Transduction heats in retinal rods: tests of the role of cGMP by pyroelectric calorimetry.

Hagins, W. A.; Ross, Philip D.; Tate, Ramon L.; Yoshikami, S.

doi:10.1073/pnas.86.4.1224

Cited by 21 publications

(10 citation statements)

References 30 publications

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“…We compute the PDE* scale of Fig. 1 using the turnover number of 500 and the -11.2 Kcal/mol enthalpy of cGMP hydrolysis (17). On this scale, the peak amplitude of the control trace in Fig.…”

Section: Resultsmentioning

confidence: 99%

Responses of the phototransduction cascade to dim light.

Langlois

Chen

Palczewski

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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The biochemistry of visual excitation is kinetically explored by measuring the activity of the cGMP phosphodiesterase (PDE) at light levels that activate only a few tens of rhodopsin molecules per rod. At 23°C and in the presence of ATP, the pulse of PDE activity lasts 4 s (full width at half maximum). Complementing the rod outer segments (ROS) with rhodopsin kinase (RK) and arrestin or its splice variant p44 does not significantly shorten the pulse. But when the ROS are washed, the duration of the signal doubles. Adding either arrestin or p44 back to washed ROS approximately restores the pulse width to its initial value, with p44 being 10 times more efficient than arrestin. This supports the idea that, in vivo, capping of phosphorylated R* is mostly done by p44. When myristoylated (14:0) recoverin is added to unwashed ROS, the pulse duration and amplitude increase by about 50% if the free calcium is 500 nM. This effect increases further if the calcium is raised to 1 ,uM. Whenever R* deactivation is changed-when RK is exogenously enriched or when ATP is omitted from the buffer-there is no impact on the rising slope of the PDE pulse but only on its amplitude and duration. We explain this effect as due to the unequal competition between transducin and RK for R*. The kinetic model issued from this idea fits the data well, and its prediction that enrichment with transducin should lengthen the PDE pulse is successfully validated. arrestin (7, 8). How would this difference manifest itself kinetically?A physiologically relevant measurement of PDE activity must meet two challenges. First, this activity can only be measured from suspensions of highly disrupted rod outer segment (ROS) fragments, where the loss of soluble and peripheral proteins is a major concern. During preparation of ROS, there is loss at several washing steps, especially of the highly soluble arrestin. During the experiment itself, if the ROS concentration is less than 25 ,uM rhodopsin, peripheral proteins such as transducin and PDE get spontaneously solubilized (9). Second, the pHmetric method is widely used to measure PDE activity (10), but it lacks the required sensitivity. A dark-adapted rod saturates electrically when only a few tens of its 108 rhodopsin molecules are photoactivated. At such light levels, the pH electrode detects nothing. We deal with potential losses of proteins by using a high concentration of membrane to prevent spurious solubilization of peripheral species and by supplementing the ROS with purified or recombinant proteins such as arrestin, p44, RK, and recoverin. As for the issue of sensitivity, the novel technique of time-resolved microcalorimetry (3) is used to follow the time course of PDE* at illumination levels comparable with those seen by a live, dark-adapted rod.

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Section: Resultsmentioning

confidence: 99%

Responses of the phototransduction cascade to dim light.

Langlois

Chen

Palczewski

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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“…The heat sensor is a poled poly-(vinylidene difluoride) film (Solvay & Cie, Brussels) on the back face of the thin (0.4 mm) 40 x 40 mm sample chamber. Heating causes dilation of the film; the displacement current generated is proportional to the rate of change oftemperature (19,20) and is measured with a sensitive amplifier. The impulse response is 140 ms (full width at half maximum) with a resistance-capacitance (RC) filter of 25 ms.…”

Section: Methodsmentioning

confidence: 99%

Deactivation kinetics of the transduction cascade of vision.

Vuong

Chabre

1991

Proc. Natl. Acad. Sci. U.S.A.

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The response of the retinal rod ceil to a dim flash lasts less than a second. This phototransduction is mediated by a guanine nucleotide-binding (G) protein cascade in which rhodopsin is the receptor, transducin is the G-protein, and the cGMP-specific phosphodiesterase (PDE) is the effector. Photoexcited rhodopsin activates transducin which in turn activates PDE. For this underlying biochemistry to be kinetically compatible with the photoresponse, both transducin and PDE must be deactivated in subsecond times. We report here direct measurements of their deactivation kinetics. The rate of heat release when transducin and PDE hydrolyze, respectively, GTP and cGMP was measured using time-resolved microcalorimetry. With only GTP present, the heat pulse comes from the activation of transducin and its subsequent deactivation by endogenous GTP hydrolysis. The nonhydrolyzable analog guanine 5'-[y-thioltriphosphate was used to disdngush between these two processes: about 40% of the total heat is due to activation. From the time course of the deactivation heat, the active lifetime of transducin is <1 s at 22C. With both GTP and cGMP present, the highly amplified hydrolytic activity of the PDE is responsible for most of the heat produced; its rate of release is directly proportional to the amount of activated PDE. Measurements of this rate at low photoexcitation levels (e.g., 30 molecules of photoexcited rhodopsin per rod) provide much kinetic information about the cascade. Notably, deactivation of the PDE takes 0.6 s (at 23YC) and absolutely requires GTP hydrolysis. This concurs with the subsecond lifetime of active transducin and means that, once GTP hydrolysis has occurred, the hitherto active PDE is quickly inhibited.

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“…In full dark adaptation sodium ions and water enter the outer limb at a maximal rate, and are pumped out in the inner limb 3. The entire cytosol volume is pumped in about 15 seconds 4. This process requires a great deal of energy and a large oxygen supply.…”

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confidence: 99%