Critical roles of the S3 segment and S3-S4 linker of repeat I in activation of L-type calcium channels.

Abstract

Each ofthe four repeats (or motifs) ofvoltagegated Ion ch ls I thought to contaIn six tran1 membrane segments (S1-S6 (5,6) myotubes were prepared from newborn mice essentially following described procedures (5,7,8). Briefly, myoblasts isolated from fore-and hindlimbs were plated at a density of 2 x 104 per 35-mm Falcon Primaria dish, on which a grid of -2-mm squares was ruled. After fusion of myoblasts into myotubes had begun, some cultures were treated for 24 hr with 10 pM cytosine arabinoside. The cultures were used within 6-9 days after plating cells.Consuction of DHPR cDNAs. To construct pCAC7, the nucleotides AAG (residues 493-495) of pCAC6 (8), the expression plasmid of the rabbit skeletal muscle DHPR used in our previous studies (2,(8)(9)(10)(11), were replaced with the nucleotides CGC by site-directed mutagenesis (12). The compositions of the individual chimeric DHPRs are given below [Sk and Ca, skeletal muscle (13) and cardiac (14) DHPR, respectively; numbers in parentheses are amino acid numbers; for a region of identical sequence that flanks the joining site between the cardiac and skeletal muscle DHPRs, amino acid numbers are given as if that entire region were of cardiac sequence]: SkC15 (2) Sk

Chimeric L‐type Ca2+ channels expressed in Xenopus laevis oocytes reveal role of repeats III and IV in activation gating.

“…2A), two time constants were required for the more complex activation kinetics of chimera sHHSSs which did not reach peak current during the 350 ms pulse (Fig. 2B) in repeat III and/or IV, distinct from a previously identified sequence in repeat I (Tanabe et al 1991;Nakai et al 1994), which determine current activation. Fig.…”

“…Motifs determining current activation have been identified so far in repeat I within the transmembrane segment S3 and the extracellular S3-S4 linker (Tanabe et al 1991;Nakai et at. 1994).…”

“…The biexponential nature of the current activation observed in the present study in slowly activating chimeras clearly distinguishes the regulation of current activation by elements of repeat III and IV from previous results obtained from dysgenic mouse myotubes. In dysgenic mouse myotubes chimeras with skeletal muscle properties activate with monoexponential time course, in other words a single time constant is sufficient to describe current activation kinetics (Tanabe et al 1991;Nakai et at. 1994).…”

Swapping the I-II intracellular linker between L-type CaV1.2 and R-type CaV2.3 high-voltage gated calcium channels exchanges activation attributes

“…[35][36][37][38] Here we swapped the I-II linker between the more distantly related channels Ca V 1.2 and Ca V 2.3 and studied the resulting chimeras in absence and presence of Ca V β. A similar approach helped identify molecular determinants that contribute to the differences in inactivation between these two isoforms.…”

What Does Maurocalcine Tell Us About the Process of Excitation-Contraction Coupling?

“…In these subunits, positively charged S4 segments act as voltage-sensing mechanical transducers by altering their position within the plasma membrane as a function of voltage value (Nakai, Adams, Imoto, & Beam, 1994). How movements of other structural elements are related to these S4 repositioning is still an open but particularly important question with regard to the excitation-contraction coupling process as we will see later.…”