An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx moril-glutamate-gated chloride channel

Figures & data

An L319F mutation in TM3 reduces okaramine B sensitivity more profoundly than ivermectin sensitivity of Bombyx mori l-glutamate-gated chloride channel. The Graphical image was illustrated using PyMOL software.

Fig. 1. Structures of Okaramine B and ivermectin B1a (A) and the alignment of amino acid sequences of C. elegans and insect GluCls and insect GABA-gated chloride channels (GABACls) (B).

Notes: Accession numbers of protein sequences compared: C. elegans GluCl, AAA50785.1; B. mori GluCl, BAD16657.1; Drosophila melanogaster GluCl, AAG40735.1; Musca domestica GluCl AGW27406.1; Anopheles gambiae GluCl, AGS43092.1; B. mori GABACl, BAN92442.1; D. melanogaster GABACl, AAA28556.1; M. domestica GABACl, BAD16658.2; A. gambiae GABACl, APA16887.1. Amino acid sequences are numbered according to the B. mori GluCl when counted from the start methionine1. The position of leucine319 of BmGluCl (=methionine345 of C. elegans GluCl) is indicated by an arrow. (C) The crystal structure of the C. elegans GluCl with ivermectin bound (3RHW). The figures were generated using PyMOL software. Methionine345 corresponding to leucine319 of BmGluCl and phenylalanine319 of BmGABACl is indicated by space filling model where carbons, oxygen, nitrogen and sulfur are colored white, red, blue and yellow, respectively. Ivermectin is shown as a wire model where carbons and oxygens are colored green and red, respectively.

Table 1. pEC₅₀ and I_max values^a for l-glutamate, ivermectin and okaramine B actions on wild type and L319F mutant BmGluCl expressed in Xenopus laevis oocytes.

Compounds	Wild type		L319F Mutant
	pEC50	Imax	pEC50	Imax
l-Glutamate	3.61 ± 0.20	1.37 ± 0.12	3.54 ± 0.07	1.13 ± 0.05
Ivermectin	6.23 ± 0.09	0.24 ± 0.02	5.11 ± 0.71	0.08 ± 0.05
Okaramine B	6.25 ± 0.10	0.19 ± 0.01	ND^b	ND

^aData are represented as mean ± standard error of the mean (n = 4).

^bCould not be determined because okaramine B was inactive in the range 10–100 μM when tested on oocytes expressing the L319F BmGluCl mutant.

Fig. 2. Effects of the BmGluCl L319F mutation on the response to l-glutamate.

Notes: (A) Inward currents induced by l-glutamate in Xenopus laevis oocytes expressing the wild type and L319F mutant BmGluCl. (B) Concentration-response curve of l-glutamate for the wild type and L319F mutant BmGluCl. Data in (B) are represented as mean ± standard error (n = 4).

Fig. 3. Effects of the BmGluCl L319F mutation on the response to ivermectin.

Notes: (A) Inward currents evoked by ivermectin in Xenopus laevis oocytes expressing the wild type and L319F mutant BmGluCl. The application of ivermectin is indicated by horizontal lines. (B) Concentration-response curve of ivermectin for the wild type and L319F mutant BmGluCl. Data are represented as mean ± standard error (n = 4).

Fig. 4. Effects of the BmGluCl L319F mutation on the response to okaramine B.

Notes: (A) Inward currents evoked by okaramine B in Xenopus laevis oocytes expressing the wild type and L319F mutant BmGluCl. Application of okaramine B is indicated by horizontal lines. (B) Concentration-response curve of okaramine B for the wild type and L319F mutant BmGluCl. Data are represented as mean ± standard error (n = 4).

Fig. 5. Effects of co-application of okaramine B on the response to l-glutamate of BmGluCl expressed in Xenopus laevis oocytes.

Notes: (A) After recording a control response to 100 μM l-glutamate, 10 μM okaramine B was bath-applied at 10 μM for 1 min and then co-applied with 100 μM l-glutamate for 2 s to an oocyte expressing BmGluCl. After washing for 3 min with SOS, 100 μM l-glutamate was applied again. Application of l-glutamate and okaramine B are indicated by solid and hashed horizontal lines, respectively. (B) Effects of 10 μM okaramine B on the peak amplitude of the response to 100 μM l-glutamate of BmGluCl. The data represent mean ± standard error of the mean (n = 4). *, Difference is statistically significant by paired t test (p < 0.05).

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