THE S5-S6 LINKER OF REPEAT-I IS A CRITICAL DETERMINANT OF L-TYPE CA2+CHANNEL CONDUCTANCE

The alpha(1)-subunits of the skeletal and cardiac L-type calcium chann els (L-channels) contain nearly identical pore regions (P-regions) in each of the four internal homology repeats. In spite of this high cons ervation of the P-regions, native skeletal L-channels exhibit a unitar y conductance that is only about half that of native cardiac L-channel s. To identify structural determinants of this difference in L-channel conductance, we have characterized unitary activity in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, and chimeri c L-channel alpha(1)-subunits. Our results demonstrate that the S5-S6 linker of repeat I (IS5-IS6 linker) is a critical determinant of the d ifference in skeletal and cardiac unitary conductance. The unitary con ductances attributable to the wild-type skeletal (CAC6; similar to 14 pS) and cardiac (CARD1; similar to 25 pS) alpha(1)-subunits expressed in dysgenic myotubes are identical to those observed in native tissues . Chimeric alpha(1)-subunits containing skeletal sequence for the firs t internal repeat and all of the putative intracellular loops (SkC15), the IS5-IS6 linker and the intracellular loops (SkC51), or only the I S5-IS6 linker (SkC49) each exhibit a low, skeletal-like unitary conduc tance (less than or equal to 17 pS). Constructs in which the IS5-IS6 l inker is of cardiac origin (CARD1 and CSk9) display cardiac-like condu ctance (similar to 25 pS). Unitary conductance and the rate of channel activation are apparently independent processes, since both SkC51 and SkC49 exhibit low, skeletal-like conductance and rapid, cardiac-like rates of ensemble activation. These results demonstrate that the IS5-I S6 linker strongly influences the single channel conductance of L-chan nels in a manner that is independent from the rate of channel activati on.