Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Tuesday Speaker Abstracts

LARGE-PORE CHANNELS AS TRANSPORTERS: LESSONS FROM CONNEXIN, PANNEXIN AND CALHM1 CHANNELS Jorge E. Contreras 1 ; Pablo S Gaete 1 ; Cynthia I Fernandez 1 ; Wenjuan Jiang 2 ; Yun Luo 2 ; 1 University of California Davis , Physiology & Membrane Biology, Davis, CA, USA 2 Western University of Health Sciences, Pharmaceutical Sciences, Pomona, CA, USA Large-pore channels are a diverse group of non-conventional membrane channels characterized by their permeability to atomic ions and a broad repertoire of small molecules. Examples of vertebrate large-pore channels include those formed by connexins (Cxs), pannexins, calcium homeostasis modulators (CALHMs), and LRRC8 (also known as SWELL channels). Although they are currently emerging as important regulators in health and disease, mechanisms for molecular permeation and selectivity remain largely unexplored. Accordingly, we have recently developed a methodology that allows us to simultaneously examine the flux of atomic ions and molecules through large-pore channels. We found that connexin hemichannels, pannexin, and CALHM-1 channels display molecular transport that can be described by Michaelis-Menten kinetics, with apparent K M and V max . The observed V max , but not K M values, were sensitive to channel gating modulators (e.g., voltage and extracellular calcium). To mechanistically examine these properties, we performed molecular dynamics using equilibrated Cx26 and Cx30 hemichannels. Simulations confirmed molecular permselectivity and identified discrete binding sites and energy barriers for molecules within the pore. Calculation of the nonbonded interaction between molecules and protein residues showed that permeants form strong interactions with the N-terminal (NT) region of connexin hemichannels. Mutagenesis analysis and functional assays revealed that the NT is a critical player in the mechanisms of permselectivity. Consistently, human disease-associated mutations at the N-terminal region of Cx26 and Cx30 significantly affected kinetics of molecular transport and selectivity. Our results reveal that large-pore channels are not free diffusion pores for molecules and suggest that the presence of binding sites in the permeation pathway could also determine selectivity, similarly to transporters/carriers.

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