Background
==========

Quality of life is an important clinical outcome in assessing the efficacy of health care. It is a concept that includes many subjective elements: physical, emotional and social function, attitudes to illness, patients\' daily lives-including family interactions, Spitzer, Dobson, et al. \[[@B1]\], Cella and Tulsky \[[@B2]\].

Treatments that are designed to control symptoms are suited for Quality of Life end points. When the goal of treatment is symptom palliation rather than prolongation, the use of Quality of Life as a primary end point of survival can be considered, Winer \[[@B3]\]. As a result of the increased interest in the impact of disease and treatment on the patient\'s life and functioning, several well-validated questionnaires that measure health-related quality of life (HRQoL) have been developed, some of which formed the foundation for the questionnaire that was used in this study \[[@B4]\]\[[@B5]\]\[[@B6]\]\[[@B7]\].

Cancer is not a disease of an individual but impacts the family system. Families must confront, and attempt to realize the meaning of cancer for the patient, for each family member individually and for the family system as a whole \[[@B8]\]. The diagnosis of cancer confronts both the patient and his/her caregiver(s) with a major life crisis, therefore family therapists now refer to the \'cancer family\' and not to the \'cancer personality\' \[[@B9]\]. Furthermore, as a life-threatening disease progresses, family members may assume new roles and responsibilities. They are usually supportive or concerned for their terminally ill patients, but they are frequently opposed to the patient\'s wishes or the recommendations of the treatment team. When the patient is incompetent, conflicts of interest may arise since decision-making is a great responsibility for the family \[[@B10]\]. A relative, who feels guilty or denies the hopelessness of the situation, may press the patient for cure rather than care, or to prolong or discontinue treatment \[[@B11]\].

It is worth noting, that every patient and family has a culture. Cultural aspects of values and behaviors are the key variables, along with life experiences, socio-economic status, and personality differences, that affect the meaning of cancer for both individuals and their families, as well as how they cope with the disease \[[@B10]\]\[[@B12]\]\[[@B13]\]. Thus, it is important to mention the close bonds found in a Greek family, especially towards severe problems such as cancer \[[@B14]\]. Furthermore, when relatives know the truth and try to keep it from their patients, they are likely to transmit their fears and anxiety through non-verbal ways. The cost of deception is high. For the patient who suspects but still hopes, the state of uncertainty is harder to bear than the certainty of the knowledge \[[@B15]\]\[[@B16]\]. This attitude of non-disclosure is not only associated with Greek culture but has also been observed in Japan \[[@B17]\]\[[@B18]\]\[[@B19]\]. Denying acceptance of the disease, family members even avoid discussing about cancer \[[@B20]\]\[[@B21]\].

The objectives of this study were to investigate the attitudes, perceptions and patterns of choice in the management of terminal stage cancer patients with respect to their families through the development of a relatives\' patient management questionnaire. Typically HRQoL questionnaires assessing the patients\' perceptions and choices are used to determine choice of treatment and palliation, however, with the realization that cancer families are also affected and involved by the disease and its management their perceptions of management choices are becoming important considerations in oncology.

Methods
=======

The study took place in the Pain Relief and Palliative Care Unit, of Areteion Hospital, in University of Athens. The study was carried out between March and September 2000, with hospital ethics committee approval. During this time 400 relatives of advanced cancer patients visited the Unit, from all over Greece. The questionnaire was randomly distributed to 146 Greek relatives who approached the Unit for pain relief and the control of other cancer related symptoms. It was drawn using stratified random sampling, based upon the level of first-degree relatedness, i.e. spouse, child, sibling, parent.

Inclusion criteria were a) first-degree relative, b) mentally capable, c) informed consent.

The relatives were asked to complete the questionnaire in the outpatient unit, and were given brief instructions on how to complete the questionnaire (particularly Question 3). The initial measurement consisted of 25-item questions, composed of 6 multi-item scales, and 7 single item scales. Twelve of the scales were presented into 3 optional statements to be scored \"yes\", \"sometimes\", \"no\", and the relatives were asked to mark the appropriate answer accordingly. Question number 3 consisted of 7 factors and relatives had to mark their choice in a hierarchical order. The questionnaire was designed to be self-assessment and was formulated after a thorough review of the relevant literature, incorporating previously validated questions from other scales and measures appropriate for use in palliative care settings for the assessment of relatives\' patient management perceptions and choices \[[@B4]\]\[[@B5]\]\[[@B6]\]\[[@B7]\]. The questionnaire contained 4 sections with different aspects of consultation: information disclosure (Qs, 1, 2, 4, 8); therapy choices (Q 3); support-communication and care (Qs, 5, 6, 7, 9 and 13) and hospitalization (Qs, 10, 11, 12). Patients\' medical records provided information about demographics and disease characteristics, specifically: type of cancer, presence of metastasis, and Karnofsky performance. Similar demographic data was obtained from the relatives: age and sex, with stratification based upon degree of relatedness, spouse, child, parent, sibling; educational status, primary, high school and university; residence, rural, village and town. Multivariate analysis was conducted on these stratifications. However, no analysis was conducted for the stratification residence, as only 10 relatives did not reside in the town. Relatives were also timed during the completion of the questionnaire.

Quantitative variables are described by the mean ± standard deviation. Quantitative aspects were analyzed using *t*-test, one-way and two-way analysis of variance (ANOVA) and categorical variables by Pearson\'s chi-square, where appropriate. The reliability and validity was assessed by average inter-item and average item totals and Cronbach\'s alpha. Statistical significance was accepted with p ≤ 0.05.

Results
=======

The patients\' survival time was between 4 months to 5 years (median: 2 years) while they were off anti-cancer treatment and under palliative care for symptom control. Socio-demographic and clinical data for patients and relatives are presented in Table [1](#T1){ref-type="table"}. In all the questions, except for question number 3, the rate of answers given was 100%, the average time required to complete the questionnaire was 8--10 minutes. Regarding question number 3, 15% of responders did not complete this question.

