Outward Current in SRA 01/04 Human Lens Epithelial Cells is Reduced Following BK Channel Knockdown With RNAi

Abstract

Abstract: : Purpose: We have shown previously that inhibition of K⁺ channels in lens epithelial cells (LEC) is correlated with lenticular opacity. Electrophysiological and pharmacological studies suggest that affected K⁺ channels include calcium–activated BK and voltage–gated Kv channel subtypes. The purpose of this study was to further specify affected K⁺ channel subtypes in LECs using RNAi and patch clamp electrophysiology. Methods: SV40–immortalized SRA 01/04 human LECs were incubated in normal media (low glucose DMEM w/15% FBS) until ∼70% confluent. Cells were treated with FITC–labeled, non–specific siRNA for 4 hours to confirm transfectibility, and with channel–specific siRNAs for 6 and 24 hours (and subsequently in normal media for total incubation times of 24–72 hrs) to knock down human isoforms of the BK, Kv1.3, and Kv1.5 potassium channels. Following transfections cells were analyzed for viability using WST–1. Constitutive expression and knockdown efficiency were determined using RT–PCR and Western Blot. Effects on outward current were determined using whole–cell patch clamp electrophysiology. Results: RT–PCR and Western Blot showed constitutive expression of BK, Kv1.3, and Kv1.5 potassium channel subtypes in control SRA 01/04 human LECs. Following transfection with the FITC–labeled, non–specific siRNA nearly all SRA cells were labeled, demonstrating high transfection efficiency. Transfection with BK–specific siRNAs produced a 70% decrease in BK message after exposure to 100 nM siRNA; a concentration not affecting cell viability. Transfection with Kv–specific siRNAs produced a 40–50% reduction in Kv1.3 and Kv1.5 message under similar conditions. After 48 to 72 hours, whole–cell patch clamp analysis of SRAs showed a 70% reduction (P < 0.05) in membrane outward current density (39.1 ± 32.0 pA/pF (n = 7) vs. 139.2 ± 122.7 pA/pF (n = 16) in BK channel knockdowns, but no change (P > 0.05) in Kv1.3 (n = 5) or Kv1.5 (n = 7) channel knockdowns. Conclusions: These results indicate that large–conductance, calcium–activated potassium channels (BK channels) underlie the majority of the voltage–dependent, outward current in the SRA 01/04 human LEC. Some types of drug–induced cataractogenesis may result from compound interactions with BK potassium channels in the lens.

Keywords: ion channels • cataract • electrophysiology: non-clinical