Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.
BACKGROUND
The pH and voltage-regulated Slo3 K(+) channel, a homologue of the Ca(2+)- and voltage-regulated Slo1 K(+) channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels.


RESULTS
To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm.


CONCLUSIONS
These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.
Yang
Cheng-Tao
CT
Department of Anesthesiology, Washington University School of Medicine, St Louis, MO, USA.
Zeng
Xu-Hui
XH
Xia
Xiao-Ming
XM
Lingle
Christopher J
CJ
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits / Q000737:chemistry / Q000378:metabolism*; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D008297:Male; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013093:Spermatogonia / Q000378:metabolism; D014982:Xenopus laevis
C410183:KCNMB4 protein, human; C494843:Kcnmb4 protein, mouse; C110627:Kcnu1 protein, mouse; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D009419:Nerve Tissue Proteins