A novel voltage-clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels

Abstract

Large-pore channels permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, their mechanisms of molecular permeation and selectivity remain mostly unexplored. Combining fluorescence microscopy and electrophysiology, we developed a novel technique that allows kinetic analysis of molecular permeation through connexin and CALHM1 channels in Xenopus oocytes rendered translucent. Using this methodology, we found that (1) molecular flux through these channels saturates at low micromolar concentrations, (2) kinetic parameters of molecular transport are sensitive to modulators of channel gating, (3) molecular transport and ionic currents can be differentially affected by mutation and gating, and (4) N-terminal regions of these channels control transport kinetics and permselectivity. Our methodology allows analysis of how human disease–causing mutations affect kinetic properties and permselectivity of molecular signaling and enables the study of molecular mechanisms, including selectivity and saturability, of molecular transport in other large-pore channels.

Connexin and Pannexin Large-Pore Channels in Microcirculation and Neurovascular Coupling Function

“…The role and participation of Cxs and Panxs large-pore channels are still under experimental observation. For instance, recent results display a transport behavior with a molecular permeability at a negative potential (i.e., resting membrane potential) in the absence or reduced atomic ion conductance [6]. The physiological and pathophysiological roles of Cxs and Panxs could be essential in homeostatic functions of the microcirculation.…”

“…Connexin (Cxs) hemichannels (also termed connexons) are the hexameric single-membrane subunits of GJ contributed by each cell (Figure 1). GJ channels and Cxs hemichannels are permeable to ions and small molecules (<1.5 kDa) [4][5][6][7][8][9][10]. Twenty connexin isoforms are expressed in mammalian cells, and each connexin isoform is referred to according to its molecular weight (i.e., Cx43 has a molecular weight of 43 kDa) [11].…”

“…Tissues and cells, including the vascular microcirculation, often express more than one connexin isoform [12][13][14]. Hemichannels can be organized by an association of one (homomeric channels) or a combination (heteromeric channels) of additional connexin isoforms, which normally differ in unitary conductance, ion/molecule permeability, and regulation [4,6,8,11,15,16]. The activity of connexin proteins is regulated by several factors including: voltage, pH, carbon monoxide, and extracellular-intracellular Ca 2+ [4,11,[17][18][19][20][21][22].…”

“…Connexin post-translational modifications such as phosphorylation or S-nitrosylation play a role in keeping the function and homeostasis of the tissue [23][24][25][26][27]. Cxs hemichannels also promote ion permeation (i.e., Ca 2+ ) [6,9,15,[28][29][30] and molecule release (ATP, D-serine, prostaglandins, nitric oxide, etc.) to the milieu, playing a vital role in cell function activity [15,[31][32][33][34][35].…”

A new window into large-pore channels

“…Despite their physiological importance, little is known about how these channels transport molecules. In this issue of JGP, Gaete et al describe a new assay to analyze the kinetic properties and permselectivity of this diverse family of channels (1).…”