::: {#T1 .table-wrap}
::: {.caption}
######

Socio-demographic characteristics
:::

                         **Relatives\' Age (mean, ± s.d)**   **Range**
  ---------------------- ----------------------------------- ----------- -------
                                                             **N**       **%**
  **Sex**                Male                                39          27
                         Female                              107         73
  **Education**          Primary                             22          15
                         High School                         73          50
                         University                          51          35
  **Patients\' Sex**     Male                                88          60
                         Female                              58          40

                         **Patients\' Age (mean, ± s.d)**    **Range**
                         67 ± 12                             30--98

                                                             **N**       **%**
  **Cancer Locations**   Lung                                38          27
                         Pancreatic                          18          12
                         Rectum                              15          10
                         Bladder                             14          10
                         Other                               61          42
:::

Descriptive statistics for the questionnaire are presented in Table [2](#T2){ref-type="table"}, for all questions except question 3. Some points of interest that derive from Table [2](#T2){ref-type="table"} include the following. First, a small percentage (23%) of relatives believe that diagnosis and prognosis should be revealed to the patients, while the majority (56% and 51% respectively) of the participants believe that only \"sometimes\" they would agree to do so. However, they are of the opinion that the patients should be given information on the possible treatment choices (71%) and the complications or side effects of the treatment (53%). A large percentage (81.5%) would like a health care team to be involved in caring for their patients, and 76% would like physicians from various specialties to co-operate, when this is judged necessary, and 78% of relatives would like the assistance of a social worker. However, only 44% would choose psychological support from specialists, even though 34% answered that they \"sometimes\" prefer the cooperation with psychologists. Several family members (67%), indicated that they would prefer to inform their patients of the palliative nature of the treatment, but only 44% would discuss with them their fears on death, with 28% \"sometimes\" choosing to discuss about it and 28% not. Only 30% chose the intensive care unit for a patient in the terminal stage, while 57% preferred the patients to stay at home with the provision of the appropriate care. It is worth mentioning that close communication and support are variables, which are now starting to be considered seriously from the vast majority of the relatives (96%).

::: {#T2 .table-wrap}
::: {.caption}
######

Relatives\' attitudes towards their patients\' quality of life
:::

  **Questionnaire items**                                                                                **Percentage (%) of Relatives\'**
  --------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------- --------- ------------------- --------- ------------ ---------
                                                                                **Responding \'yes\'**              ***n***   **\'sometimes\'**   ***n***   **\'no\'**   ***n***
  1\) Do you believe that patient should fully be informed
   a) for the diagnosis?                                                                                23%                                 34        56%                 82        20.5%        30
   b) for the prognosis of the disease?                                                                                23%                                 34        51%                 74        26%          38
  2\) Do you agree the patient to be informed
   a) for the possible treatment choices?                                                                                71%                                 104       24%                 35        5%           7
   b) for the percentage effectiveness?                                                                                51%                                 74        37%                 54        12%          18
   c) for the complications or the side-effects of the treatment?                                                                                53%                                 77        33%                 48        15%          21
  4\) Do you believe that patient should be informed before amputative/disfiguring operations?                                                                    68.5%                               100       21%                 31        10%          15
  5\) Would you like for the caring and treatment of cancer patients to be involved
   a) the health care team?                                                                                81.5%                               119       16%                 24        2.%          3
   b) physicians from various specialties Cooperating as necessary?                                                                                76%                                 111       12%                 17        12%          18
  6\) Would you like to cooperate with psychological support specialists?                                                                                44%                                 64        34%                 50        22%          32
  7\) Would you like to cooperate with a social worker?                                                                                78%                                 114       19%                 28        3%           4
  8\) In the final stage of the patient would you prefer to inform him/her for the palliative nature of the treatment?                                            67%                                 98        24%                 35        9%           13
  9\) Would you like to discuss with the patient his/her fears on death?                                                                                44%                                 64        28%                 41        28%          41
  10\) In order to prolong the life of cancer patient for a few days in the final stage, would you choose the intensive care unit?                                30%                                 44        1 30%               44        39%          58
  11\) Do you believe that the advanced cancer patient when he/she receives the appropriate care should:
   a) be hospitalized                                                                                32%                                 47        30%                 44        38%          55
   b) remain at home                                                                                57%                                 83        23%                 34        20%          29
  12\) According to your opinion, the dying patient should:
   a) remain at hospital?                                                                                34%                                 50        34%                 50        31.5^\*^     46
   b) die at home?                                                                                41%                                 60        29%                 42        30%          44
  13\) In order to prolong the life of cancer patient for a few days in the final stage, is communication and support from the relatives and friends important?   96%                                 140       4%                  6         0            0
:::

The results of question number 3 \"choice of the medical treatment\" are presented in Table [3](#T3){ref-type="table"} (the distribution of factors influencing choice of treatment in a hierarchical order), and the percentage first choice for each of the items is depicted in Figure [1](#F1){ref-type="fig"}. According to the participants, the first choice on the hierarchy is the factor \" expectance of survival\" (28%), while only an 18.5% chose the factor of \"long term quality of life\" as first in the hierarchy. The most common second factor is \"expected effectiveness\" (28%). The \"long-term quality of life\", depicts third place (19%), while 18.5% chose the factor of \"expected effectiveness\". The patients\' \"short-term quality of life\" comes forth in the hierarchy (21%), while the factor of \"possible complications and side effects\" comes fifth (20.5%). The latter, also occupies the sixth place in the hierarchical ranking (23%). As the most common lowest ranked factor was, \"consequences in the patients\' sexuality\" (63%).

![First choices from each factor in \"choice of the medical treatment\".](1472-684X-1-3-1){#F1}

::: {#T3 .table-wrap}
::: {.caption}
######

Ranking variables of treatment choices
:::

                **Expectance of survival**   **Expected Effectiveness**   **Complications Side-effects**   **Consequences in sexuality**   **Patient\'s Choice**   **Short term QoL**   **Long term QoL**
  ------------- ---------------------------- ---------------------------- -------------------------------- ------------------------------- ----------------------- -------------------- -------------------
  **Choices**
  **1st**       41 (28%)                     24(16%)                      4 (3%)                           0                               18(12%)                 8 (5.5%)             27(18.5%)
  **2nd**       20 (14%)                     41 (28%)                     5 (3%)                           0                               11(7.5%)                20(13.5%)            27(18.5%)
  **3rd**       20 (14%)                     27(18.5%)                    17(12%)                          0                               18(12%)                 12 (8%)              28 (19%)
  **4th**       23 (16%)                     12 (8%)                      26(18%)                          4 (3%)                          17(11.5%)               31 (21%)             11(7.5%)
  **5th**       5 (3%)                       17(11.5%)                    30 (20.5%)                       2 (1%)                          24(16%)                 28(19%)              17(11.5%)
  **6th**       12 (8%)                      2 (1%)                       34 (23%)                         26(18%)                         28(19%)                 14 (9.5%)            7 (5%)
  **7th**       3 (2%)                       0                            6 (4%)                           92 (63%)                        8 (5.5%)                11 (7.5%)            4 (3%)
  **missing**   22 (15%)                     22(15%)                      22(15%)                          22 (15%)                        22(15%)                 22(15%)              22(15%)
:::

Reliability of the three different aspects of consultation: information disclosure (Qs, 1, 2, 4, 8), support-communication and care (Qs, 5, 6, 7, 9 and 13) and hospitalisation (Qs, 10, 11, 12) (scales) were assessed by average inter-item correlation, average item total correlation and Cronbach\'s alpha. This initial analysis prompted shortening of the questionnaire via the removal of questions 6 and 13 from the support-communication and care group, and question 12 from the hospitalisation group. This shortening of the questionnaire resulted in improving the average all item total and inter-item correlations to between 0.62--0.70. The internal-consistency reliability coefficients, Cronbach\'s alpha correlations, for the three scales were: information disclosure (Qs, 1, 2, 4, 8) 0.66, support-communication and care (Qs, 5, 6 and 7) 0.69 and hospitalisation (Qs, 10 and 11) 0.50. Using these shortened scales the construct validity revealed that the items were sensibly related to the domains that contained them (convergent validity correlations between 0.6--0.86) and had no association with items from different domains (discriminate validity). This reappraisal of the questionnaire has resulted in the generation of a 21-item scale consisting of 5 multi-item and 5 single-item scales, which demonstrates acceptable validity in assessing relatives\' perceptions and choices for the palliative management of terminal stage cancer patients.

