Retinal Ganglion Cell Inward Rectifier Potassium Channel (of Kir) And Hyperpolarization-activated H Channel (i_h)

Inwardly rectifying potassium channels(Kir channels) are important for neuronal signaling and membrane excitability.In the present work we characterized,for the first time,Kir channels in rat retinal ganglion cells(RGCs),the output neurons in the retina,using immunocytochemical and patch clamp techniques.Various subunits of Kir channels(Kir1.1,2.1,2.3,3.1,3.2,3.3) were expressed in RGCs,but with distinct subcellular localization.Kirl.1 was mainly expressed in axons of RGCs.Kir2.1 and Kir2.3 were both present in somata of RGCs.Whereas staining for Kir3.1 was profoundly present in an endoplasmic reticulum-like structure and Kir3.2 was strongly expressed in the cytoplasm and the cytomembrane of somata,dendrites and axons of RGCs,faint,sparse labeling for Kir3.3 was seen in the cytomembrane. Immunoreactivity for Kir4.1 and Kir4.2 was not detectable in RGCs.Whole-cell currents mediated by Kir channels were recorded in isolated RGCs,showing full activation in less than 10 ms,no inactivation,and being significantly suppressed by 300μM Ba²⁺.Unlike retinal horizontal cells and bipolar cells,these currents were mainly mediated by G-protein coupled Kir3(GIRK) channels,as evidenced by the fact that GDP_βS and GTP_γS included in the pipette solution markedly decreased and increased the currents respectively.Furthermore,the GIRK channels were likely coupled to GABA_B receptors,since baclofen considerably increased the Kir currents and the increased currents were suppressed by Ba²⁺.These characteristics of the Kir currents provide more versatility for signaling of RGCs.Dopamine is an important neurotransmitter and neuromodulator in the vertebrate retina and it is thought that dopamine is involved in the light-dark adaptation by regulating the functions of many retinal cells.In the present study,we examined the effects dopamine on rat retinal ganglion cells(RGCs) using whole-cell patch-clamp technique.Dopamine(5-10μM) depolarized the membrane potential and decreased the input resistance of RGCs.Dopamine also reduced the intrinsic excitability of these cells.The D1-like dopamine receptor agonist SKF38393(10μM) mimicked the dopamine effects whilst no significant changes in membrane properties and excitability were observed when the D2-1ike receptor agonist quinpirole(10μM) was applied.Additionally,the D1-like receptor antagonist SCH23390(10μM),but not the D2-1ike receptor antagonist sulpiride(10μM),blocked the dopamine effects on rat RGCs.Furthermore,the dopamine actions on membrane properties and excitability were mimicked by application of 8-Br-cAMP(200μM) and blocked by preincubation of KT5720(1μM),indicating the involvement of cAMP and protein kinase A(PKA).The membrane potential depolarization and the decrease of the input resistance induced by DA suggest that some ion conductances could be modulated by DA.In most cases,we noticed a voltage "sag" while injecting a hyperpolarization current to the recorded RGCs.It has been shown that,the voltage "sag" induced by hyperpolarization current indicates the presence of the hyperpolarization-activated nonspecific cation channel(I_h).The voltage "sag" became more significant in the presence of DA raises a possibility that Ih is modulated by DA,To test this possibility, we first characterize the I_h current in RGCs.ZD7288,a specific Ih channel blocker, could block the I_h current in RGCs.We then test effect of DA on I_h and found DA increased the I_h current through the D1 receptor,which could be mimicked by application of 8-Br-cAMP and attenuated by preapplication of KT5720.To test whether the hyperpolarization-activated nonspecific cation channel(I_h) was involved in the dopamine effects,ZD7288 was used.Preincubation of ZD7288 (20-30μM) blocked the effects of dopamine on membrane potential,input resistance and excitability of rat RGCs.These data suggest that I_h may contribute to the dopamine-induced modulation of the membrane properties and excitability of rat RGCs.