Characterization of Hemi Gap Junction Channels in Zebrafish Horizontal Cells

Abstract

Purpose:: Hemi gap junction (HGJ) channels of horizontal cells (HCs) formed by a subset of connexins (Cxs) may participate in the negative feedback from HCs to cones. So far several Cxs have been cloned and identified from zebrafish retina and some have been shown to possess hemichannel-forming properties in heterologous expression systems. However, little is known about native HGJ channels in zebrafish HCs. To better understand the putative roles for HGJ channels in vivo, here we examined the electrophysiological, biophysical and pharmacological properties of hemichannels of zebrafish HCs.

Methods:: Solitary HCs were isolated from zebrafish retinas and macroscopic currents were recorded with a conventional whole-cell voltage clamp mode. The single-channel properties of HGJ channels were investigated using an outside-out patch configuration. During the recordings, potassium channels of HCs were blocked by adding potassium channel blockers into intracellular and extracellular solutions.

Results:: In Ca²⁺-free medium, slowly activating inward and outward hemichannel currents were elicited by 10-s voltage pulses stepping from a holding potential of 0 mV. Outward currents elicited by depolarizing to +60 mV were 273 ± 23 pA (n=10, mean ± SE), while inward currents induced by hyperpolarizing to -60 mV were 52 ± 5 pA (n=10). Increasing the external Ca²⁺ from 0 mM to 2.5 mM substantially suppressed both inward and outward currents to 19 ± 5% (n=5) and 12 ± 5% (n=5) of control, respectively. In addition, external application of 200 µM carbenoxolone reversibly reduced outward currents, while external application of 200 µM quinine enhanced the outward currents. However, neither of these treatments affected inward HGJ currents. Furthermore, single-channel recordings from outside-out patches show that the unitary conductance of hemichannels was 47 ± 4 pS (n=5) at a holding potential of -20 mV, whereas it was 70 ± 5 pS (n=5) at 20 mV.

Conclusions:: Our results demonstrate that functional HGJ channels are expressed in zebrafish HCs. The general properties of zebrafish HGJ outward currents are consistent with those of other fish, whereas inward HGJ hemichannel currents induced by hyperpolarization have not been previously described. In addition, the different biophysical and pharmacological properties of outward currents and inward currents suggest that their channels could be composed of different Cxs.

Keywords: horizontal cells • electrophysiology: non-clinical • ion channels