Mechanism and function of the Ca2+-gated cation channel in sarcoplasmic reticulum vesicles.

Abstract

The mechanism and function of the Ca 2$ -gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring both choline influx and Ca 2$ efflux. The choline influx was measured by the light scattering method using a stopped flow apparatus. The Ca 2$ efflux was measured by the Millipore filtration method using 46 Ca. Kinetic analysis of the rates of choline influx and Ca 2$ efflux led to the following results. 1. The Ca 2$ -activated choline influx was dependent on the choline concentration. It increased with increase of choline concentration in a cooperative manner ( nH = 2.6) and was saturable at high choline concentrations. The apparent dissociation constant for choline was 0.39 M. 2. Caffeine increased the Ca 2$ -activated choline influx according to the Michaelis-Menten equation at low concentrations of choline, whereas the action of caffeine became cooperative ( nH = 1.9) at higher concentrations of choline, suggesting that there is a cooperative interaction between channels. 3. The Ca 2$ -activated choline influx was inhibited competitively by Ca 2$ . One Ca 2$ competes with 2 cholines. The inhibition constant for Ca 2$ was 22 μ m and the dissociation constant of choline was 0.28 m , suggesting that the competition site is the transport site of choline. 4. Various polyvalent cations also blocked the channel. The apparent inhibition constants for the inhibitors were as follows: La 2$ 0.01 μ m , Co 2$ 7μ m , Mn 2$ 8 μ m , Mg 2$ 14 μ m , Ni 2$ 19 μ m , Ca 2$ 22 μ m , Ba 2 30 μ m , and Sr 2$ 40 μ m . 5. The inhibition of Ca 2$ -activated choline influx by Mg 2$ or Co 2$ was competitive with respect to choline. One Mg 2$ or Ca 2$ competes with 2 cholines. The apparent dissociation constant of choline was 0.31 m , suggesting that the competition site is also the transport site of choline. 6. An organic Ca 2$ antagonist, nicardipine, blocked the Ca 2$ -gated cation channel with a dissociation constant of 5.4 μ m . Ruthenium red and arsenazo III also blocked the channel with apparent dissociation constants of 0.07 μ m and 16 μ m , respectively. 7. The channel was also blocked by a fluorescent cyanine dye, diS-C 2 -(5), and by quinine, which are potent inhibitors of the Ca 2$ -activated K $ channel in red blood cell membrane; the apparent inhibition constants were 4.4μm and 240 μ m , respectively. However, blockers of the K $ channel in nerve membrane, such as TEA and 3,4-diaminopyridine, were not effective. 8. When SR vesicles were modified with N , N ′-dicyclohexylcarbodiimide (DCCD), both Ca 2$ -activated choline influx and Ca 2$ -activated Ca 2$ release from SR vesicles were blocked. The apparent inhibition constant of DCCD was 80 μ m . The blockade was prevented by Ca 2$ . The Ca 2$ concentration required for half-maximal protection was about 1 m m . These results suggest that two cholines are simultaneously transported by one channel and there exists a positive homotropic cooperativity between some channels. In addition, from the pharmacological data, we concluded that the so-called Ca 2$ -gated cation channel is the Ca 2$ channel existing in sarcoplasmic reticulum.