Affinity purification of the photoreceptor cGMP-gated cation channel

Hurwitz, Richard L.; Holcombe, Vien N.

doi:10.1016/s0021-9258(18)92925-x

Cited by 51 publications

(3 citation statements)

References 18 publications

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“…While several investigators have used diltiazem as a tool to characterize the cGMPgated channel of rod photoreceptors (Koch and Kaupp, 1985;Stern, Kaupp, and MacLeish, 1986;Koch et al, 1987;Nicol, Schnetkamp, Saimi, Cragoe, and Bownds, 1987;Kaupp et al, 1989;Caretta et al, 1991 ;Hurwitz and Holcombe, 1991 ;Quandt et al, 1991), the mechanism by which/-c/s-diltiazem blocks the cGMP-gated channels of rods and cones remained unknown. The experiments presented here suggest that the rod and cone cGMP-gated channels each possess a single class of binding site for diltiazem, located on the cytoplasmic surface of the channel, and that the occupation of a single site is sufficient to block the channel.…”

Section: Discussionmentioning

confidence: 99%

Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

Haynes

1992

The Journal of General Physiology

148

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Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of/-c/s-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 ~.M diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra-and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.

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Section: Discussionmentioning

confidence: 99%

Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

Haynes

1992

The Journal of General Physiology

148

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“…It seems possible, therefore, that the channel protein undergoes posttranslational or cotranslational modification in the native cell but not in the expression system, an interpretation that could account for differences between native and reconstituted cloned cng channels. Posttranslational modification could involve proteolysis, as suggested for cng channels from photoreceptors (Hurwitz and Holcombe, 1991;Molday, Molday, Dos&, Clark-Lewis, IUing, Cook, Eismann, and Kaupp, 1991), and it may also influence the affinity of the channel for cyclic nucleotides.…”

Section: Activation By Cyclic Nucleotidesmentioning

confidence: 99%

“…This selectivity, together with the strong voltage sensitivity observed with all blockers, may indicate that inhibition by diltiazem is not "nonspecific" but that it involves interaction of the blocker with a binding site in the channel. Apparently, binding of diltiazem to the channel protein involves a distinct domain, different from the cGMP-binding domain, because after proteolytic treatment of cng channels purified from photoreceptors, sensitivity to l-cis-diltiazem was lost while activation by cGMP was sustained (Hurwitz and Holcombe, 1991). Interestingly, blockage of Ca channels by diltiazem has the inverse stereoselectivity, with d-cis-being eight times more potent than 1-cis-diltiazem (Reynolds and Snyder, 1988).…”

Section: Inhibition By Organic Blockersmentioning

confidence: 99%

Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.

Frings

Lynch

Lindemann

1992

The Journal of General Physiology

185

142

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Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 ~M for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca 2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.

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Gating of retinal rod cation channel by different nucleotides: Comparative study of unitary currents

1992

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Single channels are observed after incorporation of native vesicles from bovine rod outer segment membranes into planar lipid bilayers. The activity of a single channel in the presence of cGMP is compared to that induced by the analog 8-bromo-cGMP and by cAMP. At +80 mV, K0.5 is about 3 microM for 8Br-cGMP, 18 microM for cGMP and 740 microM for cAMP. In cAMP, the amplitude of the current is smaller than in cGMP or 8Br-cGMP and depends on the filter cut-off frequency. The open/closed transition rates of the channel are slightly slower with 8Br-cGMP than with cGMP while they are 5 to 10 times faster with cAMP. Addition of Ni2+ ions to either cGMP or cAMP increases the open probability: the open/closed transition rates and amplitude of the current in cAMP are then comparable to those in cGMP. A dual effect of the addition of cAMP on the cGMP- or 8Br-cGMP-dependent activity previously reported (Furman, R.E., Tanaka, J.C. 1989. Biochemistry 28:2785-2788) is observed with a single channel: addition of subthreshold cAMP concentrations to cGMP (or to 8Br-cGMP) markedly increases Po; addition of cAMP concentrations higher than about 70 microM progressively accelerates the kinetics and reduces the amplitude to values observed in cAMP alone. The results are discussed in relation with the model previously proposed to account for the existence of four current levels (Ildefonse, M., Bennett, N. 1991. J. Membrane Biol. 123:133-147).

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Affinity purification of the photoreceptor cGMP-gated cation channel

Cited by 51 publications

References 18 publications

Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.

Gating of retinal rod cation channel by different nucleotides: Comparative study of unitary currents

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