Saturday, October 29, 2011
Hall 1-2 (San Jose Convention Center)
The senses of hearing and balance rely on signal transduction by ion channels of mechanosensory hair cells. The ability to examine channel proteins outside of the hair cell membrane enables controlled characterization of ion channel structure and function. To this end, we cloned the Xenopus inner ear BK channel (large conductance calcium-activated potassium channel; Big K; Maxi K; slo; KCa1.1; KCNMA1), and developed methods for heterologous expression of the alpha and beta subunits in cell lines. We selected the BK channel because of its role in the electrical tuning of inner ear hair cells. We cloned the Xenopus transmembrane alpha (XBK-a, 3600 bp) and ancillary beta (XBK-b; 618 bp) BK channel subunits from inner ear template RNA with RT-PCR and specific primers. The XBK-a subunit shares 92% amino acid identity with mouse and human homologs; the XBK-b subunit shares 41% and 43% amino acid identity with mouse and human sequences, respectively. With these clones, we generated two spectrally distinct fluorescent fusion constructs that produce translated proteins with XBK-b and XBK-a attached to the N-terminus of m-Cherry (Clontech) and GFP (Clontech), respectively. Both proteins were successfully expressed in Xenopus laevis kidney (A6; ATCC CCL-102) and Chinese hamster ovary cells (CHO; ATCC CCL-61) using lipid-mediated transfection and biolistics (particle bombardment). Only biolistics resulted in cells that simultaneously expressed both fluorescent constructs. This research establishes the foundation for electrophysiological studies aimed at clarifying the role of the BK channel in inner ear disorders. Research Support: NSF (HRD-0031446; IGERT-0504304); NIH (R01 DC008136; P50GM068762).