Role of the pore region in voltage dependent calcium channels for drug interaction and channel kinetics / eingereicht von Richard L. Kraus

• VerfasserIn: Kraus, Richard L.
• Place / Publishing House: 1998
• Year of Publication: 1998
• Language: English
• Physical Description: III, 63 Bl.; Ill., graph. Darst.

eng: I could provide evidence that the pore region of voltage gated calcium channels emerge as structures not only important for cation permeation through the channel but also plays an important role for (1) pharmacology and (2) pathophysiology. (1) Synthesizing a novel photoreactive diltiazem- derivative, ["3H]benziazem, I could identify the BTZ binding domain within the #alpha#1 subunit of partially purified L-type calcium channels by antibody mapping and demonstrate that the binding sites for all three major chemical classes of calcium antagonists (dihydropyridines DHPs; phenylalkylamines, PAAs and BTZs) are located in close proximity within the pore-forming region of the #alpha#1 subunit. I further investigated single amino acid residues involved in high affinity BTZ binding in the transmembrane segment IIIS6 of the channel pore. Using alanine scanning mutagenesis 19 residues of segment IIIS6 were mutated to alanine. Mutant #alpha#1 subunits were coexpressed, in X. laevis oocytes and drug sensitivity to 100 #mu#M (+)-cis diltiazem, was assayed using the two- electrode voltage-clamp technique. Some amino acids are involved in BTZ as well as PAA- binding suggesting overlapping binding sites. Results of drug binding studies to partially purified calcium channels could not consistently be interpreted according to a former postulated allosteric model. Therefore we suggest allosteric and additional steric interactions to play an important role between channel bound drug molecules. (2) Mutations inducing amino acid substitutions within the calcium channel pore lead to neuronal disorders. We have introduced four single familial hemiplegic migraine (FBM) mutations reported for the human #alpha#1A subunit into the conserved rabbit isoform and investigated possible changes in channel function. Mutant and wild type rabbit #alpha#1A were coexpressed in X. laevis oocytes and calcium channel currents were analyzed. We could show that mutations associated with FHM affect kinetics of neuronal N-type calcium channel involved in neurotransmitter release.