Multivariate analysis of the socio-demographic data was conducted for all remaining scales. For question 3, higher scores were placed on all factors except \"patient\'s long term quality of life\" ranked in the order University \> High School \> Primary (0.001 \> p \> 0.02, ANOVA). A similar trend, Child \> Spouse, was observed for relatedness (as only one relative was a parent and 9 were siblings theses were not included in the analysis) with significance reached in \"consequences in patients sexuality\", \"the patients choice\" and \"patient\'s short term quality of life\" (p= 0.007, 0.003 and 0.009, respectively; *t*-test).

With respect to relatedness, statistical significantly more child than spouse favored \"yes\" to questions 1b) \"for the prognosis of the disease\", 2b) \"for the percentage of effectiveness\", 4) \"amputative/disfiguring operations\" and 5b) \"physicians from various specialties\" (p = 0.001, 0.036, 0.06 and 0.05 respectively, chi-square). Stratification in relation to the sex of the relative demonstrated significance towards men answering \"yes\" to Ib) \"for the prognosis of the disease\", 10) \"final stage\.... intensive care unit\" and 11b) \"remain at home\" (p = 0.012, 0.016 and 0.048 respectively). For women answering \"yes\" 2a) \"for possible treatment choices\" and 4) \"amputative/disfiguring operations\" (p = 0.037 and 0.010 respectively). As for educational status there was a clearly more pronounced firm \"yes\" to various questions with higher educational status (University \> High school \> Primary) for questions 2c) \" complications or side-effects of the treatment\", 4) \"amputative/disfiguring operations\", 5a) \"the health care team\", 5b) \"physicians from various specialties\", 7) \"cooperate with social worker\", 8) \"inform him/her for the palliative nature of the treatment\", 11b) \"remain at home\" (p = 0.028, 0.001, 0.017, 0.018, 0.024, 0.007 and 0.002 respectively); however the reverse rank order (University \< High school \< Primary) was observed for 10) \"final stage\....intensive care unit\" and 11a) \"be hospitalized\" (p = 0.001).

Discussion
==========

Traditionally, studies concerning cancer have focused on the effects of diagnosis, prognosis, treatment or the course of the disease on patients\' well-being which refers to psychological as well as social and physical well-being, components referred to as quality of life, Bloom \[[@B22]\], Lewis and Bloom \[[@B23]\], Meyerowitz \[[@B24]\], Penman, Bloom et al \[[@B25]\]. Most clinicians recognize that cancer is painful for patients and their families, but surprisingly there is little research to document this effect. Most of the studies in this area examine the quality of life according to patient assessments, Covinsky, Goldman et al \[[@B26]\].

In this article the assessment and validation of a questionnaire designed to assess terminal stage cancer patient family perceptions and choices in patient management (information disclosure, therapy choices, hospitalization, support-communication and care) including aspects regarding end-of-life and quality-of-life decisions are presented. The goals of palliative treatment are to improve patients\' functioning and their quality of life \[[@B15]\], input from relatives may offer additional measures in this respect.

The questionnaire was well accepted by all the relatives that approached the unit. On average, it required 8--10 minutes to complete, and relatives appeared to be pleased that they could have the opportunity to participate in a study examining their patients\' quality of life. Following completion of the survey, the reliability and validity of the questionnaire was assessed by average inter-item and average item totals and Cronbach\'s alpha. Re-appraisal of both the support-communication and care and the hospitalization sections indicated that by shortening the initial 25-item scale to a 21-item scale generated a questionnaire that demonstrates acceptable validity and reliability.

In assessing the benefits of the treatment of a fatal illness, health care professionals need to know about the quality of survival. Withholding the truth from a patient appears common in Greece \[[@B27]\], as reported in other nations Niimi \[[@B17]\], \[[@B18]\]\[[@B19]\]; with more information given in the U.S.A and U.K \[[@B28]\]\[[@B29]\]. Many studies suggest that most patients would like to be informed about the diagnosis and prognosis of their disease \[[@B30]\], and evidence suggests that there is little benefit in withholding such disclosure \[[@B17]\]\[[@B18]\]\[[@B31]\]. Frequently, it seems that the caregivers take all the responsibilities or even decide on the patient\'s behalf, and the suffering person remains in ignorance \[[@B32]\]. This is apparent in the present study, where the percentage of the relatives choosing the disclosure of diagnosis or prognosis to their patients is small (23%, and 23% respectively). However, a large percentage (56%) of the relatives believe that only sometimes the diagnosis should be revealed, while 51% indicated that only sometimes the prognosis should be announced.

Multivariate analysis demonstrated that there was a significant trend (p = 0.001) for children of patients over spouse in disclosure of \"prognosis of disease\". A similar trend was observed with male relatives over females (p = 0.012). Furthermore, a trend towards a higher educational status also favored this response (however, this was not significant, p = 0.065), and that for patients\' with cancer devoid of metastasis there was also a significant trend towards disclose (p = 0.028). This was similarly matched by the response to question 3 \"patients long term quality of life\", where only in stratification towards patients with cancer (and not metastasis, p = 0.28) was there a significantly greater interest for hierachical importance. In this respect \"expectance of survival\" (p = 0.008), disclosure \"of the prognosis of disease\" (p = 0.028) and \"the involvement of physicians from various specialties cooperating as necessary\" (p = 0.016) all added support to relatives\' interest and involvement in the decision making process aimed at cure in non-metastatic patients.

Despite the fact that they confront such a dilemma, they are still fairly certain as to their opinion with other aspects such as their attitudes about informing their patients for the possible treatment choices (71%) and for amputative/disfiguring operations (68.5%). Additionally, 53% of the relatives would choose the patient to be aware of the complications or the side effects of the treatment. Again with these latter two aspects there was a statistically significant trend towards relatives with a higher educational status indicating favorably (p = 0.001 and 0.028 respectively). Still, although in most European countries doctors tend to reveal the truth directly to the patient \[[@B27]\]\[[@B28]\]\[[@B29]\] without the family\'s consent, it is still not common practice yet in Greece, possibly as a result of the strong family bonds.

