An electrophysiological and spectroscopic study of the properties and structure of biological calcium channels. Investigations of a model ion channel.
The N- and C-terminally protected peptide N-acetyl-Asp-Phe-Ala-Asn-Arg-Val-Leu-Leu-Ser-Leu-Phe-Thr-Ile-Glu-Met-Leu -Leu-Lys-Met-Leu-NH2, closely based on the sequence of the putative S2 membrane spanning helix of domain II of the dihydropyridine receptor calcium channel of the T-system of skeletal muscle, residues 465-486 (Tanabe et al. (1987) Nature 328, 313-318) has been synthesised. Conductance measurements in planar lipid bilayers show that the peptide is capable of inducing the transmembrane passage of calcium and barium ions, in preference to monovalent cations. No anion conductance is observed. 1H-NMR spectroscopy demonstrates that in an amphilic solvent, methanol, the peptide forms highly stable structures characterised by very slow exchange with solvent of peptide N-H protons. Double-quantum filtered phase-sensitive COSY shows that, on the basis of NH-CH alpha scalar coupling constants, most peptide torsion angles are appropriate to an overall alpha-helical conformation; the presence of some alpha-helix is also supported by CD measurements. Most side-chain connectivities have been identified in a DIPSI-TOCSY experiment. This evidence has been used to construct a low-resolution model of the ion-conducting channel of the muscle T-system dihydropyridine receptor from the sequences of the four homologous putative channel-lining stretches. It is characterised by an association of acidic residues at the putative extra-membranous face of the channel, followed by a predominantly hydrophobic band. The next prominent feature of the model is an ordered array of four acidic residues (glutamates 100, 478, 846 and 1164), followed by four lysines (104, 482, 850 and 1168) which may play a gating role.
Reid
D G
DG
Analytical Sciences Department, SmithKline Beecham Pharmaceuticals Ltd., Welwyn, UK.
MacLachlan
L K
LK
Salter
C J
CJ
Saunders
M J
MJ
Jane
S D
SD
Lee
A G
AG
Tremeer
E J
EJ
Salisbury
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000502:physiology*; D004594:Electrophysiology / Q000379:methods; D008051:Lipid Bilayers; D009682:Magnetic Resonance Spectroscopy; D008564:Membrane Potentials; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000502:physiology*
D015220:Calcium Channels; D008051:Lipid Bilayers; D010455:Peptides; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D011978:Receptors, Nicotinic