Innexins form two types of channels

Abstract

Injury to the central nervous system triggers glial calcium waves in both vertebrates and invertebrates. In vertebrates the pannexin1 ATP-release channel appears to provide for calcium wave initiation and propagation. The innexins, which form invertebrate gap junctions and have sequence similarity with the pannexins, are candidates to form non-junctional membrane channels. Two leech innexins previously demonstrated in glia were expressed in frog oocytes. In addition to making gap junctions, innexins also formed non-junctional membrane channels with properties similar to those of pannexons. In addition, carbenoxolone reversibly blocked the loss of carboxyfluorescein dye into the bath from the giant glial cells in the connectives of the leech nerve cord, which are known to express the innexins we assayed.

Figure 1

Macroscopic membrane currents. (a): The membrane potential of an Hminx2--expressing oocyte was held at the potentials indicated and 5 mV hyperpolarizing voltage pulses to test membrane resistance were applied (top trace) at a rate of 6/min. The corresponding currents are shown in the bottom trace. (b) Voltage dependence of membrane conductance (g_m) of oocytes expressing Hminx2 ( ),Hminx3 ( ) or uninjected oocytes (◽). Means ±SD (n=4, 5, or 5, respectively) are plotted. The membrane potential was held at the respective voltages for 5 minutes and conductance was determined with 5 mV depolarizing pulses. (c) & (d): Application of CO₂ to the bath solution reduced membrane currents of oocytes expressing Hminx2 (c) or Hminx3 (d). (e): Addition of 100 μM carbenoxolone increased membrane resistance and blocked the current in an oocyte expressing Hminx2. Note the slow time course of recovery. (f) ATP release mediated by Hminx2is inhibited by carbenoxolone. Release of ATP to the extracellular medium by oocytes was measured by luminometry using a luciferase assay. Depolarization of the cells with high potassium solution (KGlu) resulted in a small ATP release from uninjected oocytes and a significantly larger release from Hminx2injected oocytes. Carbenoxolone attenuated this larger release in a dose dependent fashion. Means ± SEM are given (n=5). ***= p< 0001, **=p<0.001, *=p<0.05.

Figure 2

Single channel currents of leech Hminx2. (a) The membrane potential was held at positive potential to activate the channel before shifting to –50 mV. Like human pannexin 1 channels, the channel has a large unit conductance (~500 pS in 150 mM KCl) and exhibits many subconductance levels (compare to Figure 1 in Bao et al., 2004). Histogram shows distribution of current amplitudes, with intermediate peaks at subconductance levels. (b) Patch clamp recording from a membrane patch of an oocyte exogenously expressing Hminx2. After activation of the channels at +20 mV, the membrane potential was held at −50 mV. At this potential, channel activity continued for a period of time and then subsided, as is typical for this holding potential. Negative pressure (~40 mbar) was applied by suction to the patch pipette during the time indicated by the line. That the patch contained at least three channels can be seen by the steps during negative pressure application.

Figure 3

Carbenoxolone reversibly inhibits 6- carboxyfluorescein dye loss from the connective glial cell. (a) Representative time course of dye loss from a 1 mm piece of 6-carboxyfluorescein (6-CF) injected connective glial cell (see Supplemental Figure 1b). Carbenoxolone (CBX, 10μM) was added and washed out as indicated. (b) Quantification of average dye loss before, during, and after CBX treatment. (*= p<.01, **= p<0.001, n=6) Error bars represent SEM. (c): A schematic diagram showing that innexins share the roles both of pannexins in forming channels to the extracellular space and of connexins in forming gap junctions.