The Makino Lab

Research

The rate of cGMP synthesis modulates visual transduction

In retinal rods, cGMP is the second messenger that links photon capture by rhodopsin on internal disk membranes to ion channel activity on the plasma membrane. Rhodopsin photoexcitation leads to the hydrolysis of cGMP and subsequent closure of cGMP-gated channels, curtailing the entry of Na+ and Ca2+. During response recovery, retina-specific guanylyl cyclases (retGCs) replenish cGMP, reopen the channels, and restore the influx of cations. To facilitate the recovery, guanylyl cyclase activating proteins (GCAPs) sense the decrease in Ca2+ caused by illumination and greatly stimulate the rate of cGMP synthesis.

All vertebrate rods use at least two GCAPs: GCAP1 and GCAP2. Why are 2 types of GCAPS necessary? Working closely with the Dizhoor laboratory at Salus University, we explored the basis for the dual system by using electrophysiological methods to study rods of mutant mice that lack one or both GCAPs. Deletion of GCAP2 did not change the amplitude of the single photon response, but slowed its recovery (Fig. 1). Elimination of GCAP1 caused the photon response to rise for twice as long to an amplitude that was twice as large. Although knockout of GCAP2 did not affect GCAP1 expression, knockout of GCAP1 did cause an up-regulation of GCAP2 as detected by immunofluorescence and Western blot. The overexpression of GCAP2 resulted in acceleration of the response recovery rather than the slowdown that was expected from the loss of GCAP1 alone.

Figure 1. The single photon responses of wild-type control (black trace), GCAP1 knockout (blue), GCAP2 knockout (orange), and GCAPs1&2 knockout (gray) mouse rods.

Thus the two GCAPs regulate retGCs sequentially (Fig. 2). Because of its lower Ca2+ affinity, GCAP1 is the first responder and acts to limit response amplitude. GCAP2, with a higher Ca2+ affinity, does not assist retGCs until Ca2+ concentration declines even further, which happens somewhat during a single photon response, but to a greater extent with bright light. GCAP2 stimulation of retGCs provides for a faster response recovery. Together, the two GCAPs tune the operating range and temporal resolution of rod photoreceptors.

Figure 2. Relay model for GCAPs regulation of retGCs activity. The physiological free Ca2+ concentration ranges from 250 nM in the dark to 20 nM in the light in a mouse rod. In the dark, both GCAPs bind Ca2+ and suppress retGC activity. Under illumination, GCAP1 responds first to the light-induced Ca2+ decrease, releasing its bound Ca2+ and allowing Mg2+ to take its place. With Mg2+ bound, GCAP1 stimulates retGC activity. As Ca2+ concentration falls further, GCAP2 follows suit. With the reopening of the cGMP-gated channels, Ca2+ concentration rises, GCAPs rebind Ca2+ and retGC activity returns to its basal level.

 

The Sharma and Duda laboratories at Salus University discovered that bicarbonate ions stimulate cGMP synthesis by retGC, independent of GCAPs. In single cell recordings, we discovered that bicarbonate operated synergistically with GCAPs and low Ca2+ in rods and in cones. As a result, bicarbonate boosted the size of the maximal flash response, reduced relative sensitivity to flashes and altered response kinetics (Fig. 3).

Figure 3. Bicarbonate increased the maximal flash response amplitude and quickened flash response kinetics in a salamander rod. Flash responses were recorded first in MOPS/Ringer’s, then in the presence of 50 mM bicarbonate, and finally during a wash with MOPS/Ringer’s.

Rods do not express carbonic anhydrase, suggesting that modulation of visual transduction involves bicarbonate produced exogenously. We found that only rods retaining an intact spherule were affected by bicarbonate, indicating that uptake occurred in the inner segment, near the synaptic ending. In contrast, cones took up bicarbonate at their outer segments as well as at their inner segments. Moreover, cones did express carbonic anhydrase, which meant that they were affected by endogenous as well as exogenous bicarbonate.


Figure 4. Pathway for bicarbonate through rods and cones. Bicarbonate enters rods and cones at their synapses (1). Cones are also able to take up bicarbonate at their outer segments, perhaps by reversed action of a bicarbonate/Cl- exchanger (2). Carbon dioxide is able to pass through the plasma membrane and in cones, but not in rods, gets converted to bicarbonate by carbonic anhydrase. Bicarbonate is removed from rods and cones by a bicarbonate/Cl- exchanger located in their outer segments.


