The membrane protein Pannexin1 forms two open-channel conformations depending on the mode of activation

Abstract

Pannexin1 (Panx1) participates in several signaling events that involve adenosine triphosphate (ATP) release, including the innate immune response, ciliary beat in airway epithelia, and oxygen supply in the vasculature. The view that Panx1 forms a large ATP release channel has been challenged by the association of a low-conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K(+)), Panx1 formed a high-conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K(+). The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single-particle electron microscopic analysis revealed that K(+) stimulated the formation of channels with a larger pore diameter than those formed in the absence of K(+). These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties.

Fig. 1

ATP release from uninjected and Panx1-expressing oocytes. All colored bars represent data obtained under voltage-clamp conditions; white bars are data from unclamped cells. ATP in the media was measured as luciferase luminescence.(A) ATP release from uninjected oocytes or Panx1-expressing oocytes in the presence of oocyte Ringer solution (OR), 150 mM potassium gluconate (KGlu) solution, or to holding the membrane potential at +40 mV under voltage-clamp conditions. Data are shown as means ± SD. N is indicated above the bars. (B) Membrane currents and ATP release in uninjected and Panx1-expressing oocytes with the membrane potential held at +40 mV. Blue = uninjected and red = Panx1-injected oocytes. (C) ATP release from Panx1-expressing oocytes induced by KGlu with and without holding the membrane potential under voltage-clamp conditions at −60, 0, or +40 mV. Voltage-clamp conditions are indicated with blue lines below the graph and blue bars in the graph. Open bars are data from oocytes that were not voltage clamped. The presence of 150 mM KGlu or 100 µM carbenoxolone (CBX) is indicated. Data are shown as means ± SD. N=5 for each measurement. (D) Membrane currents of oocytes used in experiments shown in C. The voltage traces (top) start with the resting potential followed by the holding potential at +40 mV. Blue current traces: membrane potential stepped from the resting membrane potential to +40 mV. Red current traces: membrane potential stepped from the resting membrane potential to 0 mV in the presence of 150 mM KGlu.

Fig. 2

K⁺-induced ATP release from Panx1-expressing oocytes. (A) Dose-dependent induction of ATP release from Panx1-expressing oocytes. Panx1-expressing oocytes were exposed for 20 minutes to Ringer solutions with increased K⁺ (replacing Na⁺) and aliquots of the supernatant were collected for ATP measurements with the luciferase assay.. (B) Reversibility of K+-induced ATP release. ATP was measured from the same oocytes before, immediately after the K⁺ stimulus, and 20 or 40 minutes after replacement of the K⁺ stimulus with regular oocyte Ringer solution (OR, wash). (C) ATP release from oocytes expressing Cx46 in the presence of oocyte Ringer solution (OR), or holding the membrane potential at 0 mV under voltage-clamp conditions in the presence or absence of CBX (200 µM). For all panels, data are shown as means ± SD. (N=5 for each measurement)

Fig. 3

Single-channel recordings of Panx1 channels with K⁺ or Na⁺ in the extracellular solution. (A) An inside-out membrane patch exposed to high extracellular [K⁺] and clamped at – 100 mV exhibited a maximal conductance of ~500 pS. The fully open and fully closed states are indicated by solid lines; the dashed lines indicate levels of three major subconductances. The current amplitude determined by the solid lines (i_max) was used to calculate the maximal conductance (g_max) of the Panx1 channels contained in different patches. (B) An outside-out patch exposed to a low extracellular [K⁺] (OR) and clamped at + 50 mV containing two channels that opened to the current levels O₁ and O₂. A segment of the record with single channel activity is shown at extended scale (5×) as indicated. The pA/sec scale applies to the main recordings in A (20 pA) and B (10 pA). (C) Current record from an outside-out patch held at +100 mV and sequentially exposed to high K⁺ solution (150 mM KGlu), normal OR (NaCl), and then high K⁺ solution.

Fig. 4

Modification by thiol reaction of Panx1 channels activated by K⁺ or by voltage. (A) Current traces recorded from an oocyte expressing wild-type Panx1 (wtPanx1). The membrane potential was held at −50 mV and 5 mV voltage pulses were applied at a rate of 0.1 Hz. Lower bar indicates KGlu (85 mM, K⁺); upper bar indicates presence of 100 µM MMB. KGlu and MMB were washed out and solution was replaced with oocyte Ringer solution, allowing, the membrane currents and conductance to return to baseline. After changing the gain (upward arrow), the pulse amplitude was increased to 100 mV (downward arrow) to stimulate voltage-activated Panx1 channel currents. Bar indicates the presence of 100 µM MBB. (B) Quantitative analysis of current inhibition by MBB for K⁺-activated and voltage-induced Panx1 channel currents. The number of measurements (N) is indicated above the bars. Data are shown as means ± SD. (C) Current traces from oocytes expressing Panx1^T62C,C426S channels. Recording conditions were the same as in (A). Upper trace shows the effect of 1 mM MTSET on K⁺-activated Panx1 current. Lower trace shows the effect on voltage-activated current. Because of the reactivity of the thiol groups in K⁺ with MTSET, different oocytes were used for testing the effects on K⁺-activated and voltage-activated Panx1 currents. (D) Quantitative analysis of current inhibition by 1 mM MTSET for K⁺-activated and voltage-induced Panx1^T62C,C426S channel currents. Data are shown as means ± SD. N is indicated above the bars. (E) Current traces of oocytes expressing wtPanx1. Recording conditions were the same as in (A). Panx1 was activated by K⁺ as indicated, and MTSET (1 mM) was applied as shown by the bar.

Fig. 5

EM image analysis of negatively stained Panx1 oligomers. (A) Preparations purified from baculovirus-infected Sf9 cells as detected on a Sypro Ruby-stained protein gel (Gel Stain) and a corresponding Western Blot (WB). (B, C) Two representative low dose electron micrographs for uranyl acetate-stained purified Panx1 channels diluted in water (B, unstimulated condition) and in KCl 50 mM (C, stimulated condition). These micrographs were recorded with a standard low dose imaging protocol (47) to minimize electron beam damage and preserve higher resolution structural features. The scale bar represents 50 nm. The insets on the top of the micrographs are three-fold enlargements of the encircled particles. The rectangle indicates one channel with the cytoplasmic view of the Panx1 channel facing up, and the circle indicates a channel with the extracellular view facing up. (D) Representative class averages (Class Avg) and hexagonal-symmetrized averages (Hexagonal Avg) of cytoplasmic and extracellular surface views of unstimulated and stimulated Panx1 channels. Shown also are averages and standard deviations from 10 measurements of the channel and pore diameters of 4–5 class averages for both views from the two conditions. (E) Scatter plot of the measurements shown in (D, n=10 for each measurement). +/−KCl Cyto Ptcl, stimulated and unstimulated cytoplasmic particle outer diameter; +/− KCl Extracell Ptcl, stimulated and unstimulated extracellular particle outer diameter