The health care team-patient relationship is a triangle not a dyad, consisting of the health care professionals, the patient and the family. Each part supports the relationship between the other two, and each is affected by what else happens in the triangle \[[@B8]\]\[[@B33]\]. Hence, the involvement of health care teams is very important for the care and treatment of the patients. A high percentage (81.5%) of responders indicated \"yes\", especially the higher educated (p = 0.017), and a high percentage of relatives preferred to cooperate with a social worker (78%, again correlating with the more higher educated (p = 0.024)). The response to the involvement of a psychologist (44%), however, was somewhat contradictory, although only 22% were against it. Cancer affects the family in many ways and it is important for them to have effective co-operation with health care professionals in order to receive care. In the final stage, 67% of family members would prefer to inform patients about the palliative nature of the treatment (p = 0.07 for the higher educated). This is probably due to the fact that the relatives try to preserve the patients\' quality of life until their death.

Ongoing open communication between cancer patients and family members is essential, both between each other and with health care professionals. Patients and family members need to feel supported in their expressions. The health care team can help by letting family members know that emotional stress and behavioral changes are not uncommon during illness, that these and other problems can be overcome with patience and support \[[@B22]\]. It is undoubtedly true, that social support can help people to cope with the psychological effects of cancer and help promote physical recovery; when this support is not available or is withheld the patients\' situation is aggravated \[[@B34]\]. The aspect that arises from the study is the fact that the vast majority of the caregivers give a special emphasis on communication between patients and the social environment (family, friends), and the support they receive with respect to disclosure of treatment choices, the involvement of health care teams, cooperation with social workers and physicians from various specialties, all of which demonstrated a stronger positive correlation with a higher educational status. Such communication provides support to the family -- between health care professionals, patients and relatives -- and the means to overcome the dilemma between disclosing information to the patient on prognosis, diagnosis and the treatment choice.

Conclusions
===========

The study argues that more attention needs to be paid to family members of cancer patients, as they are the primary caregivers in order to have more open-communication with their patients, and to be more empathic and realize the welfare of their patients\' quality of life. Good family interactions can help cancer patients adjust to diagnosis and treatment. Family relationships appear to be particularly important because the family is involved with the patient during the illness. The dynamics of family interactions can set the stage of social interactions with friends and health care providers. After all, cancer is a family illness and disrupts the relationship between them and the health care team.

Competing interests
===================

None declared

Authors\' contributions
=======================

All authors participated in the design of the study, conceived of the study, and participated in its design, coordination and statistical analysis. Finally, all authors read and approved the final manuscript.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Background
==========

Nociceptors are specialized primary afferent neurons and the first cells in the series of neurons that lead to the sensation of pain \[[@B1]-[@B8]\]. The receptors in these cells can be activated by different noxious chemical or physical stimuli \[[@B9]-[@B11]\]. The essential functions of nociceptors include the transduction of noxious stimuli into depolarizations that trigger action potentials, conduction of action potentials from peripheral sensory sites to synapses in the central nervous system, and conversion of action potentials into neurotransmitter release at presynaptic terminals, all of which depend on ion channels \[[@B6],[@B12]-[@B16]\]. Recent expression cloning has led to the identification of the first pain sensory receptor. The cloned receptor is called VR1 (vanilloid receptor subtype 1) \[[@B9],[@B10]\]. The nucleotide sequence of VR1 predicts a protein of 838 amino acids with a molecular mass of 95 kDa. The predicted topological organization consists of six transmembrane domains with a hydrophobic loop between the fifth and sixth domain which lines the ion conducting pore \[[@B17]\]. VR1 has been expressed heterologously in several cell lines and has intrinsic sensitivity to thermal stimuli and to capsaicin (a pungent extract of the *Capsicum* pepper family) \[[@B18]\]. VR1 does not discriminate among monovalent cations \[[@B19]\]; however, it exhibits a notable preference for divalent cations with a permeability sequence of Ca^2+^ \> Mg^2+^ \> Na^+^ ≈ K^+^ ≈ Cs^+^\[[@B9]\]. Ca^2+^ is especially important to VR1 function, as extracellular Ca^2+^ mediates desensitization \[[@B20],[@B21]\], a process which enables a neuron to adapt to specific stimuli by diminishing its overall response to a particular chemical or physical signal. Although not activated by voltage alone, VR1 currents show outward rectification and a region of negative resistance in the current-voltage relation.

The VR1 channel is a member of the superfamily of ion channels with six membrane-spanning domains, with highest homology to the *trp* family of ion channels. For those ion channels within this superfamily for which stoichiometry has been directly examined, all have been shown to be composed of four six-transmembrane domain subunits or pseudosubunits, with auxiliary subunits sometimes present as well \[[@B22]\]. An initial characterization of VR1 channels expressed in Cos and CHO cells has recently revealed that, under certain conditions, they run as multimers on pseudo-native (PFO) gels, with tetramers being one of the primary bands observed \[[@B23]\]. Thus, like other six membrane spanning domain channels, VR1 almost certainly forms as a tetramer; whether it combines with homologous subunits to form heteromeric channels remains to be determined.

In this study we have examined the electrophysiological and biochemical properties of VR1 expressed in *Xenopus* oocytes. We found that its apparent affinity for the ligand capsaicin is comparable to that observed by others. When examined for size on denaturing gels, we found that the monomer appeared to be a doublet and that there was a band that corresponded to roughly twice the molecular weight of the monomer bands. Through site-directed mutagenesis, we determined that the doublet represented unglycosylated and glycosylated forms of the VR1 subunit monomer and identified the glycosylation site as N604. Next, using a VR1 subunit engineered to be of different size, we show that the larger band on the gel represented dimerized subunits. Several mechanisms underlying dimerization were examined and ruled out. Since VR1 likely forms as a tetramer, the strong interaction we observed between pairs of subunits raises the question of whether this subunit interaction is involved in VR1 function.

Results
=======

Electrophysiological and biochemical properties of VR1
------------------------------------------------------

We expressed VR1 channels in *Xenopus* oocytes in order to characterize their electrophysiological and biochemical properties. Using outside-out patch-clamp recordings, we studied the responses of the channels to capsaicin. Figure [1A](#F1){ref-type="fig"} depicts a current family obtained using a saturating (4 μM) capsaicin concentration when the voltage was stepped from a holding potential of 0 mV to from -100 to +100 mV. A current-voltage relation measured from these currents (Figure [1B](#F1){ref-type="fig"}) shows the pronounced outward rectification exhibited by VR1. This rectification has been shown to be independent of external divalent cations \[[@B24]\], and is likely due to a combination of rectification in unitary conductance \[[@B9]\] and voltage-dependent gating \[[@B24]\]. Application of a lower concentration of capsaicin (0.5 μM) activated a smaller fraction of the channels in this patch, giving a smaller overall current (Figure [1C](#F1){ref-type="fig"}). By plotting the normalized currents in the presence of 4 μM and 0.5 μM capsaicin on the same graph (Figure [1D](#F1){ref-type="fig"}), we show that the voltage dependence remains unaltered under both experimental conditions. Figure [1E](#F1){ref-type="fig"} shows a dose-response relation for activation of VR1 channels by capsaicin for the same patch. Fits of dose-response relations with the Hill equation (see Experimental Procedures) yielded values of n = 1.8 ± 0.06 and a K~½~ = 614 ± 110 nM (mean of 5 patches). These values are similar to those previously reported \[[@B20],[@B24]\]. These data indicate that in our system VR1 RNA is expressed as a functional protein with characteristics similar to those reported by other groups.

