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 Caand 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 b subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct b subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels. Methodology/Principal Findings: To examine the ability of the KCNMB family of auxiliary b subunits to regulate Slo3 function, we co-expressed Slo3 and each b subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The b4 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 YFPtagged Slo3 or biotin labeled Slo3. Neither b1, b2, nor b3 mimicked the ability of b4 to increase surface expression, although biochemical tests suggested that all four b subunits are competent to coassemble with Slo3. Fluorescence microscopy from b4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that b4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that b4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm. Conclusions/Significance: These results argue that, for native mouse Slo3 channels, the b4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 a subunit. Citation: Yang C-T, Zeng X-H, Xia X-M, Lingle CJ (2009) Interactions between b Subunits of the KCNMB Family and Slo3: b4 Selectively Modulates Slo3 Expression and Function. PLoS ONE 4(7): e6135. doi:10.1371/journal.pone.0006135 Editor: David E. Clapham, Harvard Medical School, United States of America Received May 1, 2009; Accepted June 1, 2009; Published July 3, 2009 Copyright: 2009 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by National Institutes of Health grants GM068580 and GM081748 to C.L. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (XHZ); [email protected] (CJL)