Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels are activated in response to the direct binding of cyclic nucleotides to an intracellular domain. This domain is thought to contain a beta roll and two alpha helices, designated the B and C helices. To probe the conformational changes occurring in the ligand-binding domain during channel activation, we used the substituted cysteine accessibility method (SCAM). We found that a residue in the beta roll, C505, is more accessible in unliganded channels than in liganded channels, whereas a residue in the C helix, G597C, is more accessible in closed channels than in open channels. These results support a molecular mechanism for channel activation in which the ligand initially binds to the beta roll, followed by an opening allosteric transition involving the relative movement of the C helix toward the beta roll.

Amino-acid sequence of squid myosin heavy chain.

This work describes the determination of the cDNA sequence encoding the myosin heavy chain (MHC) of the squid, Loligo pealei. To date, the amino-acid sequence of the MHC of calcium-regulated myosins is known only for two closely related species of scallops. We have determined the sequence of the entire coding region of the muscle MHC of squid, a cephalopod, and compared it with the MHC of scallops, which are pelecypods, and to other regulated and non-regulated myosins. Residues present in the MHC of only regulated myosins have been identified. The 6504 base pair (bp) sequence contains an open reading frame of 5805 nucleotides, which encodes 1935 amino acids. The sequence includes 697 bps of 3' untranslated sequence and 2 bps of 5' untranslated sequence. The deduced amino-acid sequence shows the squid MHC to be 72-73% identical and 86-87% similar to the calcium-regulated scallop MHCs cloned previously. In contrast, the squid MHC sequence is only 54-55% identical and 74% similar to skeletal MHCs of non-regulated myosins such as human fast skeletal embryonic and human perinatal skeletal muscle, and 39-40% identical and 60-62% similar to smooth muscle MHC of rabbit uterus muscle and chicken gizzard muscle, respectively. We have also detected two isoforms of the MHC in squid that appear to be spliced variants of a single myosin gene. These isoforms differ in the sequence encoding the surface loop at the nucleotide binding site. Taken together, our data may help to identify more precisely the residues that are crucial in regulated myosins.