Internal membrane systems

The next question to arise is, how does excitation at the cell surface cause release of calcium ions inside the fibre? The first step in the solution of this problem was the demonstration by A. F. Huxley and R. E. Taylor that there is a specific inward-conducting mechanism located at the Z line in frog sartorius muscles. (We shall examine the striation pattern in detail later. But here it is worth noting that the markedly birefringent A bands alternate with the less birefringent I bands, and that a thin dense line, the Z line, bisects the I bands. Fig. 10.7 shows the whole pattern.)

The muscle fibres were viewed by polarized light microscopy so as to make the striation pattern visible. They were stimulated by passing depolarizing

Fig. 10.3. The effect of local depolarizations on a frog muscle fibre. When the electrode is opposite certain active spots in the I band, as shown here, contraction ensues. Based on Huxley and Taylor (1958).

current through an external microelectrode applied to the fibre surface. The stimulus was effective only when the electrode was positioned at certain 'active spots' located on the fibre surface in rows opposite the Z lines. In these cases the A bands adjacent to the I band opposite the electrode were drawn together, as is shown in Fig. 10.3.

At first it was thought that the inward-conducting mechanism was the Z line itself, but on repeating the experiments with crab muscle fibres it was found that the 'active spots' were localized not at the Z line but near the boundary between the A and I bands. This suggests that there is some transverse structure located at the Z lines in frog muscles and at the A—I boundary in crab muscles.

Such a structure was found in the electron microscopic examination of the sarcoplasmic reticulum in various skeletal muscles. This consists of a network of vesicular elements surrounding the myofibrils (Fig. 10.4). At the Z lines in frog muscles, and at the A—I boundaries in most other striated muscles (including crab muscles), are structures known as 'triads', in which a central tubular element is situated between two vesicular elements. These central elements of the triads are in fact tubules which run transversely across the fibre and are known as the transverse tubular system or T system. There is no continuity between the insides of the tubules of the T system and the vesicles of the sarcoplasmic reticulum, although their membranes are in close contact. The T system tubules are invaginations of the cell surface membrane. They open to the exterior at a limited number of sites corresponding to Huxley and Taylor's 'active spots'.

The sarcoplasmic reticulum consists of a series of membrane-bound sacs between the myofibrils. Vesicles formed from these sacs can be isolated from homogenised muscle by differential centrifugation. Their most interesting property is that they will accumulate calcium ions against a concentration gradient, by means of a 'calcium pump' which requires energy from the splitting

Fig. 10.4. The internal membrane systems of a frog sartorius muscle fibre. From Peachey (1965).

of ATP for its activity. The pump itself is a calicum—magnesium-activated ATPase, molecular weight about 110000, which is firmly bound in the sar-coplasmic reticulum membrane. The pump serves to maintain the calcium ion concentration in the sarcoplasm of the living muscle at its low resting level, and to produce relaxation of the muscle after a contraction.

How is calcium released from the sarcoplasmic reticulum on excitation? High-quality electron micrographs by Clara Franzini-Armstrong and her colleagues show that at the triads the T tubule and its adjacent sarcoplasmic reticulum sac are connected by an array of structures called 'feet'. These are anchored in the sarcoplasmic reticulum membrane and consist of four sub-units. The T tubule membrane contains particles grouped in fours ('tetrads') that are positioned opposite the feet.

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Ryanodine receptor

Fig. 10.5. Schematic diagram to show the membrane topology of the ryanodine receptor in skeletal muscle. The receptor is a calcium-release channel anchored in the sarcoplasmic reticulum (SR) membrane. Only one of its four identical subunits is shown. Each subunit is closely associated with a voltage-gated calcium channel (the dihydropyridine receptor) in the T-tubule membrane, which probably acts primarily as a voltage sensor. From Takeshima etal. (1989), reprinted with permission from Nature (vol. 339), copyright 1989 Macmillan Magazines Limited.

Molecular cloning shows that these structures are two sets of ion channels. The tetrads are groups of four voltage-gated calcium channels, also known as DHP receptors, since they will bind dihydropyridine. The feet are ryanodine receptors, calcium-release channels that open to allow calcium ions to flow out of the sarcoplasmic reticulum into the sarcoplasm surrounding the myofibrils. Each ryanodine receptor contains four subunits, large protein chains with over 5000 amino acid residues, so the whole complex has a molecular weight of over 2 million. All the DHP receptor tetrads are closely applied to ryanodine receptors (Fig. 10.5), but about half of the ryanodine receptors have no associated DHP receptors.

The likely sequence of events in the coupling process is as follows (Fig. 10.6). Depolarization of the cell surface membrane spreads down the T tubule. This activates channels of the ryanodine receptors. We may assume that the S4 segments of the DHP receptors are the sensors for the voltage change, but just

Action potential —>- Cel I su rface membrane

Action potential —>- Cel I su rface membrane

Fig. 10.6. Schematic diagram of the coupling process in skeletal muscle. The depolarization from the cell surface membrane spreads down the T-tubule. The DHP (dihydropyridine) receptors respond to this by opening the calcium-release channels (ryanodine receptors) in the sarcoplasmic reticulum membrane. Calcium ions then flow down their concentration gradient from the sarcoplasmic reticulum into the sarcoplasm, where they activate the contractile apparatus. On relaxation the calcium ions are pumped back into the sarcoplasmic reticulum by the ATP-driven calcium pump.

Fig. 10.6. Schematic diagram of the coupling process in skeletal muscle. The depolarization from the cell surface membrane spreads down the T-tubule. The DHP (dihydropyridine) receptors respond to this by opening the calcium-release channels (ryanodine receptors) in the sarcoplasmic reticulum membrane. Calcium ions then flow down their concentration gradient from the sarcoplasmic reticulum into the sarcoplasm, where they activate the contractile apparatus. On relaxation the calcium ions are pumped back into the sarcoplasmic reticulum by the ATP-driven calcium pump.

how the two channels are coupled is yet to be determined. Calcium ions flow out of the sarcoplasmic reticulum via the ryanodine receptor channels. The released calcium then activates the contractile mechanism and the muscle contracts. On repolarization the DHP receptors return to their resting conformation and this closes the ryanodine receptor channels. The calcium pump pumps calcium back into the sarcoplasmic reticulum so the muscle relaxes.

How does the calcium activate the contractile mechanism? Before answering this question we must first see how the contractile mechanism itself works.

Fig. 10.7. The striation pattern of a vertebrate skeletal muscle fibre as seen by electron microscopy of thin sections (a), and its interpretation as two sets of interdigitating filaments (b). Photograph for (a) supplied by Dr H. E. Huxley.
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