The M34A mutant of Connexin26 reveals active conductance states in pore-suspending membranes

Abstract

Connexin26 (Cx26) is a member of the connexin family, the building blocks for gap junction intercellular channels. These dodecameric assemblies are involved in gap junction-mediated cellcell communication allowing the passage of ions and small molecules between two neighboring cells. Mutations in Cx26 lead to the disruption of gap junction-mediated intercellular communication with consequences such as hearing loss and skin disorders. We show here that a mutant of Cx26, M34A, forms an active hemichannel in lipid bilayer experiments. A comparison with the Cx26 wild-type is presented. Two different techniques using micro/nano-structured substrates for the formation of poresuspending lipid membranes are used. We reconstituted the Cx26 wild-type and Cx26M34A into artificial lipid bilayers and observed single channel activity for each technique, with conductance levels of around 35, 70 and 165 pS for the wildtype. The conductance levels of Cx26M34A were found at around 45 and 70 pS.

Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins

“…Although the biochemical evidence provided strong indications that Na V Sp1p is a folded, tetrameric protein, it was essential to determine whether Na V Sp1p retained any functional ion channel properties. Therefore, we reconstituted purified Na V Sp1p into 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) giant unilamellar vesicles (GUVs) created by electroformation (44,45) and assayed the properties of the reconstituted channels using planar patch clamp technology (46,47). Measurement of the reversal potential of bilayers containing hundreds of channels under asymmetric NaCl conditions yielded E rev ¼ 53.69 AE 8.05 mV (n ¼ 6) (Fig.…”

“…Lipid bilayer experiments performed using a planar patch clamp system (Port-a-Patch, Nanion Technologies GmbH) on Na V Sp1p, Na V Ae1p, or Na V Ab1p were incorporated into GUVs using procedures described previously (44,45). To determine Na þ , K þ , and Ca 2þ permeabilities, the reversal potential was measured in asymmetric conditions where the internal solution contained 10 mM Na-Hepes, 200 mM NaCl, pH 7.0 (adjusted with NaOH) and the external solution contained 10 mM Hepes, 160 mM CaCl 2 , pH 7.0 (adjusted with HCl) or 10 mM Hepes, 110 mM KCl, pH 7.0 (adjusted with KOH).…”

Permeation of Calcium through Purified Connexin 26 Hemichannels

“…Recombinant Cx26 HCs expressed in Sf9 insect cells has been used for structural studies without parallel functional analysis (37)(38)(39), with exception of a recent publication focused on single-HC recordings (40). Previous electron microscopy results clearly point to Cx26 purified as HCs (hexamers), as opposed to GJCs (dodecamers) (37)(38)(39)41), and our measurements of hydrodynamic radius by dynamic light scattering and apparent molecular mass by size exclusion chromatography fully agree with that notion.…”

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

“…Such an amount of protein is, however, sufficient to monitor single channel activities of the proteins. The method has already been applied to reconstitute Cx26 in GUVs (24). To obtain larger protein amounts, Streicher et al (46) reconstituted integrins into GUVs by briefly drying preformed proteoliposomes on ITO coated-glass slides.…”

“…Solubilization, Purification, and Western Blot Analysis-P. pastoris membrane pellets were resuspended for 2 h at 4°C in 1 mM bicarbonate buffer, pH 8.0, containing 1 mM PMSF and homogenized by sonification for 30 s. The purification protocol was adapted from a previously reported procedure (24). First, the membrane fragments were solubilized for 2 h at 4°C in detergent buffer containing 3% dodecyl maltoside (DDM), 1 M NaCl, 1 mM PMSF, 0.005% NaN 3 , and 10 mM HEPES, pH 7.4.…”