Transduction Channels of Rod and Cone Photoreceptors |
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Despite the experimental evidence, a rigorous test of the involvement of CNG channels in synaptic transmission is missing. Such a test will be technically challenging since it requires the selective ablation of synaptic CNG channels.
THE MOLECULAR COMPOSITION OF CNG CHANNELS
Scientists successfully purified a protein from bovine retina that functionally reconstituted CNG channel activity in artificial liposomes [8]. Partial amino acid sequences derived from the purified protein led to the discovery and functional in vitro expression of the first CNG channel gene in 1989 [9]. The CNG channel protein displays sequence similarity to voltage-gated K+, Na+, and Ca2+ channels, and it was categorized into the same gene superfamily [10]. It contains six transmembrane segments (S1–S6) and a pore-forming stretch of about 20 amino acids (“p region”) that is located between transmembrane segments 5 and 6. The C-terminal region harbors a binding domain for nucleoside 3′,5′-cyclic monophosphates (cNMPs). On heterologous expression, the CNG channel protein displays several of the key properties of the native rod CNG channel but differs in the gating kinetics and the sensitivity to L-cis-diltiazem, a substance that efficiently blocks native channels. The simplest explanation for these functional differences is that the native channel comprises several distinct subunits. In fact, speculation about additional subunits was already stimulated by the presence of a second, larger protein (240 kDa in sodium dodecyl sulfate [SDS]-polyacrylamide gels) in the purified channel preparation (e.g., [11]). Subsequent studies finally identified the 240-kDa protein as a second subunit of rod CNG channels. Coexpression of both channel proteins, later termed CNGA1 (the first subunit) and CNGB1 (the second subunit), recapitulated the properties of native rod CNG channels [12, 13]. CNGB1 exhibits a bipartite structure with a membraneintegrated part that is homologous to CNGA1 (“β′ part”) and a large cytoplasmic N-terminal domain homologous to glutamic acid-rich proteins (GARPs) expressed in rod photoreceptors. The gene encoding the A subunit of cone photoreceptors (CNGA3a) was identified using a cloning strategy that exploited the sequence similarity to the gene encoding CNGA1 [14]. The cone B subunit (CNGB3) was identified by two different approaches. Just before the release of the full sequence of the human genome in 2001, the missing gene was identified in a human expressed sequence tag (EST) database due to its similarity to other CNG channel subunits [15]. At the same time, a genomic region containing CNGB3 was found to be responsible for inherited color blindness among inhabitants of Pingelap, an isolated atoll in the west Pacific [16, 17]. Unlike the counterpart in rod photoreceptors, CNGB3 does not display a bipartite structure and comprises only the membrane-spanning core segment common to all CNG channel subunits.
While CNGA1 or CNGA3 form functional channels in heterologous expression systems if expressed alone, CNGB1 or CNGB3 fail to form functional homomeric channels.
a The human genome is comprised of six different CNG channel genes: CNGA1–4, CNGB1, and CNGB3. CNGA2, CNGA4, and CNGB1 encode for the CNG channel in olfactory receptor neurons. CNGB2 has not been assigned.
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Due to this observation, CNGA1 and CNGA3 are often referred to as principal subunits and CNGB1 and CNGB3 as modulatory subunits.
How CNGA and CNGB subunits assemble was first understood for the rod CNG channel. The rod CNG channel is a tetramer composed of three CNGA1 subunits and one CNGB1 subunit [18–20]. In contrast, CNG channels in cones adopt a more symmetrical arrangement with two copies of each subunit type, CNGA3 and CNGB3 [21]. It is likely that the four CNG channel subunits arrange such that the p region of each subunit is oriented toward the center of the complex, each contributing to a central membrane-spanning pore. Such an arrangement has been demonstrated for voltagegated K+ channels using x-ray crystallography and is consistent with low-resolution cryoelectron microscopic images of native rod CNG channels [22, 23].
THE BASIC ACTIVATION PROPERTIES OF CNG CHANNELS
The CNG channels of rods and cones respond to both cAMP and cGMP but display higher sensitivity to cGMP [13, 24–26] (Fig. 2A). Moreover, cAMP is only a partial agonist for photoreceptor CNG channels, with saturating cAMP concentrations
Fig. 2. Ligand selectivity of rod and cone cyclic nucleotide-gated (CNG) channels. A Heterologously expressed bovine rod (black line) and human cone (gray line) CNG channels are activated by micromolar cyclic 3′,5′-guanosine monophosphate (cGMP) concentrations. In contrast, even millimolar cyclic adenosine monophosphate (cAMP) concentrations activate only a fraction of the maximal cGMP-induced current through both rod and cone CNG channels (data were obtained at a membrane voltage of +80 mV [13, 26]). B Molecular modeling predicts ten interactions between the ligand-binding site of CNG channels and cGMP. C The ligand-binding site of CNG channel subunits is predicted to fold into three α-helices and eight β-strands. Shown is the amino acid sequence of the ligand-binding site in bovine CNGA1. Boxed residues are predicted to interact with cGMP.