Camphor

According to PubChem, the best source of of campor is the Camphor tree Cinnamomum camphora. Rosemary Rosmarinus officinalis L. may contain 10-20% camphor.   Camphor is only one of many terpenes found in basil, or Ocimum species. Scientists from South Africa have published an interesting review on the historic use of camphor throughout the world.    Natural camphor, the (+) form, is a distillate of  camphor laurel tree (Cinnamomum camphora)  wood.  Examples of other trees that produce camphor were provided.  Camphor is also present in Crataegus berries.  A study was cited of these berries used to treat orthostatic hypotension.   [1] 

Some chemical players in Sherkheli (2013)

These are the TrpV3 ligands and why the authors were so concerned with cysteine amino acids in the TrpV3 channel

2-APB and DHC are agonists of TrpV3. Cysteine is an amino acid with an -SH side chain (circled) that can oxidize to sulfenic acid. This will eventually form disulfide bonds (cystine) within the protein. Mutating cysteines to serines preserves the hydropilicity why removing the reactivity.

Introduction to TrpV3 [2]

The TRPV3 receptor/ion channel has regions that respond to different stimuli

  • The heat sensor is located in the 6th transmembrane helix and adjacent extracellular loop within the pore region.
  • A region that responds to 2-aminoethyoxy diphenyl borinate (2-APB) or camphor is another region of interest in this study. [2]   His 426 and Arg696 are crucial for TRPV3 responsiveness to 2-APB. These mutants show normal responses to camphor whereas sensitivity to 2-APB is completely lost. [2]
  • Other background references demonstrated that camphor, not 2-APB, desensitizes TrpV3. [2]

This publication explored which amino acids were involved in the different responses of ligands binding to the same general region.  These scientists were interested in cysteine resides (C612 andC619) in the pore region of  channel [2] The mutated these cysteines to serines.  Instead of having a -SH side chain, these amino acids had an -OH.   Both side chains are hydrophilic.  The -SH group can react with oxygen and other compounds.  -SH reacts with one oxygen to form cysteine sulfenic acid -SOH.  -SOH can react with -SH to form protein and mixed disulfides.

Sherkheli and coworkers expressed the human TrpV3 channel in frog oocytes.  Frog oocytes are rather large and easy to insert electrodes to make measurements of the currents produced by ions flowing through ion channels like TrpV3. [2] The methods used for culturing and transfecting the oocytes [2] will not be discussed on this post.  Two-electrode voltage-clamp recordings were performed to obtain current responses to the chemicals.  Two electrodes are used to measure the potential difference across the oocyte membrane.  A current is then passed to set the potential difference at a certain level.  Current across a channel(s) is measured by the additional current required to keep the voltage at the clamped number.  Ligands were diluted to the final concentration in the standard extracellular saline (SES) solution  Ca2+.

SES: 115 mM NaCl,2.5 mM KCl, 1.8 mM, CaCl2, 200 μM flufenamic acid, 10mM HEPES, pH 7.4) 

Ca2+-free: 115 mM NaCl, 2.5 m M KCl, 10 mM HEPES, 10 mM EGTA, pH 7.4

Ligands were usually applied for 10-20 seconds. [2]  As a general note, potassium is high and sodium low inside the cell.   TrpV3 is a Ca2+ channel that can conduct Na+.

Sherkheli Fig 1

Agonist is added at each horizontal bar.  Note that in the presence of Ca2+ the current gets greater with each subsequent addition of 2-APB.  Without Ca2+ the current is consistent.

Note that in a Ca2+ free medium TrpV3 slowly shuts down with successive additions of camphor.  Also note the increase in current as the temperature is raised.

Sherkheli Fig 2

TRPV3 was considered a target of many terpenes by the scientists.  They were interested in determining if 2w-ABP and DHC bind to the same or different sites.  A DHC alone did not produce a current.  Together with 2-APB it elicited a current greater than 2-APB alone. B. Potentiations of 2-APB was. also seen with camphor .(3 mM)

Figure 2 from [2]

The current-magnitudes increased to

  • 294±37 % (p<0.008, n=6) for camphor (5 mM) plus 2-APB (3 mM) compared to 2-APB alone.
  •  194±31% (p<0.01, n=6) for dihydrocarveol (2 mM) compared to 2-APB alone.
  • 46±25%, (p<0.49, n=6) for camphor and HHC in combination compared tgo 2-APB alone
  • 30±12 % camphor alone
  • 70±9%, DHC alone

According to the authors this cross-potentiation property supports the idea that 2-APB and terpenoids interact at different domains on TRPV3 and hence activate the channel through independent mechanisms. [2] If some of this seems artificial, we have TRPV3 gene knockout studies from 2005 [3] to fall back on. These results showed lack of response to innocuous heat, noxious heat, and camphor. Trpv3 appeared to be expressed in keratinocytes rather than sensory neurons. [3]

Sherkheli Fig 3 and 4 probing thiols

(A) Putative topology of TRPV3.

(A) Putative topology of TRPV3. His 426 required for 2-APB binding occurs before the first trans membrane domain and after the four red dot ankyrin repeats. Arg 696 is in the cytosol right after the 6th transmembrane domain. Val686-Trp752 is responsible for temperature gating shown in Figure 1D. This region is the 6th transmembrane helix (652-676) UniProt (B) Exact sequence of the pore-loop of TRPV3 showing conserved cysteines mutated to checkcamphor sensitivity. The scientists are making a point that these cysteines are close enough to form a disulfide bond. (C) Many TrpV family members have potentially reactive cysteines in this external loop region that guards the mouth of the channel.
channels.

The hypothesis was that TrpV3 with the above cysteines would behave differently in response to the agonists

From reference 2. All experiments were carried out under Ca-free SES at room temperature (~23°C). Data are expressed as mean ± SEM of six independent experiments
Point-mutants and loss of camphor sensitivity of Trp3.

The voltage across the oocyte membrane was clamped at +40 and -40 mV. The conductance in response to camphor was less than that of DHC and 2-APS in the wild type (non mutated) and C612S mutant 4ith the cysteine to serine substitution at position 612. We can conclude that there is no such thing as a Cys612 and Cys619. Cys612 might be reacting with other stuff like the xytene in the glutathione tripeptide or something completely different. Structural studies of TrpV3 [4] add more insight, but will have to be covered in a different post.

references

  1. Chen, W., Vermaak, I., & Viljoen, A. (2013). Camphor–a fumigant during the Black Death and a coveted fragrant wood in ancient Egypt and Babylon–a review. Molecules (Basel, Switzerland), 18(5), 5434–5454. PMC free article.
  2. Sherkheli MA, Vogt-Eisele AK, Weber K, Hatt H. Camphor modulates TRPV3 cation channels activity by interacting with critical pore-region cysteine residues. Pak J Pharm Sci. 2013 May;26(3):431-8. PMC free article
  3. Moqrich A, Hwang SW, Earley TJ, Petrus MJ, Murray AN, Spencer KS, Andahazy M, Story GM, Patapoutian A. (2005)Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin. Science. 2005 Mar 4;307(5714):1468-72.  PMC free article
  4. Billen, B., Brams, M., Debaveye, S., Remeeva, A., Alpizar, Y. A., Waelkens, E., Kreir, M., Brüggemann, A., Talavera, K., Nilius, B., Voets, T., & Ulens, C. (2015). Different ligands of the TRPV3 cation channel cause distinct conformational changes as revealed by intrinsic tryptophan fluorescence quenching. The Journal of biological chemistry, 290(20), 12964–12974. PMC free article

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