![**VR1 forms functional channels in Xenopus oocytes.** (A) Current family activated by a saturating capsaicin concentration (4 μM) in an outside-out patch obtained from stepping the voltage from a holding potential of 0 mV to between -100 and +100 mV. For scale bar see (C). (B) Current-voltage relation for currents activated by 4 μM capsaicin. Data were normalized to the value of the current at -100 mV. (C) Current family activated by a sub-saturating capsaicin concentration (0.5 μM). (D) Current-voltage relation for currents activated by 0.5 μM capsaicin (red) with data from currents obtained with 4 μM capsaicin shown also (blue). Data were normalized to the value of the current at -100 mV. (E) Dose-response relation for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K~½~ = 440 nM, I~max~ = 567 pA, and n = 2.2. Filled circles represent actual data values. All data in this figure are from the same patch.](1471-2202-3-4-1){#F1}

To examine the size of VR1 with SDS/PAGE, we constructed a VR1 subunit with a FLAG epitope tag on its extreme C-terminal end (Figure [2A](#F2){ref-type="fig"}). By using both this C-terminal FLAG epitope and an N-terminal epitope for which a commercial antibody is available, we could determine whether the band(s) we observed corresponded to full-length VR1 subunits. When oocytes injected with VR1 RNA were examined with SDS/PAGE and Western blot, three bands were apparent (Figures [2B,2C,2D](#F2){ref-type="fig"}). Two of these bands were seen as a doublet at about 80 kDa and 84 kDa (best seen in Figure [2D](#F2){ref-type="fig"}). A third band of about 200 kDa was also observed. The size of these bands was the same for blots probed with the N-terminal antibody (Figure [2B](#F2){ref-type="fig"}) or the FLAG antibody (Figure [2C](#F2){ref-type="fig"}), indicating that they represent full-length VR1 subunits. For the remainder of this study we used the FLAG antibody due to the lower background it produced.

![**VR1 is glycosylated at N604.** (A) Cartoon (not to scale) of proposed subunit topology for a single VR1 subunit. Shown are the epitopes for the N-terminal and FLAG antibodies used in Western blot experiments, and the consensus sequence for glycosylation, located just distal to the fifth transmembrane domain at position 604. The red circle depicts the approximate localization of this consensus sequence. The line parting from the circle points to the sequence of this N-glycosylation site and the yellow box shows the asparagine which was substituted for a serine in order to produce the glycosylation mutant (N604S). (B) Western blot of oocytes expressing VR1 probed with the N-terminal antibody. Uninjected oocytes and oocytes expressing VR1 were prepared as described in Experimental Procedures. The monomer was observed as a doublet at 80 kDa and at 84 kDa in VR1-injected oocytes and a third band of 200 kDa was also observed in these oocytes. No bands were observed in the uninjected oocytes. (C) Western blot of oocytes expressing VR1 probed with the FLAG antibody. Bands are as in (B). (D) Western blot of oocytes expressing VR1 or N604S. Uninjected oocytes and oocytes expressing VR1 or N604S were prepared as described in Experimental Procedures. The monomer doublet in the VR1 is not present in the N604S mutant; only the 80 kDa band is observed. No bands were observed in the uninjected oocytes. This blot was probed with the FLAG antibody.](1471-2202-3-4-2){#F2}

Identification of the glycosylation site in VR1
-----------------------------------------------

A doublet of monomers has been previously observed for VR1 expressed in CHO cells \[[@B23]\]. In that case, treatment of cells with peptide-*N*-glycosidase F eliminated the larger monomer band, suggesting that it represented a glycosylated form of VR1. Although the difference in apparent size in that study was 19 kDa and we see a difference of only 4 kDa (Figure [2C](#F2){ref-type="fig"}), we wondered whether glycosylation might explain our doublet as well. We therefore examined the predicted amino acid sequence of VR1 in order to identify potential glycosylation sites (see Experimental Procedures). Figure [2A](#F2){ref-type="fig"} depicts the proposed topology of the six transmembrane domains of VR1. A consensus sequence for N-linked glycosylation is located just distal to the fifth transmembrane domain at position 604. We introduced a point mutation to change the asparagine at position 604 into a serine (N604S). If indeed N604 is a glycosylation site, channels expressed from this N604S construct would be expected to lack glycosylation. Figure [2D](#F2){ref-type="fig"} shows that in N604S channels the upper band of the doublet has been eliminated. These data indicate that wild-type VR1 channels expressed in *Xenopus* oocytes are present in both unglycosylated and glycosylated forms and that the N-linked glycosylation occurs at position 604.

Once we determined that the N604S mutation efficiently removed the glycosylated form of the monomer, we tested to be certain that N604S channels were present in the plasma membrane and had electrophysiological characteristics similar to those observed in wild-type channels. We performed patch-clamp recordings on oocytes injected with N604S RNA. The results of these experiments are shown in Figure [3](#F3){ref-type="fig"}. Like for wild-type VR1 channels, capsaicin activated N604S channels in a concentration-dependent manner (Figures [3A, 3C](#F3){ref-type="fig"}, and [3E](#F3){ref-type="fig"}). Current voltage relations at both a saturating (Figure [3B](#F3){ref-type="fig"}) and a subsaturating (Figure [3D](#F3){ref-type="fig"}) concentration of capsaicin show that the voltage dependence of N604S channels is similar to that of wild-type VR1 channels (in Figure [3](#F3){ref-type="fig"} compare data from N604S channels \[blue and red\] to data from wild-type VR1 channels \[green\]): the ratio of the current at +100 mV to that at -100 mV is not statistically different from wild-type VR1. Furthermore, the dose-response relation in Figure [3E](#F3){ref-type="fig"} shows that the apparent affinity of N604S channels for capsaicin is not significantly different from wild-type channels (t-test, p \> 0.05), with n = 2.3 ± 0.3 and K~½~ = 780 ± 71 nM (mean of 5 patches). These data indicate that eliminating the N-glycosylation site of the VR1 channel gives rise to a functional protein electrophysiologically similar to wild-type VR1.