Recent Publications

Full listing of citations on PubMed

PAPERS

Makino, C.L., Duda, T., Pertzev, A., Isayama, T., Geva, P., Sandberg, M.A. and Sharma, R.K. (2019) Modes of accessing bicarbonate for the regulation of membrane guanylate cyclase (ROS-GC) in retinal rods and cones. eNeuro, 6(1). PMCID: PMC6378327.

Duda, T., Pertzev, A., Makino, C.L. & Sharma, R.K. (2016) Bicarbonate and Ca2+ sensing modulators activate photoreceptor ROS-GC1 synergistically. Frontiers in Molecular Neuroscience 9: 5.

Duda, T., Wen, X.-H., Isayama, T., Sharma, R.K. and Makino, C.L. (2015) Bicarbonate modulates photoreceptor guanylate cyclase (ROS-GC) catalytic activity. Journal of Biological Chemistry 290: 11052-11060.

Isayama, T., Chen, Y., Kono, M., Fabre, E., Slavsky, M., DeGrip, W.J., Ma, J.-X., Crouch, R.K. and Makino, C.L. (2014) Co-expression of three opsins in cone photoreceptors of the salamander, Ambystoma tigrinum. Journal of Comparative Neurology 522: 2249-2265. PMCID: PMC3997598.

Makino, C.L., Wen, X.-H., Olshevskaya, E.V., Peshenko, I.V., Savchenko, A.B. and Dizhoor, A.M. (2012) Enzymatic relay mechanism stimulates cyclic GMP synthesis in rod photoresponse: biochemical and physiological study in guanylyl cyclase activating protein 1 knockout mice. PLoS ONE 7: e47637. PMCID: PMC3474714.

Makino, C.L., Wen, X.-H., Michaud, N.A., Covington, H.I., DiBenedetto, E., Hamm, H.E., Lem, J., and Caruso, G. (2012) Rhodopsin expression level affects rod outer segment morphology and photoresponse kinetics. PLoS ONE 7: e37832. PMCID: PMC3360601.

Wen, X.-H., Duda, T., Pertzev, A., Venkataraman, V., Makino, C.L. and Sharma, R.K. (2012) S100B serves as a Ca2+ sensor for ROS-GC1 guanylate cyclase in cones but not in rods of the murine retina. Cellular Physiology and Biochemistry 29: 417-430. PMCID: PMC3434333.

REVIEWS

Sharma, R.K., Duda, T. and Makino, C.L. (2016) Integrative signaling networks of membrane guanylate cyclases: Biochemistry and physiology. Frontiers in Molecular Neuroscience 9: 83. doi: 10.3389/fnmol.2016.00083. PMCID: PMC5023690.

Wen, X.-H., Dizhoor, A.M. and Makino, C.L. (2014) Membrane guanylyl cyclase complexes shape the photoresponses of retinal rods and cones. Frontiers in Molecular Neuroscience 7:45. PMCID: PMC4040495.

Sharma, R.K., Makino, C.L., Hicks, D. and Duda, T. (2014) ROS-GC interlocked Ca2+-sensor S100B protein signaling in cone photoreceptors: review. Frontiers in Molecular Neuroscience 7: 21. PMCID: PMC3972482.

BOOK CHAPTERS

Makino, C.L,. Duda, T., Pertzev, A. and Sharma, R.K. (2018) Experimental approaches for defining the role of the Ca2+-modulated ROS-GC system in retinal rods of mouse, in Methods in Molecular Biology 1753: Mouse Retinal Phenotyping – Methods and Protocols, ed, N Tanimoto. Humana Press, New York, pp. 129-158.

Isayama, T. and Makino, C.L. (2012) Pigment mixtures and other determinants of spectral sensitivity of vertebrate retinal photoreceptors, in Photoreceptors: Physiology, Types and Abnormalities, eds. E. Akutagawa and K Ozaki. Nova Science Publishers, pp. 1-31.

Lab Members:



Md Uddin Shahabe Talukder, Ph.D.



Brianna Kim


Past Lab Members:

Rajan Adhikari, Ph.D.
Anaid Perez
Polina Geva, Ph.D.
Amanda Kossoff
Paris Chakravarty


Links:

Faculty Profile
ResearchGate



Contact Us

Department of Pharmacology, Physiology & Biophysics
Chobanian & Avedisian School of Medicine
700 Albany Street W402B
Boston, MA 02118-2526

Phone: (617) 358-8470
e-mail: cmakino@bu.edu