Clint L. Makino, Ph.D.

Clint L. Makino, Ph.D.

Associate Professor of Physiology and Biophysics

B.S. Duke University
Ph.D. Florida State University

Phone: (617) 638-4460
Fax: (617) 638-4041
E-mail: cmakino@bu.edu
Address: see below
Link to BU Faculty Profile
Link to ORCID


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.
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.
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.

Recently, the Sharma and Duda laboratories at Salus University discovered that bicarbonate ions can stimulate cGMP synthesis by retGC, independent of GCAPs. In a collaboration with them, we discovered that bicarbonate operates synergistically with GCAPs and low Ca2+ in rods. As a result, bicarbonate boosts the size of the maximal flash response and quickens response kinetics (Fig. 3). Rods do not express carbonic anhydrase, suggesting that visual transduction is subject to modulation by bicarbonate produced exogenously.

Figure 3. Bicarbonate increases the rod’s maximal flash response amplitude. The saturating flash response in a mouse rod enlarged during perfusion of a piece of excised retina with 20 mM bicarbonate (red) and subsided as the bicarbonate was washed away (black).
Figure 3. Bicarbonate increases the rod’s maximal flash response amplitude. The saturating flash response in a mouse rod enlarged during perfusion of a piece of excised retina with 20 mM bicarbonate (red) and subsided as the bicarbonate was washed away (black).

 

Selected Publications:

Full listing of citations on PubMed

PAPERS

Duda, T., Pertzev, A., Makino, C.L. & Sharma, R.K. (2016) Bicarbonate and Ca2+ sensing modulators activate photoreceptor ROS-GC1 synergistically. Front. Mol. Neurosci. 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. J. Biol. Chem. 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. J. Comp. Neurol. 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. Cell. Physiol. Biochemistry 29: 417-430. PMCID: PMC3434333.

Miyazono, S., Isayama, T., DeLori, F.C. and Makino, C.L. (2011) Vitamin A activates rhodopsin and sensitizes it to UV light. Vis. Neurosci. 28: 485-497. PMCID: PMC3601037.

Makino, C. L., Riley, C. K., Looney, J., Crouch, R. K. and Okada, T. (2010) Binding of more than one retinoid to visual opsins. Biophy. J. 99: 2366-2373. PMCID: PMC3042582.

REVIEWS

Wen, X.-H., Dizhoor, A.M. and Makino, C.L. (2014) Membrane guanylyl cyclase complexes shape the photoresponses of retinal rods and cones. Front. Mol. Neurosci. 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. Front. Mol. Neurosci. 7: 21. PMCID: PMC3972482.

BOOK CHAPTERS

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.

MacLeish, P. R. and Makino, C. L. (2011) Photoresponses of rods and cones, in Adler’s Physiology of the Eye 11th edition, eds. P. L. Kaufman, A. Alm, L. L. Levin, S. F. E. Nilsson, J. N. Ver Hoeve and S. M. Wu. Elsevier, pp. 411-428.

Contact Us

Clint L. Makino, Ph.D.
Department of Physiology and Biophysics
Boston University School of Medicine
700 Albany Street, W402B
Boston, MA 02118-2526

Phone: (617) 638-4460
Fax: (617) 638-4041
E-mail: cmakino@bu.edu