology of the alpha subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel alpha subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1-S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers beta-subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function.
Wallner
M
M
Department of Anesthesiology, University of California Los Angeles 90095-1778, USA. Martin@cvmail.anes.ucla.edu
Meera
P
P
Toro
L
L
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000502:physiology*; D002478:Cells, Cultured; D004330:Drosophila; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D017421:Sequence Analysis
C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins