Colocalization of the Dihydropyridine Receptor, the Plasma‐Membrane Calcium ATPase Isoform 1 and the Sodium/Calcium Exchanger to the Junctional‐Membrane Domain of Transverse Tubules of Rabbit Skeletal Muscle

Abstract

The subcellular distribution of the calmodulin-stimulated plasma-membrane Ca*+-ATPase (PMCA) has been studied in rat and rabbit skeletal muscle cells by indirect (calmodulin gel overlays) and direct (Western blotting with specific antibodies) methods. It has also been studied in situ in immunocytochemistry experiments. The distribution of PMCA has been compared with that of the Na+/Ca*+ exchanger and of the dihydropyridine receptor, which has been studied by Western blotting with specific antibodies. Both PMCA and the Na+/Ca'+ exchanger had a dual localization, i.e., they were found in the plasma membrane and in the transverse-tubule fractions of the two main types of skeletal muscles studied. The pump and the exchanger were not diffusely distributed in the transverse-tubule-membrane system, but specifically confined to the membrane domain where the dihydropyridine receptor was also localized, i.e., the junctional membrane. Experiments with isoform-specific antibodies have shown that the pump isoform expressed in skeletal muscle is PMCA 1.

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

“…This reduction in the elevation of [Ca 2ϩ ] r supports the idea that NCX activity in MHN and MHS muscle fiber appears to be predominantly across the t-tubule rather than the surface sarcolemma. These results agree with previous studies showing that localization of NCX is higher in the t-tubular system than in the sarcolemma in skeletal and cardiac muscles (6,55 ] r must be considered with caution due to the lack of selectivity of KB-R7943, because there are evidences that show that it also inhibits voltage-gated Na ϩ and Ca 2ϩ channels, the inward rectifying K ϩ channels in cardiac cells (56), the transient receptor potential channels (TRPCs) in HEK293 cells (57), and RyR1 in skeletal muscle and HEK293 cells (58).…”

“…Three mammalian isoforms of the NCX protein have been discovered (NCX1, NCX2, and NCX3) (3)(4)(5). Only the NCX1 and NCX3 isoforms are expressed in skeletal muscle and are localized in the transverse tubules (t-tubules) and in the sarcolemma (4,6,7). NCX1 is expressed at high levels during embryonic development and postnatal maturation (8) then its expression is gradually reduced as the NCX3 gene becomes more highly expressed and predominates in adult skeletal muscle (8).…”

Phosphorylation of Anchoring Protein by Calmodulin Protein Kinase Associated to the Sarcoplasmic Reticulum of Rabbit Fast-Twitch Muscle

“…Blots either were overlayed with biotinylated CaM (A, D), or were immunostained with antibody to ␦ (B, E) or to ␣ CaM K II, respectively (C, F). In A and D, the additional CaM-binding proteins of molecular mass ranging from 130 to140 kDa, and found to be prominent with rat fast-twitch muscle, are identifiable with plasma membrane Ca 2ϩ -ATPase protein bands, based on previous work [33], and are explained with the presence of a varying amount of contaminant TT in the particular membrane fraction.…”

Fiber Types in Mammalian Skeletal Muscles

“…Three mammalian isoforms of the NCX protein, products of three different genes, have been cloned and appear to have very similar properties: NCX1, the predominant isoform in cardiac muscle, is also expressed in skeletal muscles, where it coexists with the other two isoforms NCX2 and NCX3. NCX is mainly localized at the t tubule and contributes to calcium extrusion after contraction (151,679). Actually, due to its lower affinity (K m ϳ3 M) and high capacity, NCX cannot have a significant impact on resting cytosolic calcium concentration (ϳ50 nM), but can contribute to export calcium during caffeine or tert-nutyl-hydroquinone (TBQ)-induced contracture (43).…”