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Norianne T Ingram, Gordon L Fain, Alapakkam P Sampath; VOLTAGE-CLAMP CURRENTS AND LIGHT RESPONSES OF MOUSE CONES. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1377.
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© ARVO (1962-2015); The Authors (2016-present)
Though much is known about ion channels in vertebrate photoreceptors, few measurements have been made from mammalian cones. We have developed methodology for voltage-clamping mouse cone photoreceptors while maintaining robust light responses. Using whole-cell recording, we have investigated photocurrents and major voltage and Ca2+-activated conductances.
Patch-clamp recordings were made from cones in retinal slices with techniques previously described. We used Gnat1-/- or Cx36-/- cones to eliminate contributions from rods through gap junctions and to improve our space clamp. Cones were identified from the position and shape of their nuclei, with confirmation by dye filling. Light responses and voltage-clamp currents were recorded conventionally.
Initial attempts to measure the reversal potential of the photocurrent gave surprisingly high positive values (about +40 mV) compared to those based on Nernst calculations (0 to +10 mV), indicating a further conductance in addition to the cGMP-gated channels. Cx36-/- cones filled with GTP-γ-S continued to show a light-sensitive current but with an anomalous waveform and voltage dependence. Since light has been shown to release Ca2+ in cones (Brockerhoff et al, J Neurosci. 23:470-480, 2003), we reasoned that this remaining response could be produced at least in part by a Ca2+-activated Cl- current. We then showed that depolarization gates an inward Ca2+ current followed by an outward Cl- current blocked by niflumic acid. Both currents were blocked by the L-type Ca2+-channel blocker isradipine, and with this treatment the light responses reversed at +11 ± 2 mV (K+ internal) and +26 ± 3 mV (Cs+ internal), with PK/PNa = 0.7 and PCs/PNa = 0.4. We also describe the voltage-dependence of the Ca2+ current and of Ih, which contributes to the cone voltage response.
We describe a new technique to record voltage-clamp currents from mouse cones, which will permit us to explore responses not only from WT cones but from cones with mutations in genes essential for cone function. We show that cone photocurrent can be produced not only by cGMP-channel gating but also by a Ca2+-activated Cl- conductance. Our characterization of voltage-gated currents will help us understand the waveform of the cone voltage response and the voltage dependence of synaptic transmission.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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