![**N604S forms functional channels, with properties like that of wild type.** (A) Current family activated by a saturating capsaicin concentration (4 μM) in an outside-out patch obtained from stepping the voltage from a holding potential of 0 mV to between -100 and +100 mV. (B) Current-voltage relation for currents activated by 4 μM capsaicin for N604S (blue) and wild-type VR1 channels (green). Data were normalized to the value of the current at-100 mV. (C) Current family activated by a sub-saturating capsaicin concentration (0.5 μM). (D) Current-voltage relation for currents activated by 0.5 μM capsaicin for N604S (red) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (E) Dose-response relations for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K~½~ = 905 nM, I~max~ = 114 pA, and n = 1.8. Filled circles represent actual data values, plotted on a double-log axis. All data in this figure is from the same patch.](1471-2202-3-4-3){#F3}

Identification of the nature of the VR1 high molecular size band
----------------------------------------------------------------

At 200 kDa, the third band observed in Figure [2](#F2){ref-type="fig"} was approximately twice the molecular weight of the monomer. We therefore suspected that it might represent a dimer of VR1 subunits. However, it could also arise from a monomer in combination with another cellular factor. To distinguish between these possibilities, we engineered a VR1 mutant in which amino acids 2--52 in the N-terminal were deleted (termed Δ2--52). This construct gave rise to a high molecular weight band of 180 kDa, 20 kDa smaller than that observed for wild-type VR1 (Figure [4](#F4){ref-type="fig"}). When coexpressed with VR1, heteromeric channels composed of both types of subunits are expected to form. If the higher molecular weight band does represent a dimer of subunits, the heteromeric VR1/Δ2--52 ought to be intermediate in size between the homomeric dimers. When equal amounts of VR1 and Δ2--52 RNA\'s were coinjected into oocytes, three bands were observed: 200, 190 and 180 kDa (Figure [4](#F4){ref-type="fig"}). The 200 kDa band represents the homomeric VR1 dimer whereas the 180 kDa band represent the homomeric Δ2--52 dimer. The middle band, at 190 kDa, is indeed intermediate in size. Our interpretation is that this 190 kDa band is composed of one wild-type VR1 subunit and one Δ2--52 subunit, indicating that the high molecular weight band represents subunit dimers. Furthermore, quantification of the intensity of the bands on the gel revealed a 1:2:1 ratio of the three bands. This ratio of the three ways in which the two types of subunit can assemble is what would be expected if assembly between the different types of subunits were random.

![**The 200 kDa band represents a dimer of VR1 subunits.** Western blot of oocytes expressing wild-type VR1, Δ2--52, or wild-type VR1 + Δ2--52. Oocytes were prepared as described in Experimental Procedures. The band observed for VR1-express ing oocytes was at 200 kDa, the band observed for the Δ2--52-expressing oocytes was at 180 kDa, while oocytes injected with a 1:1 ratio of VR1 and Δ2--52 RNA yield three bands of 200 kDa, 180 kDa, and 190 kDa. This blot was probed with the FLAG antibody.](1471-2202-3-4-4){#F4}

Next, as with N604S, we wanted to be certain that Δ2--52 expressed as a functional channel in *Xenopus* oocytes. The results of electrophysiological experiments are shown in Figure [5](#F5){ref-type="fig"}. We found Δ2--52 to form functional, capsaicin-activated channels in *Xenopus* oocytes. Although an increase in rectification was consistently observed with these channels, compared to wild-type VR1 (Figure [5B](#F5){ref-type="fig"}), the ratio of the current at +100 mV to that at -100 mV is not statistically different from wild-type VR1. Fits of dose-response relations (Figure [5E](#F5){ref-type="fig"}) with the Hill equation yielded a mean n value of 2.1 ± 0.26, and a mean K~½~ value of 1.98 ± 0.8 μM; neither n nor K~½~ were significantly different from VR1 (t-test, p \> 0.05, for 5 patches).

![**Δ2--52 forms functional, capsaicin-activated channels.** (A) Current family activated by a saturating capsaicin concentration (4 μM) in an outside-out patch obtained from stepping the voltage from a holding potential of 0 mV to between -100 and +100 mV. (B) Current-voltage relation for currents activated by 4 μM capsaicin for Δ2--52 (blue) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (C) Current family activated by a sub-saturating capsaicin concentration (0.5 μM). (D) Current-voltage relation for currents activated by 0.5 μM capsaicin for Δ2--52 (blue) and wild-type VR1 (green) channels. Data were normalized to the value of the current at -100 mV. (E) Dose-response relations for activation by capsaicin plotted on a double-log scale. The smooth curves are fits with the Hill equation with K~½~= 390 nM, I~max~ = 1630 pA, and n = 1.5. Filled circles represent actual data values. All data in this figure are from the same patch.](1471-2202-3-4-5){#F5}

Examination of putative cross-linking factors
---------------------------------------------

A previous study has reported that the presence of capsaicin and chemical cross-linkers influence the formation of multimers in VR1 \[[@B23]\]. Moreover, this study reported that cross-linking could be a Ca^2+^ mediated process, through the activation of endogenous transglutaminases. In our expression system, the presence of a dimer was seen even in the absence of capsaicin. Thus, we set out to study the factors that could be involved in the formation of this complex. Our first approach was to determine whether this dimer could be due to the presence of a disulfide bond between subunits. Although we have β-mercaptoethanol in the sample buffer for all experiments, it is possible that a disulfide bond refractory to reduction by this reagent was present. We therefore used the stronger reducing agents DTT and TCEP in the biochemical assays, and used them in various steps of the purification (see Materials and methods). Figure [6A](#F6){ref-type="fig"} shows the results for this experiment. The control lane represents the results obtained from oocytes processed under control conditions (with β-mercaptoethanol as the only reducing agent). The next two lanes shown are those of oocytes exposed to the reducing agents DTT (100 mM) and TCEP (20 mM) both during processing and in the sample buffer. We quantified the ratio of intensity of the monomer band to the dimer band. Neither DTT nor TCEP treatment produced a difference in this ratio compared to the control condition (t-test, p \> 0.05 for 3 independent experiments). These data indicate that the presence of the VR1 dimer is likely not due to an intersubunit disulfide bond.

![**VR1 dimerization is not affected by reducing agents, capsaicin, Ca^2+^ or transglutaminase inhibitors.** (A) Western Blot of the effect of reducing agents on the VR1 dimer. The addition of DTT (100 mM) and TCEP (20 mM) did not modify the ratio of monomer to dimer in VR1-expressing oocytes (p \> 0.05, for 3 independent experiments). (B) Effect of Ca^2+^ on dimer formation in VR1. The addition of Ca^2+^ to the biochemical assays (in the presence or the absence of capsaicin, lanes 1 and 2) did not modify the ratio of monomer to dimer (p \> 0.05, for 3 independent experiment). Addition of EGTA (2 mM) to the assays (lanes 3 and 4) did not modify the monomer to dimer ratio either (p \> 0.05, for 3 independent experiments). For the Ca^2+^-free condition, the expected free Ca^2+^ concentration was 0.4 nM, calculated using WebMax C version 2.1 <>. and assuming a contaminant level of 5 μM Ca^2+^ in our water. For the condition in which Ca^2+^ was present, we added 1.8 mM Ca^2+^ and no chelator to the solution (see Materials and Methods). (C) Effects of transglutaminase inhibitors on dimerization of VR1. The addition of cysteamine (20 mM) and MDC (250 μM) to oocytes did not alter the amount of dimer in relation to monomer when compared to the control lane which did not receive any treatment (p \> 0.05, for 3 independent experiments).](1471-2202-3-4-6){#F6}

We next addressed whether Ca^2+^ could affect the  ratio in our expression system. Oocytes were processed for biochemical assays under the various experimental conditions depicted in Figure [6B](#F6){ref-type="fig"}. In the first lane we show protein obtained from oocytes exposed to Ca^2+^ (1.8 mM in frog Ringer\'s solution) but not to capsaicin; both monomer and dimer bands can be observed. When capsaicin and Ca^2+^ were added together, the ratio of monomer: dimer remained unchanged in comparison to the previous experiment (p \> 0.05, for 3 independent experiments). We then tested whether eliminating Ca^2+^ from the oocyte media would affect this ratio. As seen in the last two lanes of this gel, the presence of EGTA (2 mM) did not alter the formation of the VR1 dimer, whether capsaicin was or was not present in the assay -- the  ratio did not differ from control conditions (p \> 0.05, for 3 independent experiments under the same conditions). Our data indicate that under our experimental conditions Ca^2+^ does not play a pivotal role in VR1 dimerization.

