A number of naturally occurring toxins have provided electrophysiologists with useful pharmacological tools for the study of ion channels. Many bind with high affinity and specificity to different Kvα subunits; some even distinguish between different members of the same Kv subfamily. Diochot et al. (1998) reported that the toxin, blood depressing substance (BDS), isolated from Anemonia sulcata, selectively blocked the rapidly inactivating Kv3.4 channel with an IC50 ~50 nM. Here we show BDS also inhibits Kv3.1 and Kv3.2 channels expressed in mammalian cell lines.
Recordings were made from HEK293 and B82 cells using whole cell voltage-clamp techniques. External and internal (pipette) solutions were based on Korngreen & Sakmann (2000). Voltage-gated K+ currents were evoked by stepping the cells from a holding potential (VH) of -80 mV to a variety of test potentials.
Both BDS-I and BDS-II (50-1000 nM) inhibited Kv3.1 currents in a concentration-dependent manner (Fig. 1). A single concentration of 500 nM was used in subsequent experiments as it resulted in approximately 50 % block – BDS-I: 48.1 ± 4.5 % (mean ± S.E.M., n = 5), BDS-II: 51.3 ± 5.4 % (n = 4).
Inhibition by BDS-I and -II caused a dramatic slowing in the rate of current activation (5- to 8-fold increase) at +40 mV with a positive shift in the activation threshold (~-20 mV, from approximately -10 to +10 mV, n = 4), suggesting these are ‘gating modifier’ toxins (e.g. Swartz & MacKinnon, 1997) (Fig. 2). Block did not require open channels, but was voltage dependent. With increasingly positive test potentials, the amount of block significantly reduced: e.g. +10 mV = 76.7 ± 2.7 % (n = 4), +80 mV = 21.0 ± 2.8 % (n = 4). The rate of Kv3 channel block was faster with BDS-II than BDS-I.
An endogenous current present in HEK 293 cells that were used to transiently express Kv3.2 channels was also blocked in a qualitatively similar manner by BDS. Antibody staining of these cells (Ruth Brooke, personal communication) suggests that Kv3.3 is present endogenously.
These data indicate that BDS-I and BDS-II are not selective blockers for the Kv3.4 channel.
This work is supported by the MRC.