K+ channels in Xenopus skeletal muscle

Fry, William Mark (2001) K+ channels in Xenopus skeletal muscle. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The spatial distribution and identity of K+ channels in embryonic Xenopus myocytes were investigated. Macropatch current recordings were used to determine the spatial distribution of inactivating (IIK) and noninactivating (IK) outward K+ currents as well as Na+ currents (INa) in nerve contacted Xenopus muscle cells grown in culture. Membrane patches either contained no delectable current or INa, and/or IIK or IK. More than 85% of the membrane patches which contained INa occurred within 40 μm of the nerve contact. In addition, more than 90% of the membrane patches which contained INa also contained IIK. Sites of acetylcholine receptor localization al nerve-muscle contacts, identified by labeling with tetramethylrhodamine labeled α-bungarotoxin, were also found to contain both INa (4/5 cells) and IIK (5/5 cells). These results suggest that in Xenopus muscle cells in culture the channels mediating IIK along with sodium charnels are clustered at sites of synaptic specialization. To identify K+ channels that underlie the observed K+ currents in Xenopus muscle, a combination of RT-PCR, 5' RACE and 3' RACE was employed. Three K+ channel cDNAs, designated XKv1.2', XKv1.4 and XKv1.10, were isolated. XKv1.2' and XKv1.4 cDNAs are Xenopus orthologues of known mammalian K+ channels,. however XKv1.10 is a novel Kv1 isoform. XKv1.4 mRNA appeared at stage 10.5 in whole embryos and was prominent in muscle throughout development from stage 14 to adult. XKv1.2' mRNA was detected by stage 11.5 in whole embryos, but remained at low levels in embryonic skeletal muscle (stages 14 and 21) and was absent from adult muscle. XKv1.10 mRNA was first detected at stage 21 in whole embryos, and was present in muscle from this stage onwards. All three transcripts were present in spinal cord at stage 21. When transiently transfected into HEK 293 cells, XKv1.2' produced a noninactivating K+ current similar to IK, while XKv1.4 and XKv1.10 produced an inactivating current similar to IIK. The results support the notion that the channel encoded by XKv1.2' may contribute to IK and channels encoded by Kv1.4 and XKv1.10 may contribute to IIK observed in embryonic muscle cells in culture. These K+ channels may be important in the development of skeletal muscle and the neuromuscular synapse.

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