Mechanism of calcium/calmodulin inhibition of rod cyclic nucleotide-gated channels

Abstract

Rod cyclic nucleotide-gated (CNG) channels are heterotetramers comprised of both CNGA1 and CNGB1 subunits. Calcium͞calmod-ulin (Ca 2؉ ͞CaM) binds to a site in the N-terminal region of CNGB1 subunits and inhibits the opening conformational change in CNGA1͞CNGB1 channels. Here, we show that polypeptides derived from an N-terminal region of CNGB1 form a specific interaction with polypeptides derived from a C-terminal region of CNGA1 that is distal to the cyclic nucleotide-binding domain. Deletion of the Ca 2؉ ͞CaM-binding site from the N-terminal region of CNGB1 eliminated both Ca 2؉ ͞CaM modulation of the channel and the intersubunit interaction. Furthermore, the interaction was disrupted by the presence of Ca 2؉ ͞CaM. These results suggest that Ca 2؉ ͞CaM-dependent inhibition of rod channels is caused by the direct binding of Ca 2؉ ͞CaM to a site in the N-terminal region in CNGB1, which disrupts the interaction between this region and a distal C-terminal region of CNGA1. The mechanism underlying Ca 2؉ ͞CaM modulation of rod channels is distinct from that in olfactory (CNGA2) CNG channels. C yclic nucleotide-gated (CNG) channels were first characterized in retinal rods, where they conduct a cation current in response to changes in intracellular levels of cGMP and mediate the electrical response to light (1). CNG channels also are found in olfactory neurons, where they respond to changes in internal cAMP and underlie the electrical response to odorants (2). Evidence exists for a cyclic nucleotide-dependent conductance in taste receptors (3,4). CNG channels are present in a variety of other tissues, including heart, aorta, and kidney (for a review see ref. 5).CNG channels were first cloned from retina (6) and olfactory neurons (7). Four channel subunits are arranged to form a tetramer with a central pore (8,9). Subunits have six proposed membrane-spanning domains, a pore-loop domain, and intracellular N-and C-terminal regions, a topology similar to that of the voltage-activated potassium channels (10). However, CNG channels are only weakly sensitive to membrane voltage. Instead, they contain a large C-terminal cyclic nucleotide-binding domain (CNBD) that exhibits sequence similarity with other cyclic nucleotide-binding proteins (11,12). CNG channels are activated by the direct binding of cyclic nucleotides to the CNBD (13).At present, there are six types of mammalian CNG channel genes. The genes are grouped according to sequence similarity into two subtypes, CNGA and CNGB (14). CNGA1, CNGA2, and CNGA3 subunits form functional homomeric channels when expressed alone, whereas CNGB1, CNGB3, and CNGA4 subunits do not appear to form functional homomeric channels when expressed alone. Instead, CNGB1, CNGB3, and CNGA4 subunits form heteromeric channels when coexpressed with CNGA1, CNGA2, or CNGA3 subunits (15).Native retinal rod channels comprise CNGA1 (formerly CNG1; Rod ␣) and CNGB1 (formerly CNG4; Rod ␤) subunits. CNGA1͞CNGB1 heterotetramers contain two CNGA1 subunits and two CNGB1 subunits (16,17). Compared with CNGA1 ...

Interactions between N and C termini of α1Csubunit regulate inactivation of CaV1.2 L-type Ca2+channel

“…39 The involvement of CaM in the NT-CT interaction also appears in other channels, such as CNG, where CaM binding to the NT disrupts the NT-CT interaction, leading to channel inactivation. 39,40 However, Ca 2C channels, unlike CNG or K C channels composed of 4 separate subunits (each with an NT and a CT), have only one NT and CT. The idea of an interaction between NT and CT in a 1C , and for a role of this interaction (within a hypothetical "NT-CT scaffold") 10 in channel gating and inactivation, has been already raised in the past.…”

Calcium/Calmodulin Modulation of Olfactory and Rod Cyclic Nucleotide-gated Ion Channels

“…The CaM-binding region in CNGA2 is an archetypal "1-8-14" site, characterized by hydrophobic residues at positions 1 and 14 and long chain aliphatic residues at position 8 (as underlined) (46). Rod CNGA1 subunits do not contain a Ca 2ϩ /CaM-binding site; however, CNGB1 subunits have an N-terminal site ( 682 LQELVKLFKERTEKVKEKLI 701 ) that is necessary for Ca 2ϩ / CaM binding (47)(48)(49). This site is critical for functional inhibition as its deletion in CNGB1 subunits results in heteromeric channels (after co-expression with CNGA1) that are insensitive to Ca 2ϩ /CaM (47)(48)(49).…”

“…Rod CNGA1 subunits do not contain a Ca 2ϩ /CaM-binding site; however, CNGB1 subunits have an N-terminal site ( 682 LQELVKLFKERTEKVKEKLI 701 ) that is necessary for Ca 2ϩ / CaM binding (47)(48)(49). This site is critical for functional inhibition as its deletion in CNGB1 subunits results in heteromeric channels (after co-expression with CNGA1) that are insensitive to Ca 2ϩ /CaM (47)(48)(49). Several key residues (underlined) are similar to those in the IQ type of CaM binding motifs (IQXXXRGXXXRXX(F/W)); however, the CNGB1 region is "unconventional" as it lacks the central glycine, and the final hydrophobic residue is not amphipathic and requires Ca 2ϩ for CaM binding, unlike IQ motifs (46).…”

Multiple Calmodulin-binding Sites Positively and Negatively Regulate Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL12

“…Mammalian CNGCs possess diverse sites for CaM binding, with at least nine CaMBDs found across both the N and C termini of the six different CNGC isoforms (Ungerer et al, 2011). However, to date, experiments have provided evidence of a functional role for only the N-terminal CaMBDs (Liu et al, 1994;Weitz et al, 1998;Trudeau and Zagotta, 2002;Zheng et al, 2003;Song et al, 2008), particularly the N-terminal LQ site of the regulatory CNGB1 subunit (Ungerer et al, 2011). Generally, CaM is hypothesized to function in the feedback regulation of CNGCs by binding to one or more CaMBD(s) at elevated cytosolic Ca 2+ levels and allosterically inhibiting CNGC conductance (Zheng et al, 2003;Zagotta, 2002, 2004;Bradley et al, 2001Bradley et al, , 2004Ungerer et al, 2011;Liu et al, 1994;Chen and Yau, 1994;Weitz et al, 1998;Song et al, 2008).…”