Finally, we tested whether the transglutaminase inhibitors cysteamine and monodansylcadaverine (MDC) could affect this process. As shown in Figure [6C](#F6){ref-type="fig"} (second and third lanes), the addition of these compounds for 1 hr to the solution bathing the oocytes and to the homogenization solution did not modify the  ratio when compared to the control lane (p \> 0.05, for 3 independent experiments). The concentrations of cysteamine and MDC used here are identical to those previously shown to disrupt transglutaminase-induced cross-linking of VR1 channels in other cell types (Kedei *et al*., 2001). This result comes as no surprise since transglutaminases are known to be Ca^2+^ dependent \[[@B25]\], and our previous experiment demonstrates that Ca^2+^ has no effect on the dimerization we observed.

Discussion
==========

In this study we have examined the properties of VR1 in a *Xenopus* oocyte heterologous expression system. We report the following findings: (1) Full-length VR1 runs as a doublet of monomers on SDS gels, with apparent molecular weights of 80 and 84 kDa. The smaller band represents unglycosylated subunits and the larger band represents subunits glycosylated at N604. (2) Channels engineered to lack glycosylation (N604S mutants) are correctly folded and targeted to the plasma membrane, with functional properties similar to wild-type channels. (3) A 200 kDa band is also apparent on VR1 SDS gels. By coexpressing full-length and truncated subunits we show that this band represents a dimer of subunits. (4) The interaction between the pair of subunits in the dimer band was quite strong; the intensity of the dimer band relative to monomer was not affected by capsaicin or calcium, and remained unchanged after treatment with reducing agents and transglutaminase inhibitors.

Unglycosylated and glycosylated forms of the VR1 monomer
--------------------------------------------------------

We show that, in *Xenopus* oocytes, VR1 is expressed in both glycosylated and unglycosylated forms, with \~4 kDa difference in their apparent molecular weights. The complete disappearance of the upper band of the monomer doublet for the N604S mutation strongly suggests that the extracellular linker between the fifth transmembrane domain and the P-loop of VR1 is subject to N-linked glycosylation. Moreover, since channel function did not appear to be affected by the absence of glycosylation (Figure [3](#F3){ref-type="fig"}), it appears that glycosylation at this site is not essential for correct folding and targeting of the protein to the plasma membrane.

The position of the glycosylation site in the structure of the channels is in a region of known importance in channel function. Lying between the fifth transmembrane domain and the P-loop, this region contributes to the extracellular vestibule of the ion-conducting pore \[[@B26]\]. The vestibules of ion channels are thought to influence the permeation of ions \[[@B27],[@B28]\] and in CNG channels it has been reported that elimination of the N-glycosylation site (which is in an analogous position between S5 and the P-loop) can induce changes in the apparent half-blocking constants for extracellular and intracellular Mg^2+^\[[@B29]\].

Whereas two monomer bands had been previously observed for VR1 expressed in CHO cells \[[@B23]\], a major difference between this study and ours is the difference in size between the two monomer bands (19 kDa -- about five times larger than we observe). On the other hand, Kedei *et al*. \[[@B23]\] show a doublet in channels purified from DRG cells, which express VR1 endogenously, that is similar to the one we observe. Further, unlike in CHO cells, no additional high molecular weight glycosylated bands were observed.

Dimerization of VR1
-------------------

As a member of the six-transmembrane domain superfamily of ion channels, VR1 most likely assembles into tetrameric complexes. Evidence that VR1 is capable of forming multimers has been previously reported when studied under pseudo-native conditions \[[@B23]\]. In this previous study, tetramers were the major band observed, although larger and smaller bands were also seen. Interestingly, there is precedent for an ion channel to retain some intersubunit interactions even on denaturing gels like those used here. The bacterial K^+^ channel KcsA, whose X-ray crystal structure has been solved and is definitively a tetramer \[[@B30]\], runs as a tetramer on SDS gels \[[@B31]\]. Furthermore, mutations that disrupt a known intersubunit interface at the level of the pore disrupt this biochemical tetramerization. The structural interactions that underlie tetramerization of KcsA on gels are disrupted only by heating the sample and by pH 12 \[[@B31]\], treatments that had no effect on the dimerization of VR1 we observed (data not shown). Although it is tempting to conclude that the pH- and heat-resistent dimerization we observe with VR1 results from a covalent interaction, we cannot rule out other explanations such as strong hydrophobic interactions.

What does a dimer observed on a gel mean given that the channels are almost certainly tetramers? Precedent for dimerization of limited domains of ion channels abounds. Cyclic nucleotide-gated channels, for example, appear to exhibit functional dimerization of their cyclic nucleotide-binding domains \[[@B32]\]. The \"RCK\" domain of BK channels has a dimerization domain even though BK channels, too, are tetrameric at the level of the pore \[[@B33]\]. Finally, the GluR2 ligand-binding core has a dimerization interface in the crystal structure \[[@B34]\]. Evidence, including the clustering of residues involved in receptor desensitization at this interface, suggests that the dimerization is not just crystallographic but functional. The dimerization observed in the above examples all involve ligand-binding sites. Given that VR1 is also a ligand-activated ion channel it is tempting to speculate that it may too contain such a dimer interface. Recent work identifying amino acid residues that likely comprise part of the capsaicin-binding site \[[@B35]\] suggest that capsaicin binds at the interface between transmembrane segment 3 and the cytoplasm. Could this be a point of intersubunit contact? Although the dimerization we observe may represent a native intersubunit interaction, other possibilities must be considered. For example, hydrophobic interactions can cause membrane proteins to aggregate during purification. Alternatively, a covalent interaction may link pairs of subunits. This possibility will be investigated in the future.

Materials and methods
=====================

Heterologous expression of channels in Xenopus oocytes
------------------------------------------------------

Segments of ovary were removed from anesthetized *Xenopus laevis.* After gross mechanical isolation, individual oocytes were defolliculated by incubation with collagenase 1A (1 mg/mL) in Ca^2+^-free OR2 medium (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl~2~, 5 mM HEPES, pH 7.6) for 1.5--3 hours. The cells were then rinsed and stored in frog Ringer\'s solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl~2~, 1 mM MgCl~2~, 5 mM HEPES, pH 7.6) at 14°C. Oocytes were injected with 50 nL mRNA solution within two days of harvest.

