Slow Gating of Gap Junction Channels and Calmodulin C. Peracchia, X.G. Wang, L.L. Peracchia Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642-8711, USA Received: 19 April 2000/Revised: 11 August 2000 Abstract. Certain COOH-terminus mutants of con- nexin32 (Cx32) were previously shown to form channels with unusual transjuctional voltage (V j ) sensitivity when tested heterotypically in oocytes against Cx32 wild type. Junctional conductance (G j ) slowly increased by several- fold or decreases to nearly zero with V j positive or nega- tive, respectively, at mutant side, and V j positive at mu- tant side reversed CO 2 -induced uncoupling. This sug- gested that the CO 2 -sensitive gate might be a V j -sensitive slow gate. Based on previous data for calmodulin (CaM) involvement in gap junction function, we have hypoth- esized that the slow gate could be a CaM-like pore plug- ging molecule (cork gating model). This study describes a similar behavior in heterotypic channels between Cx32 and each of four new Cx32 mutants modified in cyto- plasmic-loop and/or COOH-terminus residues. The mu- tants are: ML/NN+3R/N, 3R/N, ML/NN and ML/EE; in these mutants, N or E replace M105 and L106, and N replace R215, R219 and R220. This study also reports that inhibition of CaM expression strongly reduces V j and CO 2 sensitivities of two of the most effective mu- tants, suggesting a CaM role in slow and chemical gat- ing. Key words: Cell communication — Connexins — Gap junctions — Calmodulin — Channel gating — CO 2 Introduction Gap junctions are cell contact domains that contain chan- nels for direct cell-to-cell exchange of small cytosolic molecules. A gap junction channel is made of two hexa- meric hemichannels (connexons) composed of connexin proteins. Connexins are intramembrane proteins with four transmembrane domains, two extracellular loops, a cytoplasmic loop (CL), a short NH 2 -terminus (NT) and a COOH-terminus (CT) of variable length (reviewed in Peracchia, Lazrak & Peracchia, 1994). Transjuctional voltage (V j ) gradients and increased [Ca 2+ ] i or [H + ] i are known to reduce channel permeabil- ity (reviewed in Loewenstein, 1990; Bruzzone, White & Paul, 1996; Peracchia & Wang, 1997), but the molecular mechanisms of channel gating are still largely unknown. V j and chemical gates are believed to be two distinct gates; the former closes the channel rapidly but incom- pletely and the latter closes the channel slowly but com- pletely (Bukauskas & Peracchia, 1997). Recently, we have studied connexin mutants and chimeras, expressed in Xenopus oocytes, to identify do- mains of the rat connexin32 (Cx32) that participate in CO 2 -induced (low pH i ) channel gating. Cx32 is ex- pressed in liver, pancreas, kidney, thyroid, and mammary gland, as well as in various cells of the nervous system such as neurons, oligodendrocytes and Schwann cells (reviewed in Bruzzone et al., 1996). Several Cx32 mu- tations are relevant to the pathogenesis of the X-linked Charcot Marie Tooth demyelinating disease (CMTX; Bergoffen et al., 1993; Ionasescu, Searby & Baritt, 1994; Ressott et al., 1998). CL and the initial segment of CT (CT 1 ) appear to be relevant to the CO 2 gating sensitivity of Cx32 (Wang et al., 1996; Wang & Peracchia, 1996; Wang & Peracchia, 1997; Wang & Peracchia, 1998a), whereas NT (Wang et al., 1996) and the rest of CT (84%, Wang & Peracchia, 1997; Wang & Peracchia, 1998a) do not seem to play a role. Based on these data we have proposed that the membrane proximal domain of CT (CT 1 ), a basic and partly hydrophobic domain, may in- teract electrostatically and hydrophobically with acidic and hydrophobic residues of the initial CL domain (CL 1 ), and may inhibit chemical gating by latching CL (Wang & Peracchia, 1998b). To further probe the chemical gating mechanism of Correspondence to: C. Peracchia J. Membrane Biol. 178, 55–70 (2000) DOI: 10.1007/s002320010015 The Journal of Membrane Biology © Springer-Verlag New York Inc. 2000