It has been suggested that extracellular Ca
2+ electrostatically binds to negatively charged residues (e.g., glutamate at position 340 in bovine retinal rods) at pore region of the CNG channel α-subunit and thereby inhibits the flux of monovalent cations through the channel.
22 23 24 25 The inhibitory effects of extracellular Ca
2+ on hCNGA3 homomeric and hCNGA3/hCNGB3 heteromeric channels activated by 100 μM cGMP were assessed. Macroscopic patch currents were recorded in an inside-out configuration with pipette solutions containing various concentrations of Ca
2+.
Figures 6A and 6B , respectively, illustrate
I–
V relationships for hCNGA3 homomeric and hCNGA3/hCNGB3 heteromeric channels in the presence of Ca
2+ at concentrations of 1 × 10
-10 M (filled circle), 1 × 10
−6 M (open square), 1 × 10
−4 M (filled square), and 1.8 × 10
−3 M (open circle). The
I–
V relationship for hCNGA3 homomeric channels is essentially linear in the presence of extremely low concentrations of Ca
2+ (1 × 10
−10 M) but exhibits a marked outward rectification with Ca
2+ at concentrations of 1 × 10
−6 M or more
(Fig. 6A) . This characteristic
I–
V relationship is assumed to arise from voltage dependent blockade of channel currents by extracellular Ca
2+; inward current at negative potentials is more sensitive to inhibition compared with outward current at positive potentials.
24 25 On the other hand, the
I–
V relationship for hCNGA3/hCNGB3 heteromeric channels with high extracellular Ca
2+ concentrations is also outwardly rectified but exhibits some relaxations at potentials of ≤ ∼ −50 mV
(Fig. 6B) , which has been ascribed to relief of Ca
2+ blockade at these strongly hyperpolarized potentials.
8 The degree of Ca
2+ blockade was assessed by normalizing the current amplitude at −80 mV with reference to that at +80 mV in
I–
V relationships, recorded in the presence of 1.8 mM Ca
2+. The current ratio (expressed as %) averages 5.2 ± 2.0% (
n = 4) for hCNGA3 and 29.7 ± 3.1% for hCNGA3/hCNGB3 (
n = 4,
P < 0.01,
Fig. 6E ).