Electrophysiology
-----------------

Electrophysiological recordings and/or biochemistry were performed 4--8 days after injection. After brief exposure to a hypertonic medium, the vitelline membrane was stripped from each oocyte with forceps. Outside-out patch-clamp recordings were made using symmetrical NaCl/HEPES/EDTA solutions consisting of 130 mM NaCl, 10 mM HEPES, 1 mM EDTA and 10 mM EGTA (pH 7.2). Capsaicin was prepared as a 4 mM stock in dry ethanol and was added to the extracellular solution only. The solution bathing the extracellular surface of the patch was changed using a RSC-200 rapid solution changer (Molecular Kinetics, Pullman, WA). Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Pipettes were polished to a resistance of 0.3--1 MΩ and immediately before use were dipped in a seal glue composed of one part light mineral oil, one part heavy mineral oil, and 10% parafilm beads to promote formation of high-resistance seals \[[@B36]\]. Currents were low pass-filtered at 2 kHz and sampled at a 10 kHz with an Axopatch 200B (Axon Instruments, Union City, CA). Data were acquired and analyzed with the PULSE data acquisition software (Instrutech, Elmont, NY) and were plotted and fit using Igor Pro (Wavemetrics Inc., Lake Oswego, OR). All currents shown are difference currents in which the current in the patch in the absence of capsaicin has been subtracted. All dose-response curves were measured at +100 mV at room temperature. Smooth curves shown in dose-response relations are fits with the Hill equation:

![](1471-2202-3-4-i1.gif)

where I is the current at a given concentration of capsaicin, Imax is the maximal current, K~½~ is the concentration of half-maximal activation and n is the Hill coefficient. Current-voltage relations were plotted using the data obtained from voltage jumps from -100 to +100 mV for 100 ms from a holding potential of 0 mV. Data was normalized by dividing the values of the current at different voltages by the value of the current at -100 mV. When pooled data are discussed in the text, they represent the mean ± standard error of the mean (SEM) A Student\'s t-test (two-tailed) was performed on some data, as discussed in the text. The significance level was set at p \> 0.05.

Mutagenesis
-----------

A potential glycosylation site was identified by screening the predicted vanilloid receptor 1 (VR1) channel amino acid sequence \[[@B9]\]. A glycosylation consensus sequence of N-X-T/S was found at positions 604--606 (NNS). A point mutant was constructed as outlined below to replace the asparagine at position 604 with a serine. The mutant construct was designated as N604S.

The point mutation and deletion mutation were constructed by a method involving oligonucleotides synthesized to contain a mutation in combination with wild-type oligonucleotides in PCR amplifications of fragments of the cDNA. The product of the PCR reaction was then cut with two different restriction enzymes to generate a cassette containing the mutation. The cassette was then ligated into the channel cDNA cut with the same two restriction enzymes. After transformation of bacteria with the ligation product, single isolates were selected, and the entire region of the amplified cassettes was sequenced to check for the mutation and insure against second-site mutations. mRNA was synthesized in vitro, using a standard reverse-transcription kit (mMessage mMachine, Ambion, Austin, TX). All constructs, including VR1, were made in a background of a VR1 subunit in which the FLAG epitope (DYKDDDDK) had been spliced on to the C-terminus. The presence of this epitope was found to have no detectable effects on the electrophysiological properties of the channels (data not shown).

SDS/PAGE and Western blot
-------------------------

Oocytes were prepared after the method of Rho *et al*. \[[@B29]\]. Typically 30 oocytes were lysed by trituration in 200 μL of a solution containing 100 mM Tris-HCl, 100 mM NaCl, 0.5% Triton X-100, 0.05 mg/mL pepstatin, 0.05 mg/mL leupeptin, and 0.05 mg/mL aprotinin (pH 8.0) and the homogenate was incubated for 15 minutes on ice. The homogenate was then centrifuged at 18,400 g for ten minutes at 4°C in a Jouan CR3i centrifuge. The soluble portion of the homogenate was then transferred to a new tube for an additional centrifugation. 10 μL of supernatant was removed, mixed with 20 μL of Laemmli sample buffer containing β-mercaptoethanol (19:1) and incubated at room temperature for five minutes. The samples were then subjected to SDS/PAGE using NuPage 3--8% or 7% Tris-Acetate precast gels (Invitrogen Corp., Carlsbad, CA). Proteins were then transferred to a PVDF membrane and Western blot analysis was performed. For all blots except that shown in Figure [2B](#F2){ref-type="fig"}, M2 anti-FLAG primary antibody was used, and for the blot shown in Figure [2B](#F2){ref-type="fig"} a polyclonal antibody raised against the N-terminal sequence of VR1 (amino acids 4--21: RASLDSEESESPPQENSC) was used (Neuromics Inc., Minneapolis, MN). Chemiluminescent detection was then carried out using the SuperSignal West Femto kit (Pierce, Rockford, IL). Chemiluminescent signals were captured with the Flourchem Imager (Alpha Innotech, San Leandro, CA), which has a linear range of 4 O.D. units. Densitometry of bands on Western blots was done with the Flourchem software. For comparison of the ratio of monomer to dimer, we included both monomer bands in the monomer category. Because of the large linear range of detection of our instrument, we could compare this ratio with accuracy even if the amount of total protein varied between gels.

For SDS/PAGE experiments on disulfide bonds, the oocytes were treated as above, except that the lysis buffer contained either 100 mM DTT or 20 mM Tris (2-Carboxyethyl) Phosphine Hydrochloride (TCEP) (Pierce, Rockford, IL). To examine the effect of calcium and capsaicin on VR1 dimer formation, four different conditions were tested. For calcium-free condition, whole oocytes were rinsed three times with calcium-free frog Ringer\'s solution (96 mM NaCl, 2 mM KCl, 2 mM EGTA, 1 mM MgCl~2~, 5 mM HEPES, pH 7.6) prior to incubation with or without 10--20 μM capsaicin at room temperature for 30 minutes. For calcium- present condition, whole oocytes were bathed in frog Ringer\'s solution with or without 10--20 μM capsaicin for the same duration. The oocytes were triturated according to the method described above, except for the capsaicin-present condition, where 10--20 μM capsaicin was included in the lysis buffer. For experiments on transglutaminase inhibitors, the oocytes were bathed for one hour in frog Ringer\'s solution containing either 20 mM cysteamine or 250 μM MDC. These are expected to be saturating concentrations of the transglutaminase inhibitors. Oocytes were triturated according to the method above, except that either 20 mM cysteamine or 250 μM MDC were included in the lysis buffer.

Acknowledgments
===============

We would like to thank Dr. David Julius for his kind gift of the VR1 cDNA clone and Dr. Leon Islas for helpful discussions.