Physiological and molecular characterization of connexin hemichannels in zebrafish retinal horizontal cells

Abstract

Connexin channels mediate electrical synaptic transmission when assembled as cell-to-cell pores at gap junctions and can mediate transmembrane currents when expressed in plasma membranes as hemichannels. They are widely expressed in the vertebrate retina where in electrical synapses they are critical for transmission of visual signals. While the roles of connexins in electrical synapses are well-studied, the function and roles of connexin hemichannels in the nervous system are less well understood. Genetic deletion in zebrafish of connexin (Cx) 55.5 alters horizontal cell feedback to cones, spectral responses, and visual behavior. Here, we have characterized the properties of hemichannel currents in zebrafish retinal horizontal cells and examined the roles of two connexin isoforms, Cx55.5 and Cx52.6, that are coexpressed in these cells. We report that zebrafish horizontal cells express hemichannel currents that conduct inward current at physiological negative potentials and Ca(2+) levels. Manipulation of Cx55.5 and Cx52.6 gene expression in horizontal cells of adult zebrafish revealed that both Cx55.5 and Cx52.6 contribute to hemichannel currents; however, Cx55.5 expression is necessary for high-amplitude currents. Similarly, coexpression of Cx55.5 with Cx52.6 in oocytes increased hemichannel currents in a supra-additive manner. Taken together these results demonstrate that zebrafish horizontal cell hemichannel currents exhibit the functional characteristics necessary to contribute to synaptic feedback at the first visual synapse, that both Cx55.5 and Cx52.6 contribute to hemichannel currents, and that Cx55.5 may have an additional regulatory function enhancing the amplitude of hemichannel currents.

Fig. 1.

Voltage-gating and Ca²⁺-gating properties of low Ca²⁺-evoked hemichannel currents in cultured zebrafish retinal horizontal cells (HCs). A: photomicrographs of solitary HCs in primary cell culture (indicated by arrows) immediately after isolation (left) and 1 day later in culture (right). Scale bar = 10 μm. B: immunohistochemistry for connexin (Cx) 55.5 and Cx52.6 proteins in cultured solitary HCs. Both Cx55.5 (left) and Cx52.6 (right) antibodies produce intense and bright punctate staining on the somata and dendrites of cultured HCs. Scale bar = 5 μm. C: both outward (left) and inward (right) components of hemichannel currents were substantially blocked in 2.5 mM Ca²⁺ medium (gray traces), whereas they were greatly increased after perfusion with Ca²⁺-free medium (black traces). D: normalized current-voltage (I–V) curves in Ca²⁺-free medium (●) and in 2.5 mM Ca²⁺ medium (○). Net currents mediated by hemichannels were shown as the difference (▴) (n = 5, mean ± SE). E: normalized Ca²⁺ inhibitory curves for outward (●) and inward (○) components of hemichannel currents. Each point is an average from 4–6 cells, and smooth curves were fitted with the Hill equation. The half-maximal inhibitory concentration (IC₅₀) is 0.69 mM for the outward component and 1.58 mM for the inward component.

Fig. 2.

Single channel events of outward and inward components of hemichannel currents. A: single channel traces of outward (top) and inward (bottom) components of hemichannel currents elicited from holding potentials of 0 mV (middle) to +20 and −20 mV, respectively. Time and amplitude are indicated as the scale bar. B: all-points histogram of outward and inward current arms from A. Distribution of the digitized current values was plotted as a histogram, and data points were fit with Gaussian curves. Peaks shown in histogram indicate currents corresponding to open and closed channels. C: average unitary conductance of outward (70 ± 5 pS, open bar) and inward (47 ± 4 pS, filled bar) hemichannel current arms. ***P < 0.001 (n = 5, mean ± SE). D: averaged overall open probability of outward (0.54 ± 0.08, open bar) and inward (0.26 ± 0.06, filled bar) hemichannel current arms. *P < 0.05 (n = 5, mean ± SE).

Fig. 3.

Anti-Cx55.5 and anti-Cx52.6 morpholinos (MOs) inhibited the protein expression of Cx55.5 and Cx52.6 isoforms in the cultured HCs, respectively. A: immunostaining of Cx55.5 proteins (green) in anti-Cx55.5 MO-treated HC (top), anti-Cx52.6 MO-treated HC (middle), and control MO-treated HC (bottom). Lissamine-tagged MOs are visualized as red fluorescence. Merged images indicate that the expression of Cx55.5 protein in HCs was suppressed 4 days after the introduction of anti-Cx55.5 MO, whereas the Cx52.6 protein expression was normal. Scale bar = 10 μm. B: immunostaining of Cx52.6 proteins (green) in anti-Cx55.5 MO-treated HC (top), anti-Cx52.6 MO-treated HC (middle), and control MO-treated HC (bottom). Merged images indicate that the expression of Cx52.6 protein in HCs was suppressed 4 days after the introduction of anti-Cx52.6 MOs, whereas the Cx55.5 protein expression was normal. Scale bar = 10 μm.

Fig. 4.

Anti-Cx55.5 MOs suppressed both outward and inward current arms, whereas anti-Cx52.6 MOs selectively inhibited the outward current arm. A: compared with untreated HCs (black trace), both outward (left) and inward (right) hemichannel current arms were substantially decreased in anti-Cx55.5 MO-treated HCs (gray). B: averaged outward (left) and inward (right) current arms between untreated HCs (black bar) and anti-Cx55.5 MO-treated HCs (gray bar). ***P < 0.001 (n = 5). C: compared with untreated HCs (black trace), the outward current arm (left) was substantially decreased in anti-Cx52.6 MO-treated HCs (gray trace), as was the inward current (right). D: averaged outward (left) and inward (right) current arms between untreated HCs (black bar) and anti-Cx52.6 MO-treated HCs (gray bar). *P < 0.05 (untreated n = 9; Cx52.6 MO treated n = 12). E: compared with untreated HCs (black trace), both outward (left) and inward (right) current arms were sustained in control MO-treated HCs (gray trace). F: averaged outward (left) and inward (right) current arms between untreated HCs (black bar) and control MO-treated HCs (gray bar). There is no significant difference between these two groups (P > 0.05, n = 5).

Fig. 5.

Characterization of Cx52.6, Cx55.5, and Cx52.6/Cx55.5 hemichannel currents. A: current-voltage (I–V) relations of hemichannel currents recorded from Xenopus laevis oocytes. Mean current from 190 to 210 ms after pulse onset. All I–V relations show outward rectifying behavior with larger currents at positive potentials and smaller currents at negative potentials. B: I–V based on the I–V relations in A. Both the Cx55.5 and Cx52.6/Cx55.5 currents show an increase in conductance at negative potentials. Cx52.6 has the lowest conductance, whereas Cx52.6/Cx55.5 has the highest conductance.