Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC289
Characterisation and pharmacological activation by bithionol of human SLO2.2A K+ channels
D. Wrighton1, J. Lippiat1
1. IMSB, University of Leeds, Leeds, United Kingdom.
Slo2.2 (Slack) channels are structurally related to BKCa channels (Slo1), but rather than synergistic activation by Ca2+ and voltage they are activated by intracellular Na+ and Cl-. Functionally this class of channel (KNa) is believed to have a role in the control of neuronal excitability and spike frequency adaptation, where channel activity is coupled to Na+ during action potentials (1) or excitatory postsynaptic potentials (2). However the composition of the channels conducting the physiological current has yet to be elucidated. This has been complicated by the identification, in rat, of several N-terminal splice variants, including Slo2.2a (Slack-A), Slo2.2b (Slack-B), and Slo2.2m (Slack-M), in addition to the Slo2.1 (Slick) subunit, which also forms KNa channels (3). Bithionol, a bis-phenol antihelminthic compound, has previously been described to activate rat Slo2.2b (Slack-B) channels expressed in both Xenopus oocytes and human embryonic kidney (HEK293) cells (4), however its effect on the other subtypes has yet to be determined. Using whole cell patch clamp of HEK293 cells expressing the human variant of Slo2.2a we have investigated the properties of this homologue and it’s activation by bithionol. Exogenous hSlo2.2a currents were evoked by dialysis of intracellular solution containing Na+ 20 mM and currents reversed at EK (-83mV), indicating high K+-selectivity. Unlike rat channels, hSlo2.2a did not exhibit time-dependent activation with depolarising voltage pulses and exhibited a linear current-voltage relationship. Bithionol activated hSlo2.2a channels with an EC50 of 1.29 ± 0.12 µM (n=5). Secondly by variance of intracellular Na+, between 0-20 mM, in both high (130-150 mM) and low (1mM) [Cl-]i, we found that bithionol activation of hSlo2.2a was not additive but cooperative. With all intracellular Na+ concentrations the reversal potential of the bithionol activated current did not significantly deviate from EK (-83mV) (p>0.05, ANOVA). These findings show that human Slo2.2a are voltage-independent K+-selective channels that are likely to contribute to the background, rather than delayed-rectifier, K+ conductance. Furthermore, Na+ and bithionol activate the channels via different modulatory sites. Bithionol could potentially be used to lower excitability in Slo2.2a-expressing cells and would be particularly effective in highly active neurons where Na+ influx is frequent.
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