Voltage-sensitive chloride ion channels in Anopheles gambiae Sua-1B cells

Abstract

In this study, we performed electrophysiological analysis of Anopheles gambiae Sua-1B cells having "neuron-like" morphologies using the patch clamp method. The recorded cells (n = 79) had processes resembling axons/dendrites, with 63 % unipolar, 22 % bipolar, and 15 % multipolar. While no inward currents were observed following step depolarizations (holding potential = -80 mV), a slowly activating outward current was observed in 96 % of the cells, especially at depolarized potentials. The amplitude of the current was attenuated nearly 70 % by reducing extracellular Cl⁻ ion concentration, or by incubating with 100 μM DIDS, a known voltage-sensitive chloride channel blocker, suggesting that the current was mediated by chloride ions. No qualitative difference was found between recordings made with Cs⁺ ions in the intracellular pipette solution (inhibits K⁺ currents) and those made with normal physiological solution, indicating a deficiency of potassium channels. Additionally, recordings made with Ca²⁺-free extracellular bath solution eliminated the slowly activating outward current. A subset of cells (n = 3) lacked this current, but had outward currents with voltage-dependent properties similar to those of volume-regulated chloride channels. Taken together, our results suggest that the voltage-sensitive currents observed in the majority of Sua-1B cells are mediated primarily by chloride channels of the calcium-dependent subtype.

Insecticide sensitivity of native chloride and sodium channels in a mosquito cell line

“…When characterizing the chloride channels present in Sua1B cells, this study found the majority of channels to be VSCCs, while those Diykov et al [7] found were predominantly the CACC channel subtype. Both chloride channel subtypes were found in each study, but are thought to activate in different physiological circumstances and occur independently of one another.…”

“…Outwardly rectifying voltage sensitive currents observed in native Sua1B cells seem to be mediated either primarily by CACCs or by a combination of CACCs and volume-sensitive, outwardly-rectifying anion channels [7]. High concentrations of lindane, an organochlorine insecticide, inhibited growth of Sua1B cells, but the response did not occur in a typical concentration-dependent manner.…”

“…Two types of chloride channels previously identified in Sua1B cells [7] were characterized within this study. Outwardly rectifying voltage sensitive currents observed in native Sua1B cells seem to be mediated either primarily by CACCs or by a combination of CACCs and volume-sensitive, outwardly-rectifying anion channels [7].…”

“…The present study used established pharmacological probes, cell growth, and patch clamp techniques, to detect expression of targets in Sua1B cells, a cell line first isolated from neonate Anopheles gambiae larvae [6]. Screens focused mostly on compounds affecting VSCCs, since these are known to exist in Sua1B cells [7], and blockers of these channels are insecticidal [8]. Special attention was made to correlate ion channel effects with changes in cell growth.…”

Pharmaceutical technologies with potential application to insecticide discovery†

“…There have been a number of studies in which cultured insect cells have been treated with the molting hormone 20‐hydroxy ecdysone (20‐OH E) and which have resulted in a more spindly shape as the cells differentiate into neuron‐like structures with longer processes. In some cases, it appears that voltage‐gated sodium, calcium, potassium or chloride channels have been induced concomitant with the morphological change 37–41 . More recently, it has been shown that 20‐OH E treatment of Aedes RML12 cells led to spontaneous neuroactivity as monitored by multiwell microelectrode array, and that this could be enhanced by treatment with the GABA antagonist gabazine and especially nicotine 42 …”