The relationship between Ca²⁺ channel types and Ca²⁺-activated Cl⁻ channels properties in mouse retinal on-bipolar cell axon terminals

Abstract

Purpose: Two types of voltage-gated Ca²⁺ channels, L-type and T-type, are known to be localized in axon terminals of mammalian bipolar cells. We previously characterized a Ca²⁺-activated Cl⁻ channel (CaCC) in rod bipolar cell axon terminals of mouse retina and found that L-type Ca²⁺ channels acted as its major Ca²⁺ influx source. However, the role of T-type Ca²⁺ channel and its relationship with the CaCC remains unknown. This study was aimed to clarify the contribution of two types of voltage-gated Ca²⁺ channels to CaCC properties in on-type bipolar cell axon terminals of mouse retina.

Methods: Mice (7~8week-age, C57BL/6J) were deeply anaesthetized with 4% chloral hydrate and solitary on-type bipolar cells were obtained with enzymatic dissociation. The membrane currents of these cells were recorded by the patch-clamp technique in the whole-cell recording configuration. Statistical analysis was conducted by ANOVA and Student’s t-test. Data are represented as mean ± s.d. The differences were considered significant at P < 0.05.

Results: According to the pharmacological profile and biophysical property, on-bipolar cells of mouse retina were divided into three groups. First, on-bipolar axon terminals (7 out of 15) showed prominent L-type activated Ca²⁺-activated Cl⁻ current (I_Cl(Ca)) with a high sensitivity to nifedipine (10 µM) and a peak (20 ± 3 pA) at -30 mV. Second type (5 out of 15) possessed I_Cl(Ca) that attributed to the combination of two types of Ca²⁺ channels with a high sensitivity to nifedipine and a peak amplitude (13 ± 4 pA) at -40 mV. Third type of on-bipolar axon terminals (3 out of 15) showed prominent T-type mediated I_Cl(Ca) with a high sensitivity to mibefradil (10 µM) and a peak amplitude (5 ± 4 pA) at -55 mV. Interestingly, when the cells were depolarized by repeated train pulses, only L-type activated I_Cl(Ca) was highly enhanced in amplitude (330 ± 26 %, n=6).

Conclusions: These results demonstrate that both L-type and T-type Ca²⁺ channels contribute to I_Cl(Ca) in on-bipolar cell axon terminals of mouse retina and suggest that a dominant Ca²⁺ source of I_Cl(Ca) is L-type Ca²⁺ channel rather than T-type Ca²⁺ channel.