Translocation of an endoproteolytically cleaved maxi-K channel isoform: mechanisms to induce human myometrial cell repolarization.
Large conductance Ca(2+)- and voltage-activated K+ (maxi-K) channels modulate human myometrial smooth muscle cell (hMSMC) excitability; however, the role of individual alternatively spliced isoforms remains unclear. We have previously shown that the transcript of a human maxi-K channel isoform (mK44) is expressed predominantly in myometrial and aortic smooth muscle and forms a functional channel in heterologous expression systems. The mK44 isoform contains unique consensus motifs for both endoproteolytic cleavage and N-myristoylation, although the function of these post-translational modifications is unknown. The goal of these studies was to determine the role of post-translational modifications in regulating mK44 channel function in hMSMCs. An mK44-specific antibody indicated that this channel is localized intracellularly in hMSMCs and translocates to the cell membrane in response to increases in intracellular Ca(2+). Immunological analyses using an N-terminally myc-tagged mK44 construct demonstrated endoproteolytical cleavage of mK44 in hMSMCs resulting in membrane localization of the mK44 N-termini and intracellular retention of the pore-forming C-termini. Caffeine-induced Ca(2+) release from intracellular stores resulted in translocation of the C-termini of mK44 to the cell membrane and co-localization with its N-termini. Translocation of mK44 channels to the cell membrane was concomitant with repolarization of the hMSMCs. Endoproteolytic digest of mK44 did not occur in HEK293 cells or mouse fibroblasts. MK44 truncated at a putative N-myristoylation site did not produce current when expressed alone, but formed a functional channel when co-expressed with the N-terminus. These findings provide novel insight into cell-specific regulation of maxi-K channel function.
Korovkina
Victoria P
VP
Department of Physiology and Biophysics, Carver College of Medicine, University of Iowa, 6-432 BSB Iowa City, IA 52242, USA.
Brainard
Adam M
AM
England
Sarah K
SK
D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D005260:Female; D006801:Humans; D007424:Intracellular Fluid / Q000502:physiology; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D009215:Myometrium / Q000502:physiology*; D010447:Peptide Hydrolases / Q000502:physiology; D020033:Protein Isoforms / Q000502:physiology
D051036:Large-Conductance Calcium-Activated Potassium Channels; D020033:Protein Isoforms; D010447:Peptide Hydrolases