Pannexin (PANX) family proteins form large-pore channels that mediate purinergic signaling. We analyzed the cryo-EM structures of human PANX1 in lipid nanodiscs to elucidate the gating mechanism and its regulation by the amino terminus in phospholipids. The wild-type channel has an amino-terminal funnel in the pore, but in the presence of the inhibitor probenecid, a cytoplasmically oriented amino terminus and phospholipids obstruct the pore. Functional analysis using whole-cell patch-clamp and oocyte voltage clamp showed that PANX1 lacking the amino terminus did not open and had a dominant negative effect on channel activity, thus confirming that the amino-terminal domain played an essential role in channel opening. These observations suggest that dynamic conformational changes in the amino terminus of human PANX1 are associated with lipid movement in and out of the pore. Moreover, the data provide insight into the gating mechanism of PANX1 and, more broadly, other large-pore channels.

The cloned α1‐subunits of the smooth muscle Ca2+ channel (α1C‐b) from rabbit lung were expressed in Chinese hamster ovary cells. The effect of large depolarizations was examined using cell‐attached patch clamp techniques. After large, long‐duration depolarizations (to +80 mV, 4 s), the cloned smooth muscle Ca2+ channels were still open, and also showed slow channel closure upon repolarization. The sum of unitary channel currents revealed that the tail current seen after large conditioning depolarizations had a slower deactivation time constant compared to that seen when the cell membrane was depolarized briefly with a test step (to +40 mV), suggesting that large depolarizations transform the conformation of the Ca2+ channels to a second open state. The decay time course of the tail current induced by large conditioning depolarizations was prolonged by reducing the negativity of the repolarization step, and vice versa. Using the slow deactivating characteristic, the current‐voltage relationship was directly measured by applying a ramp pulse after a large depolarization. Its slope conductance was approximately 26 pS. Since the patch pipettes contained Ca2+ agonists, the transition of the Ca2+ channel conformation to the second, long open state during a large depolarization was distinct from that caused by Ca2+ agonists, suggesting that the cloned α1‐subunits of smooth muscle Ca2+ channels preserve the characteristic features seen in native smooth muscle Ca2+ channels. In addition, when skeletal muscle β‐subunits were coexpressed with the α1‐subunits, the long channel openings after large, long‐duration depolarizations were frequently suppressed. This phenomenon could be explained if the skeletal muscle β‐subunits increased the inactivation rate during the preconditioning depolarization.

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