MOLECULAR DETERMINANTS OF HIGH-AFFINITY PHENYLALKYLAMINE BLOCK OF L-TYPE CALCIUM CHANNELS IN TRANSMEMBRANE SEGMENT IIIS6 AND THE PORE REGION OF THE ALPHA(1) SUBUNIT

Recent studies of the phenylalkylamine binding site in the alpha(1C) s ubunit of L-type Ca2+ channels have revealed three amino acid residues in transmembrane segment IVS6 that are critical for high affinity blo ck and are unique to L-type channels, We have extended this analysis o f the phenylalkylamine binding site to amino acid residues in transmem brane segment IIIS6 and the pore region. Twenty-two consecutive amino acid residues in segment IIIS6 were mutated to alanine and the conserv ed Glu residues in the pore region of each homologous domain were muta ted to Gln. Mutant channels were expressed in tsA-201 cells along with the beta(1b) and alpha(2) delta auxiliary subunits, Assay for block o f Ba2+ current by (-)-D888 at -60 mV revealed that mutation of five am ino acid residues in segment IIIS6 and the pore region that are conser ved between L-type and non-L-type channels (Tyr(1152), Phe(1164), Val( 1165) Glu(1118) and Glu(1419)) and one L-type-specific amino acid (Ile (1153)) decreased affinity for (-)-D888 from 10-20 fold. Combination o f the four mutations in segment IIIS6 increased the IC50 for block by (-)-D888 to approximately 9 mu M, similar to the affinity of non-L-typ e Ca2+ channels for this drug. These results indicate that there are i mportant determinants of phenylalkylamine binding in both the S6 segme nts and the pore regions of domains III and IV, same of which are cons erved across the different classes of voltage-gated Ca2+ channels. A m odel of the phenylalkylamine receptor site at the interface between do mains III and IV of the alpha(1) subunit is presented.