Single-channel study of the cGMP-dependent conductance of retinal rods from incorporation of native vesicles into planar lipid bilayers

Ildefonse, M; Bennett, Nelly

doi:10.1007/bf01998084

Cited by 62 publications

(65 citation statements)

References 39 publications

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“…Membrane-bound proteins have also been shown to incorporate into lipid bilayers along with ion channels in several studies (Ildefonse & Bennett, 1991 shown (Ausiello, Stow, Cantiello, de-Almeida & Benos, 1992) to form a 700 kDa protein complex that produced six major protein bands on reducing sodium dodecyl sulphate (SDS)-polyacrylamide gels. Consequently, ion channels may form complexes with regulatory membrane-bound proteins, suggesting modular organization within the cell membrane.…”

Section: Selectivity and Permeabilitymentioning

confidence: 99%

Characterization and regulation of a chloride channel from bovine tracheal epithelium.

Duszyk

Liu

Kamosinska

et al. 1995

The Journal of Physiology

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1. The patch-clamp technique was used to characterize chloride channels from the apical membranes of bovine tracheal epithelial cells. Application of GTPyS or NaF to excised patches revealed the existence of a novel type of Cl-channel regulated by G-proteins in a membrane-delimited manner. 2. The channel had a linear current-voltage relationship, with a conductance of 100-120 pS.Its open probability was independent of voltage.3. The channel was highly anion selective (permeability ratio, PNa/Pcl = 0-06 + 004) and had the halide permeability sequence: 1> Br-> Cl-> F-, corresponding to the Eisenman I sequence. This suggested that neither ionic size nor diffusion rate determined ion permeation through the channel. 4. The mole fraction behaviour was studied using fluoride and chloride ions. Mixtures of ions produced currents that would be expected from the linear combination of the two ions acting independently, indicating relatively simple permeation through the pore and compatible with a single ion binding site. 5. The channel was inhibited by the stilbene disulphonates SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) and DNDS (4,4'-dinitrostilbene-2,2'-sulphonic acid). SITS introduced voltage dependence to channel gating and indicated the possible involvement of lysine residues in the channel permeation pathway. 6. NaF was unable to activate Cl-channels in the presence of the aluminum chelator, deferoxamine mesylate. This indicates that A13+ ions play an important role in chloride channel activation by fluoride. NaF activation was not dependent on the presence of calcium ions. 7. The channel was insensitive to alkaline phosphatase and to the specific inhibitors of protein phosphatase types I and 2A, okadaic acid and calyculin A. 8. The channels could be activated by GTPyS or by NaF in the presence of the phospholipase A2 inhibitor quinacrine, indicating that this enzyme is not involved in channel regulation.

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Section: Selectivity and Permeabilitymentioning

confidence: 99%

Characterization and regulation of a chloride channel from bovine tracheal epithelium.

Duszyk

Liu

Kamosinska

et al. 1995

The Journal of Physiology

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“…Ca 2+, Mg 2+, or the polycation spermine, from nonblocking to blocking concentrations of all three (1-200 p.M), did not potentiate. Ildefonse and Bennett (1991) have recently reported that Co s+, Ni 2+, and Fe z+ enhance the bovine rod channel response to low cGMP in planar lipid bilayers.…”

Section: To Subsaturating Cgmp By Low Concentrations Of Ni 2+mentioning

confidence: 99%

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods.

Karpen

Brown

Stryer

et al. 1993

The Journal of General Physiology

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The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMPactivated Na + currents by internal and external Mg 2+ as well as internal Ca ~+ was nearly independent ofcGMP concentration. This indicates that Mg 2÷ and Ca ~÷ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd 2÷ or Zn 2÷ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni 2+, which competes with Mg z÷ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni z÷, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extraceUular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni 2÷, Cd 2+, Zn ~+, and Mn 2÷ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl~ reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.Address reprint requests to Dr.

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“…The rod channel is cation selective but does not appreciably discriminate between monovalent alkali cations (1,4,(12)(13)(14)(15)(16)(17). Divalent cations also permeate the rod channel and thereby block the Na+ current in a voltage-dependent manner (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Ca2+ entry is of great physiologic importance because Ca2+ is part of a negative feedback mechanism that regulates the recovery of the light response and light adaptation (for review, see refs.…”

mentioning

confidence: 99%

A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca2+ blockage, and ionic selectivity.

Eismann

Müller

Heinemann

et al. 1994

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

183

123

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Ca2+ ions control the cGMP-gated channel of rod photoreceptor cells from the external and internal face. We studied ion selectivity and blockage by Ca2+ of wild-type and mutant channels in a heterologous expression system. ExternalCa2+ blocks the inward current at micromolar concentrations in a highly voltage-dependent manner. The blockage at negative membrane voltages shows a steep concentration dependence with a Hill coefficient of -2. The blockage from the internal face requires 1000-fold higher Ca2+ concentrations. Neutralization of a glutamate residue (E363) in the putative pore region between transmembrane segments H4 and H5 induces outward rectification and changes relative ion conductances but leaves relative ion permeabilities nearly unaffected. The current blockage at -80 mV requires =2000-fold higher external Ca2+ concentrations and the voltage dependence is almost abolished. These results demonstrate that E363 represents a binding site for monovalent and divalent cations and resides in the pore lumen.The cyclic nucleotide-gated channel of vertebrate rod photoreceptor cells (rod channel) belongs to a class of ligandgated channels that are directly and cooperatively opened by the binding of cyclic nucleotides (cGMP or cAMP) (1-11). The rod channel is cation selective but does not appreciably discriminate between monovalent alkali cations (1,4,(12)(13)(14)(15)(16)(17). Divalent cations also permeate the rod channel and thereby block the Na+ current in a voltage-dependent manner (15-29). Ca2+ entry is of great physiologic importance because Ca2+ is part of a negative feedback mechanism that regulates the recovery of the light response and light adaptation (for review, see refs. 30 and 31). Blockage by both external and internal Ca2+ is voltage dependent, indicating a binding site(s) for Ca2+ within the channel pore (26, 28). Internal Ca2+ decreases both the open probability and the single-channel conductance (32). External Ca2+ appears to reduce singlechannel conductance from 25 to =0.1 pS (33,34).Comparison with the protein sequences of K+ channels has led to the prediction that the pore region of the rod channel is located between transmembrane segments H4 and H5 (refs. 7, 8, and 35-37; for reviews, see refs. 38-41). The pore region of K+ channels contains two adjacent amino acid residues (Y445, G446; Fig. 1) that are missing in the respective region of the rod channel. By deletion of these residues, the Shaker K+ channel is converted into a nonselective cation channel (36). The sensitivity to blockage by divalent cations that is increased in the deletion mutant is controlled by the adjacent acidic residue D447 (36). This residue corresponds to glutamate-363 (E363) in the pore region ofthe rod channel. We demonstrate by heterologous expression of wild-type (wt) and mutant rod channels that E363 is crucial for binding of both monovalent and divalent cations and resides inside the pore lumen. MATERIALS AND METHODSSite-Directed Mutagenesis and in Vitro Transcription. The point mutations K346Q, E36...

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Single-channel study of the cGMP-dependent conductance of retinal rods from incorporation of native vesicles into planar lipid bilayers

Cited by 62 publications

References 39 publications

Characterization and regulation of a chloride channel from bovine tracheal epithelium.

Characterization and regulation of a chloride channel from bovine tracheal epithelium.

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods.

A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca2+ blockage, and ionic selectivity.

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