Neuronal large conductance calcium-activated potassium (BKCa) channels act as coincidence detectors. A single (Slo) gene encodes all four heterogeneous α-subunits that shape the channel pore. Alternative exon splicing of Slo mRNA provides the variation in characteristics required for BKCa to show alternative activity in different cellular environments (Shipston, 2001). It has been suggested that BKCa channel activity could be regulated by changes in oxygen tension (Lewis et al. 2002). The aim of this research was to determine whether hypoxia (20 mmHg) regulates the activity of alternative splice variants (mbr5 and STREX) of BKCa channel α-subunits using the single channel excised inside-out patch clamp recording technique.
All data are expressed as means ± S.E.M. and ANOVA or Student’s unpaired t test was used to examine statistical differences, and P < 0.05 was considered significant.
In patch recordings from mbr5-expressing HEK-293 cells, hypoxia did not cause a significant change in single channel amplitude or channel open probability (Po) in the presence of 0.1, 1 or 10 µM free calcium. In experiments using the STREX splice variant expressed in the HEK-293 cell line, changes in channel activity in response to hypoxia were observed. For example in 1 mM free calcium at +60 mV, Po was reduced from control values of 0.57 ± 0.04 (n = 12) in normoxic solutions to 0.24 ± 0.06 (n = 15) after 8 min exposure to hypoxic solution (Student’s paired t test, P < 0.05). The inhibition of channel activity was reversed upon washout. Similar responses were observed in 0.1 mM free calcium. No change in Po was observed in response to hypoxia in the presence of 10 µM free calcium (n = 8).
In the STREX-expressing corticotroph cell line AtT20, endogenous BKCachannel activity was also inhibited upon application of hypoxic solutions. In 1 µM free calcium at +20 mV, Po was reduced from control values of 0.56 ± 0.03 (n = 10) to 0.11 ± 0.02 (n = 10) (Student’s paired t test, P < 0.05). Similar reductions in Po were observed in 0.1 µM free calcium. This reduction was not observed in 10 µM free calcium (n = 8).
In conclusion, these data indicate that the mbr5 and STREX splice variants exhibit differential sensitivity to changes in oxygen tension. The inhibition of STREX variant activity by hypoxic solutions appears to be calcium dependent and further experiments will be required to elucidate the site and mechanism of channel modulation.
This work was supported by the University of Stathclyde, the Robert Gordon University and The Wellcome Trust.