RIM1/2-Mediated Facilitation of Cav1.4 Channel Opening Is Required for Ca2+-Stimulated Release in Mouse Rod Photoreceptors

J Neurosci. 2015 Sep 23;35(38):13133-47. doi: 10.1523/JNEUROSCI.0658-15.2015.

Abstract

Night blindness can result from impaired photoreceptor function and a subset of cases have been linked to dysfunction of Cav1.4 calcium channels and in turn compromised synaptic transmission. Here, we show that active zone proteins RIM1/2 are important regulators of Cav1.4 channel function in mouse rod photoreceptors and thus synaptic activity. The conditional double knock-out (cdko) of RIM1 and RIM2 from rods starting a few weeks after birth did not change Cav1.4 protein expression at rod ribbon synapses nor was the morphology of the ribbon altered. Heterologous overexpression of RIM2 with Cav1.4 had no significant influence on current density when examined with BaCl2 as the charge carrier. Nonetheless, whole-cell voltage-clamp recordings from cdko rods revealed a profound reduction in Ca(2+) currents. Concomitantly, we observed a 4-fold reduction in spontaneous miniature release events from the cdko rod terminals and an almost complete absence of evoked responses when monitoring changes in membrane incorporation after strong step depolarizations. Under control conditions, 49 and 83 vesicles were released with 0.2 and 1 s depolarizations, respectively, which is close to the maximal number of vesicles estimated to be docked at the base of the ribbon active zone, but without RIM1/2, only a few vesicles were stimulated for release after a 1 s stimulation. In conclusion, our study shows that RIM1/2 potently enhance the influx of Ca(2+) into rod terminals through Cav1.4 channels, which is vitally important for the release of vesicles from the rod ribbon. Significance statement: Active zone scaffolding proteins are thought to bring multiple components involved in Ca(2+)-dependent exocytosis into functional interactions. We show that removal of scaffolding proteins RIM1/2 from rod photoreceptor ribbon synapses causes a dramatic loss of Ca(2+) influx through Cav1.4 channels and a correlated reduction in evoked release, yet the channels remain localized to synaptic ribbons in a normal fashion. Our findings strongly argue that RIM1/2 facilitate Ca(2+) entry and in turn Ca(2+) evoked release by modulating Cav1.4 channel openings; however, RIM1/2 are not needed for the retention of Cav1.4 at the synapse. In summary, a key function of RIM1/2 at rod ribbons is to enhance Cav1.4 channel activity, possibly through direct or indirect modulation of the channel.

Keywords: CaV1.4; RIM; evoked release; mouse rod photoreceptor; night blindness; ribbon synapse.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aspartic Acid / pharmacology
  • Barium Compounds / pharmacology
  • Biophysical Phenomena / drug effects
  • Biophysical Phenomena / genetics*
  • Calcium / metabolism*
  • Calcium Channels / genetics
  • Calcium Channels / metabolism*
  • Calcium Channels, L-Type
  • Chlorides / pharmacology
  • Excitatory Amino Acid Agents / pharmacology
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism*
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / genetics*
  • HEK293 Cells
  • Humans
  • Membrane Potentials / drug effects
  • Membrane Potentials / genetics
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Presynaptic Terminals / metabolism
  • Presynaptic Terminals / ultrastructure
  • Retina / cytology
  • Retinal Rod Photoreceptor Cells / drug effects
  • Retinal Rod Photoreceptor Cells / physiology*
  • Synaptic Transmission / drug effects
  • Synaptic Transmission / genetics
  • Synaptic Vesicles / drug effects
  • Synaptic Vesicles / metabolism
  • Synaptic Vesicles / ultrastructure
  • rab3 GTP-Binding Proteins / genetics
  • rab3 GTP-Binding Proteins / metabolism*

Substances

  • Barium Compounds
  • Cacna1f protein, mouse
  • Calcium Channels
  • Calcium Channels, L-Type
  • Chlorides
  • Excitatory Amino Acid Agents
  • Rims1 protein, mouse
  • benzyloxyaspartate
  • barium chloride
  • Aspartic Acid
  • GTP-Binding Proteins
  • Rim2 protein, mouse
  • rab3 GTP-Binding Proteins
  • Calcium